Reviews in Aquaculture (2016) 0, 1–11 doi: 10.1111/raq.

12145

Cryopreservation and vitrification of fish semen: a review

with special emphasis on marine species

n Valdebenito2 and
Caio Magnotti1, Vinicius Cerqueira1, Manuel Lee-Estevez3, Jorge G. Farias3, Iva
El
ıas Figueroa 2,3

polis, Brazil
1 Aquaculture Department, Universidade Federal de Santa Catarina, Floriano
2 School of Aquaculture, Catholic University of Temuco, Temuco, Chile
3 Department of Chemical Engineering, Faculty of Engineering and Sciences, Universidad de La Frontera, Temuco, Chile

Correspondence
El
ıas Figueroa, School of Aquaculture, Catholic
University of Temuco, Temuco, Chile.
Email: efigueroa@uct.cl
Received 14 November 2015; accepted 23 Jan-
uary 2016.
Abstract
Semen conservation methods have been extensively studied for fish species over
the last 60 years. Cryopreservation techniques can be divided into two types: one
with slow cooling rates like traditional freezing and the other with ultrarapid rates
as used in vitrification. These protocols increase the time over which semen sam-
ples can be used for reproduction or sperm quality analysis. Marine fish possess
greater resistance than freshwater species to variations in osmolality. This favours
the application of these methods, which produce better results after thawing and
offer high biotechnological potential for aquaculture. In the last 15 years, sperm
cryopreservation studies have been carried out in 32 marine species, but there are
few analyses of the effects of freezing on the morphology and physiology of sperm
cells. The object of this review was to provide recent data on semen cryopreserva-
tion in marine fish species and to suggest variables which may be applied in future
research.
Key words: cryoprotectant, extender, milt, slow freezing, spermatozoa.

asynchronic gonad maturity between males and females;

Introduction
Developing gamete conservation programmes will be very
useful in future reproduction studies of fish of commercial
interest. Gamete conservation is an important tool for fish
reproduction and of great interest for fish-farming. It can
be used routinely in reproduction laboratories (Maria &
Carneiro 2012). Growing interest in improving the tech-
nique has led to an increase in the number of studies on
this subject (Carolsfeld et al. 2003; Tiersch 2011; Maria &
Carneiro 2012). It is thus now possible to start using pre-
served semen in the routine reproduction of fish and appli-
cation to aquaculture practice (Maria & Carneiro 2012).
Gamete preservation by cooling to 4°C can be used for
short-term storage (Ubilla et al. 2014), while cryopreserva-
tion is used for the long term (Oliveira et al. 2007). The lat-
ter can be done either by slow freezing or by a very rapid
process called vitrification (Merino et al. 2012; Cuevas-
Uribe et al. 2013; Figueroa et al. 2015a). These techniques
present various advantages: conservation of genetic mate-
rial from wild animals originating from remote locations or
where access is difficult; elimination of problems caused by
use of gametes from animals selected for improvement pro-
grammes or subject to genetic manipulation (tetraploids,
clones, transgenic material); reduction in the costs and risks
of transporting live animals; and establishment of hybridis-
ation programmes using fish with different reproduction
periods (Suquet et al. 2000; Linhart et al. 2005; Tiersch
et al. 2007; Maria et al. 2009; Gwo 2011).
The first work done on freezing fish semen was done in
1953 by Blaxter, who made it possible to cross different
populations of Atlantic herring (Clupea harengus) which
spawn at different times of year (Suquet et al. 2000; Liu
et al. 2006). Semen preservation protocols have been deter-
mined experimentally with more than 200 species of fish
(Billard & Zhang 2001; Gwo 2011), of which approximately
40 are marine species (Suquet et al. 2000; Gwo 2011).
Cryopreservation of marine fish semen normally presents
better results for motility rate and fertilising capacity after
thawing in species like sea bass and turbot, with mean
embryo survival of 80%, than in freshwater species like
Atlantic salmon, rainbow trout and carp which present
embryo survival of around 65% with cryopreserved semen

© 2016 Wiley Publishing Asia Pty Ltd 1

C. Magnotti et al.
(Scott & Baynes 1980; Suquet et al. 2000; Gwo 2011). These
results are probably related with the fact that spermatozoa
from marine species are adapted to high osmotic pressures
(Cuevas-Uribe et al. 2013).
In recent years, due to the rapid development of aquacul-
ture and the conservation problems facing some fish spe-
cies, cryopreservation has played an important role in
freezing gametes to protect fish of high economic and bio-
logical value. Among salmonids, these include rainbow
trout (Oncorhynchus mykiss), brown trout (Salmo trutta),
brook trout (Salvelinus fontinalis) and charr
(Salvelinus alpinus) (Lahnsteiner et al. 1996; Cabrita et al.
1998; Mart
ınez-P
aramo et al. 2009). Other species of com-
mercial interest which need protection are the sturgeons,
such as beluga (Huso huso), sterlet (Acipenser ruthenus),
pallid (Scaphirhynchus albus) Siberian, European and Rus-
sian (Aythya baeri, Acipenser sturio, Acipenser guelden-
staedtii) and the shortnose (Acipenser brevirostrum)
(Glogowski et al. 2002; Horv
ath et al. 2005, 2006, 2008;
Cabrita et al. 2010); also catfish like the African and Euro-
pean catfish (Clarias gariepinus, Silurus glanis) and carps
like the common (Cyprinus carpio) and silver carp
(Hypophthalmichthys molitrix) (Horv
ath & Urbanyi 2000;
Suquet et al. 2000; Jenkins & Draugelis-Dale, 2006; Alvarez
et al. 2008; Maisse et al. 2008; Viveiros & Komen 2008;
Cabrita et al. 2010).
However, most of these studies are designed to select
technical parameters, analysing sperm viability and quality
to obtain greater viability than in studies carried out on
freshwater fish. Various factors such as the freezing or
thawing rate, the effects of the concentration of cryoprotec-
tants on fertilisation, the equilibrium time and warming
methods have not been measured, reported or rigorously
tested in some species (Liu et al. 2006; Gwo 2011). Accord-
ing to Suquet et al. (2000), this trend is in line with world
patterns: there is a lack of research giving compete
descriptions of the morphological and metabolic changes
generated by semen conservation techniques. Gwo (2011)
notes that most works are still carried out experimentally,
based on results obtained by trial and error, and that no
standardised protocol has been produced. Many unresolved
questions therefore still remain about techniques for cryop-
reserving the spermatozoa of marine fish of economic and
environmental interest (Carolsfeld et al. 2003).
Cryopreservation protocols
Cryopreservation is the conservation of biological material
in liquid nitrogen at -196°C, which theoretically allows its
viability to be maintained indefinitely (Carolsfeld et al.
2003; Bakhach 2009). At this temperature, the structure
and functioning of live cells and tissues are maintained,
keeping them genetically viable and reversibly inactive,
2 © 2016 Wiley Publishing Asia Pty Ltd
from a metabolic perspective (Pegg 2007; Bakhach 2009).
This method can be used by reducing the temperature of
the material either slowly (traditional method) or rapidly
(vitrification) (Merino et al. 2012; Cuevas-Uribe et al.
2013; Figueroa et al. 2015a). The practical difference
between them is that the common method of cryopreserva-
tion causes ice crystals to form in the extracellular medium,
while no crystals are formed in vitrification (Cuevas-Uribe
et al. 2013). Between 2000 and 2015, the parameters for
cryopreservation by slow freezing and vitrification were
studied in 32 species of marine fish of commercial interest
or in danger of extinction (Table 1).
Slow freezing
Maria and Carneiro (2012) cite the normal sequence of
events in cryopreserving fish semen as including (i) semen
collection, (ii) microscopic assessment of semen quality,
(iii) addition of diluents and cryoprotectants, (iv) packag-
ing, (v) freezing and storage, (vi) thawing and (vii) fertilisa-
tion and monitoring the development of embryo and larva
stages. According to Gwo (2011), the possible variables
which may influence the fertilising capacity of cryopre-
served spermatozoa are (i) diluents, (ii) dilution, (iii) cry-
oprotectants, (iv) concentration of cryoprotectants, (v)
equilibrium time, (vi) freezing speed, (vii) freezing method,
(viii) thawing temperature, (ix) sperm quality, (x) oocyte
quality and (xi) sperm/oocyte ratio. These eleven points
listed by Gwo (2011) are important, as different fish species
tend to present different sperm sensitivities to dilution
levels and the substances employed in semen cryopreserva-
tion, for each of which specific protocols need to be
developed (Murgas et al. 2007; Tiersch et al. 2007). Suquet
et al. (2000) present general tables showing the diluents
and cryoprotectants most frequently used for marine
species, while Cabrita et al. (2010), Gwo (2011) and Fig-
ueroa et al. (2015a) present updated tables and results of
fertility with the protocols and formula of each diluent, cry-
oprotectants and the use of seminal plasma, but mainly for
freshwater species.
The first step in developing a cryopreservation protocol
is to choose the best composition of the diluent, generally a
saline or glucose solution with suitable osmolality contain-
ing a cryoprotection agent (Viveiros et al. 2012). The most
important characteristics of a diluent are that it should not
initiate sperm motility (Gwo 2011), and that it should be
sterile and remain stable throughout storage. The purpose
of this solution is to dilute the semen, which in some fish
presents high viscosity and low volume. In recent years,
most of the diluents used in marine fish contained salts,
Tris buffer (pH stabiliser), small quantities of monosaccha-
rides or disaccharides (glucose, saccharose, fructose,
lactose, trehalose, etc.) and protein (bovine serum albumin
Reviews in Aquaculture (2016) 0, 1–11
 Table 1 Extender composition, cryoprotectant medium and dilution used to ‘slow freezing’ and ‘ultra-rapid cooling’ sperm of marine fish from 2000 to 2015

Common name Species Extender Extender composition Cryoprotectant (v:v) Authors

Slow freezing
Pacific bluefin tuna Thunnus orientalis – 171.12 mM NaCl 20% DMSO or 1:9 Gwo et al. (2005)
10–20% Gly or
10% Methanol
Atlantic cod Gadus morhua – 137 mM NaCl, 11 mM KCl, 4 mM Na2HPO4 7H2O; pH 7.7 10% PG 1:3 Rideout et al. (2004)

© 2016 Wiley Publishing Asia Pty Ltd

Mounib 125 mM sucrose, 100 mM KHCO3, 6.5 mM reduced 10% PG 1:3 Rideout et al. (2004)

Reviews in Aquaculture (2016) 0, 1–11

glutathione
Haddock Melanogrammus – 137 mM NaCl, 11 mM KCl, 4 mM Na2HPO4 7H2O; pH 7.7 10% PG 1:3 Rideout et al. (2004)
aeglefinus
Mounib 125 mM sucrose, 100 mM KHCO3, 6.5 mM reduced 10% PG 1:3 Rideout et al. (2004)
glutathione
ocean Pout Macrozoarces – 1.45 mM CaCl2, 20% DMSO 1:3 Yao et al. (2000)
americanus 0.84 mM MgSO4,
10.25 mM KHCO3,
183 mM NaCl,
0.15 mM glucose
Longtooth grouper Epinephelus LG-ASP2 9.00 g L 1 NaCl, 0.10 g L 1 KCl, 0.06 g L 1 CaCl2, 0.08 g 10% Gly 1:2 Lim and Le (2013)
bruneus L 1 MgCl2, 0.50 g L 1 NaHCO3, 0,02 g L 1 Albumin; pH
7,8, 325,8 mOsMol kg 1
Orange-spotted Epinephelus – 150 mM NaCl; pH 7.5–8, 275 mOsMol kg 1 15–20% Trehalose 1:1 Peatpisut and Bart
grouper coioides (2010)†
Malaba grouper Epinephelus – 300 mM glucose 20% DMSO 1:9 Gwo and Ohta (2008)
malabaricus
– 150 mM NaCl 20% DMSO 1:9 Gwo and Ohta (2008)
1
Dusky grouper Epinephelus – 6.50 g L 1 NaCl, 3.00 g L 1 KCl, 0.30 g L CaCl2, 0.20 g 5% DMSO 1:3 Sanches et al. (2008)
marginatus L 1 NaHCO3; pH 7.8, 157 mOsMol kg 1
– 171.12 mM NaCl, 1% BSA 10% DMSO 1:9 Cabrita et al. (2009)‡
Kelp grouper Epinephelus – 15% trehalose No cryoprotectant 1:2 or 1: 4 Miyaki et al. (2005)
moara
Seven-band grouper Epinephelus FBS 95% Foetal Bovine Serum 5% DMSO 1:49 Koh et al. (2010)
septemfasciatus
1 1 1
ES1-3 60.00 g L glucose, 10.00 g L NaCl, 0.50 g L NaHCO3 10% DMSO or 1:1 Tian et al. (2013)‡
10% PG
Mutton snapper Lutjanus analis – 7.89 g L 1 NaCl, 1.19 g L 1 KCl, 0.22 g L 1 CaCl2, 0.73 g 10% DMSO 1:3 Sanches et al. (2013)
L 1 MgCl2, 0.08 g L 1 NaH2PO4, 0.84 g L 1 NaHCO3; pH
8.2, 172 mOsMol kg 1
Red snapper Lutjanus Ringer 7.50 g L 1 NaCl, 0.20 g L 1 NaHCO3, 0.20 g L 1 KCL, 10% DMSO 1:1 Vuthiphandchai
argentimaculatus solution 0.20 g L 1 CaCL2 2H2O, 5.00 g L 1 glucose; pH 7.9, 315 et al. (2009)
mOsmol kg 1
Cryopreservation and vitrification in sperm fish

3
 4
Table 1 (continued)

Common name Species Extender Extender composition Cryoprotectant (v:v) Authors

Red snapper Lutjanus C-F HBSS 5.26 g L 1 NaCl, 0.26 g L 1 KCl, 0.33 g L 1 NaHCO3, 0. g 10% DMSO 1:3 Riley et al. (2004)
campechanus L 1 Na2HPO4, 0.04 g L 1 KH2PO4, 0.13 g L 1 MgSO4
C. Magnotti et al.

7H20, 0.66 g L 1 glucose; 200 mosMol kg 1

Lane snapper Lutjanus synagris 1% Riger 6.50 g L 1 NaCl, 3.00 g L 1 KCl, 0.20 g L 1 NaHCO3, 10% DMSO + 8% 1:2 Gaita

n-Espitia et al.
solution 0.30 g L 1 CaCl2; pH 8.0-8.5 egg yolk/milk (2013)
– 7.89 g L 1 NaCl, 1.19 g L 1 KCl, 0.22 g L 1 CaCl2, 0.73 g 10% DMSO 1:3 Sanches et al. (2015)
L 1 MgCl2, 0.08 g L 1 NaH2PO4, 0.84 g L 1 NaHCO3; pH
8.2, 172 mOsMol kg 1
Atlantic halibut Hippoglossus MTE 70 mM NaCl, 1.5 mM KCl, 2.7 mM CaCl2, 25 mM NaHCO3, 10% DMSO or 1:3 Babiak et al. (2008)
hippoglossus 6.1 nM MgCl, 200 mM glucose, 10 mg L 1 BSA; pH 8.1, 10% DMA or 10%
400 mOsMol kg 1 Methanol
KS 100 mM KHCO3, 125 mM sucrose; 315 mOsMol kg 1 10–15% DMSO 1:3 Ding et al. (2011)
HBSS HBSS, 273 mOsm (Sigma, H-1387) 10–15% DMSO 1:3 Ding et al. (2011)
Summer flounder Paralichthys Hanker 7.25 g L 1 NaCl, 0.38 g L 1 KCl, 0.18 g L 1 CaCl2, 1.00 g 15% DMSO or 1:3 Liu et al. (2015)
dentatus extender L 1 NaHCO3, 0.23 g L 1 MgSO4 7H2O, 0.41 g L 1 15% PG
NaH2PO4 H2O, 1.00 g L 1 glucose
Flounder Paralichthys ASW 24.72 g L 1 NaCl, 0.67 g L 1 KCl, 1.36 g L 1 CaCl2 2H2O, 12% Gly 1:2 Zhang et al. (2003)
olivaceus seawater 4.66 g L 1 MgCl2 6H2O, 6.29 g L 1 MgSO4 7H2O, 0.18 g
L 1 NaHCO3; pH 8.2, 205 mOsmol kg 1
Brazilian flounder Paralichthys TS-2 110 mM Sucrose, 100 mM KHCO3, 10 mM Tris-Cl; pH 8.2, 10% DMSO 1:3 Lanes et al. (2008)
orbignyanus 335 mOsMol kg 1
Winter flounder Pseudopleuronectes – 137 mM NaCl, 11 mM KCl, 4 mM Na2HPO4 7H2O; pH 7.7 PG 1:3 Rideout et al. (2003)
americanus
Mounib 125 mM sucrose, 100 mM KHCO3, 6.5 mM reduced PG 1:3 Rideout et al. (2003)
glutathione
Turbot Scophthalmus TS-2 110 mM Sucrose, 100 mM KHCO3 and 10 mM Tris-Cl; pH 10% DMSO 1:3 Chen et al. (2004)
maximus 8.2, 335 mOsMol kg 1
Mounib modified 125 mM sucrose, 100 mM KHCO3, 6.5 mM reduced 10% DMSO 1:1 Chereguini et al.
glutathione, 1% BSA (2003)
Spotted halibut Verasper TS-2 110 mM Sucrose, 100 mM KHCO3 and 10 mM Tris-Cl; pH 13.3% DMSO 1:2 Tian et al. (2008)
variegatus 8.2, 335 mOsMol kg 1 or 13.3% PG
Red sea bream Pagrus major Hank’s extenders 8.00 g L 1 NaCl, 0.40 g L 1 KCl, 0.14 g L 1 CaCl2, 0.10 g 15% DMSO 1:3 Chen et al. (2010)
L 1 MgSO4 7H2O, 0.10 g L 1 MgCl2 6H2O, 0.06 g L 1
Na2HPO4 12H2O, 0.35 g L 1 NaHCO3, 1.00 g L 1 glucose
Cortland 7.25 g L 1 NaCl, 0.38 g L 1 KCl, 0.18 g L 1 CaCL2, 1.00 g 15–20% DMSO 1:3 Liu et al. (2006)
L 1 NaHCO3, 0.41 g L 1 Na2HPO4, 0.23 g L 1 MgSO4 or 9-12% EG
7H20, 1.00 g L 1 glucose; pH 7,0
Gilthead sea Sparus aurata – 1% NaCl, 10 mg L 1 BSA; 300 mOsMol kg 1 5% DMSO 1:6 Cabrita et al. (2005)
bream
– 1% NaCl 5% DMSO or 1:6 Fabbrocini et al.
10% PG or 10% EG (2000)

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Reviews in Aquaculture (2016) 0, 1–11
 Table 1 (continued)

Common name Species Extender Extender composition Cryoprotectant (v:v) Authors

– 1% NaCl 5% DMSO 1:6 Fabbrocini et al.

(2015)
Sea perch Lateolabrax Riger solution 60.35 mM NaCl, 1.80 mM NaH2PO4, 3 mM NaHCO3, 10%DMSO 1:1 Ji et al. (2004)
japonicus modified 5.23 mM KCl, 1.3 mM CaCl2 2H2O, 1.13 mM MgCl2 6H2O,

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Reviews in Aquaculture (2016) 0, 1–11
55.55 mM glucose
Fat snook Centropomus – 7.89 g L 1 NaCl, 1.19 g L 1 KCl, 0.22 g L 1 CaCl2, 0.73 g 10% DMSO – Tiba et al. (2009)
parallelus L 1 MgCl2, 0.081 g L 1 NaH2PO4, 0.84 g L 1 NaHCO3; pH
8.2, 172 mOsMol kg 1
Common snook Centropomus ASW seawater 24.72 g L 1 NaCl, 0.67 g L 1 KCl, 1.36 g L 1 CaCl2 2H2O, 5–10% DMSO or 5% 1:3 Tiersch et al. (2004)
undecimalis 4.66 g L 1 MgCl2 6H2O, 6.29 g L 1 MgSO4 7H2O, 0.18 g Methanol
L 1 NaHCO3; pH 8.2, 205 mOsmol kg 1
European sea Dicentrachus – 1% NaCl; pH 5.4, 297 mOsMol kg 1 10% EG 1:6 Sansone et al. (2002)
bass labrax
NAM + taurine 59.83 mM NaCl, 1.47 mM KCl, 12.91 mM MgCl2, 3.51 mM 10% DMSO 1:6 aramo
Mart
ınez-P

or hypotaurine CaCl2, 20 mM NaHCO3, 0.44 mM glucose, 1% BSA, et al. (2013)
1 m1 mM hypotaurine; pH 7.7 taurine or 1 m1 mM
hypotaurine; pH 7.7 hypotaurine; pH 7.7
Patagonian blenny Eleginops maclovinus Stopmiltâ 1.5 DMSO + 0.4M 1:4 E. Figueroa et al.,
glucose + 2% BSA unpublished data)
Golden kingklip Genypterus blacodes Stopmiltâ 1.2M DMSO + 0.3M 1:3 E. Figueroa et al.,
sucrose + 1% BSA unpublished data
Southern hake Merluccius australis Stopmiltâ 1.2M DMSO + 0.3M 1:3 E. Figueroa et al.,
sucrose + 2% BSA unpublished data
Ultra-rapid cooling
(Vitrification)
Red snapper Lutjanus C-F HBSS 5.26 g L 1 NaCl, 0.26 g L 1 KCl, 0.33 g L 1 NaHCO3, 15% DMSO + 15%EG + 1:1 Cuevas-Uribe
campechanus 0.04 g L 1 Na2HPO4, 0.04 g L 1 KH2PO4, 0.13 g L 1 10% Gly + 1% X-1000tm + et al. (2013)
MgSO4 7H20, 0.66 g L 1 glucose; 200 mOsMol kg 1 1% Z-1000tm
Spotted sea trout Cynoscion C-F HBSS 5.26 g L 1 NaCl, 0.26 g L 1 KCl, 0.33 g L 1 NaHCO3, 15% DMSO + 15%EG + 1:1 Cuevas-Uribe
nebulosus 0.04 g L 1 Na2HPO4, 0.04 g L 1 KH2PO4, 0.13 g L 1 10% Gly + 1% X-1000tm + et al. (2013)
MgSO4 7H20, 0.66 g L 1 glucose; 200 mOsMol kg 1 1% Z-1000tm
Red drum Sciaenops ocellatus C-F HBSS 5.26 g L 1 NaCl, 0.26 g L 1 KCl, 0.33 g L 1 NaHCO3, 15% DMSO + 15%EG + 1:1 Cuevas-Uribe
0.04 g L 1 Na2HPO4, 0.04 g L 1 KH2PO4, 0.13 g L 1 10% Gly + 1% X-1000tm + et al. (2013)
MgSO4 7H20, 0.66 g L 1 glucose; 200 mOsMol kg 1 1% Z-1000tm

†sex-reversed. ‡normal and sex-reversed. BSA, bovine serum albumin; DMA, Dimethylamine; DMSO, Dimethyl sulphoxide; EG, Etylene glycol; Gly, Glycerol; PG, Propylene glycol.
Cryopreservation and vitrification in sperm fish

5
C. Magnotti et al.
– BSA) with pH of 7.0–8.2 and osmolality of 205–400
mOsMol kg 1 (Table 1). The objective of adding an easily
metabolised sugar to the diluent is to enable the spermato-
zoa to obtain energy, which is found naturally in the
seminal plasma of mammals, insects, nematodes and fish
(Gregory 1968; Bozkurt et al. 2009; Dziewulska et al.
2010;). BSA acts as an antioxidant, eliminating the free rad-
icals generated by oxidative stress, and thus protecting the
cell membrane from lipids peroxidation and also interact-
ing directly with membranes preventing sperm cells from
sticking with each other (clumping) (Lewis et al. 1997).
Some other substances can be added to improve cell pro-
tection against the effects of freezing, physiological post-
thawing parameters and sperm activation. Organic acids at
low concentration, 1 mM of taurine or hypotaurine, will
increase motility, velocity and linearity in the movement of
European sea bass (Dicentrarchus labrax) spermatozoa cry-
opreserved in non-activating medium (NAM) and 10%
DMSO postactivation, analysed with CASA (computer-
assisted sperm analysis) (Mart
ınez-P
aramo et al. 2013).
Antioxidant substances like ascorbic acid, vitamin E,
reduced glutathione, reduced methionine, carnitine and
uric acid can be added to eliminate free radicals of the
superoxide, peroxide and hydroxide types which lead to
peroxidation of phospholipids in the mitochondria, caus-
ing death of the sperm cell (Ciereszko & Dabrowski 1995;
Anghel et al. 2010). Their effectiveness in freshwater teleost
fish was proved by Lahnsteiner (2009), Lahnsteiner et al.
(2010), Lahnsteiner and Mansour (2010) and Ubilla and
Valdebenito (2011).
The function of the cryoprotectant is to protect the cell
against the formation of intracellular ice crystals and
excessive dehydration which produce irreversible damage
in the plasma and nuclear membranes (damage to DNA)
and alterations in cell organelles (Cabrita et al. 2005).
Cryoprotectants can be divided into two groups: intracel-
lular action and extracellular action. Those in the first
group are capable of permeating the cell membrane; they
are generally of low molecular weight (MW: 400 Da), not
electrolytic and highly soluble in water (Bakhach 2009;
Cuevas-Uribe & Tiersch 2011). The most common com-
pounds for semen cryopreservation are dimethyl sulphox-
ide (DMSO), dimethyl acetamide (DMA), glycerol (Gly),
1-2 propanediol, ethylene glycon (EG), methyl glycol (2-
methoxyethanol), propylene glycol (PG), methanol
(MeOH) and 2.3-butanediol (BD). However, most of
these substances are toxic and their concentration and
equilibrium time (interaction with the cell) may have neg-
ative effects on sperm physiology, such as osmotic shock
or biochemical problems (Tiersch 2011). According to
Suquet et al. (2000) and Liu et al. (2006), dimethyl
sulphoxide (DMSO) is considered to produce good results
6 © 2016 Wiley Publishing Asia Pty Ltd
when used as a cryoprotectant in most fish species. In the
research published over the last 15 years, DMSO has also
been tested and approved in most marine species studied,
as has the cryoprotectant propylene glycol (PG). The con-
centrations which presented the best results in most of
the investigations were in the range of 5–15% of the final
volume of the samples, with predominance at 10%
(Table 1).
The second group consists of non-permeable substances
of high MW which may be monosaccharide, disaccharide
or polysaccharide sugars, macromolecules like polyvinyl
pyrrolidone (PVP) and hydroxyethyl starch (HES)
(Bakhach 2009), lipoproteins or proteins derived from
milk, egg yolk and vegetable oils (Moraes et al. 1998). The
macromolecules have two functions: to increase the osmo-
lality of the extracellular space, causing dehydration of the
cells during freezing, and to prevent excessive osmotic
expansion during thawing (Cuevas-Uribe & Tiersch 2011;
Fig. 1). The mechanism by which sugars act to bio-stabilise
the cell and as an energy substrate (for example glucose,
trehalose, sucrose, etc.) and the modulation effected by
their concentration still need to be elucidated and should
be systematically evaluated (Cuevas-Uribe et al. 2013;
Ciereszko et al. 2014).
When seminal plasma is incorporated into cryopreserva-
tion process, it reduces the harmful effects of other
cryoprotectants and exerts a cryoprotectant and antioxi-
dant activity which protects sperm function in fish (Alvarez
et al. 2008; Figueroa et al. 2015b). A series antioxidant sub-
stances are found in the seminal plasma of freshwater fish
(ascorbic acid, carnitine, glutathione, methionine, taco-
pheryl acid and uric acid) as well as oxidative defence
enzymes (catalase, glutathione reductase, peroxidase and
superoxide dismutase) which could help preserving the
motility and fertilising capacity of this cells (Lahnsteiner
2007; Lahnsteiner 2009; Lahnsteiner & Mansour 2010;
Lahnsteiner et al. 2010).
Lahnsteiner et al. (2010) and Lahnsteiner and Mansour
(2010) mention that uric acid is the principal antioxidant
found in the semen of burbot (Lota lota), perch (Perca flu-
viatilis), bleak (Alburnus alburnos) and brown trout
(Salmo trutta) and that it can increase sperm motility and
membrane integrity. Liu et al. (1995) suggest that antioxi-
dant protection for mammal spermatozoa is also related
with proteins from seminal plasma, and Sasaki et al. (1996)
attribute it to low molecular weight protein fractions.
According to Lahnsteiner (2007), lipoproteins from the
seminal plasma of rainbow trout are able to maintain the
lipid composition of the cytoplasm membrane and increase
the cryostability of spermatozoa. However, the antioxidant
capacity of seminal plasma is insufficient to prevent
peroxidation of the lipids during freezing, and it must be
Reviews in Aquaculture (2016) 0, 1–11
 Cryopreservation and vitrification in sperm fish

Slow
(a)
Cooling Dehydraon

4°C –196°C

Ultrarapid
(b)
Dehydraon Cooling

Figure 1 (a) Balanced freezing implies using cryoprotectants and slow freezing to produce dehydration and cell contraction. Permeable cryoprotec-
tants (○) lower the freezing point of the solution and extend the dehydration time during freezing thus minimising osmotic shock and avoiding exces-
sive cell dehydration. Non-permeable cryoprotectants (●) assist cell dehydration and stabilise the membrane during cryopreservation. Once the first
extracellular ice crystals form ( ) as the temperature falls, more water is incorporated into the crystals. These grow, creating a hypertonic condition
which produces osmotic dehydration. The combination of the increase in intracellular solutes and the reduction in temperature increases the viscosity
of the solution until the eutectic temperature ( 40 °C) is reached. At this point, the remaining unfrozen solution solidifies (partial vitrification). (b)
Unbalanced vitrification involves the use of high concentrations of cryoprotectants to dehydrate the cell and replace intracellular water before cooling
starts. Ultrarapid cooling prevents the formation of ice crystals in the cells and the surrounding medium during freezing. The result is that the solution
solidifies in the vitreous state, with no formation of crystals (total vitrification).

supplemented by the diluent and other cryoprotectants
(Figueroa et al. 2015a,b).
Ultrarapid cooling or vitrification
The vitrification method consists of direct immersion of
small volume samples of semen in liquid nitrogen; it has
been applied successfully in the spermatozoa of fish like
rainbow trout and Atlantic salmon (Merino et al. 2011,
2012; Cuevas-Uribe et al. 2013; Figueroa et al. 2013,
2015a). However, to date only Cuevas-Uribe et al. (2013)
have applied the technique to the semen of marine fish
(Table 1).
Saragusty and Arav (2010) report that the main advan-
tages of this technique over traditional cryopreservation are
simplicity and speed. Vitrification does not require spe-
cialised equipment and can easily be applied in procedures
in production plants and in field work in areas of difficult
access. In addition to the eleven points mentioned above as
critical for semen cryopreservation, several others are very
important: (i) the number of steps for adding and remov-
ing the cryoprotectant; (ii) the technique used for vitrifica-
tion (affecting the freezing rate); (iii) the viscosity and
volume of the samples (Yavin & Arav 2007); (iv) absolute
pressure (high hydrostatic pressures reduce the homoge-
nous nucleation temperature and increase the temperature
of vitreous transition) (Rabin & Steif 2009). The basic
difference between vitrification and slow cryopreservation
is that during direct immersion in liquid nitrogen, the vis-
cosity of the medium is increased and the water molecules
in the sample cannot become organised to form a crys-
talline structure, but constitute an amorphous form of solid
(vitrified) water (Cuevas-Uribe & Tiersch 2011).
The object is to ensure that the vitreous transition occurs
as quickly as possible through rapid cooling or by increas-
ing the concentration of cryoprotectants (Cuevas-Uribe &
Tiersch 2011). However, increasing the concentration of
cryoprotectants generates toxic effects on the cells such as
osmotic shock, modifications in cell membrane permeabil-
ity and high rates of DNA fragmentation. As a rule, the
most recommended system for cryopreservation is a mix-
ture of cryoprotectants with different characteristics. They
can then be used in low concentrations to ensure good vit-
reous formation with low toxicity due to the sum of the
properties of the cryoprotectants (Saragusty & Arav 2010).
For example, human serum albumin + 0.25 M of sucrose is
used successfully as a non-permeable cryoprotectant in the
vitrification of human sperm by ultra-freezing (Isachenko
et al. 2008).
Cuevas-Uribe et al. (2013) tested eight cryoprotectants
in different concentrations and found that mixing perme-
able and non-permeable substances produces better results
than using just one cryoprotectant in a high concentration.
In these experiments, the mixture of 15% DMSO + 15%

Reviews in Aquaculture (2016) 0, 1–11

© 2016 Wiley Publishing Asia Pty Ltd 7
C. Magnotti et al.
EG + 10% Gly + 1% X-1000TM (X) + 1% Z-1000TM (Z)
resulted in the highest rate of vitrification, motility and
membrane integrity in the semen of red snapper (Lut-
janus campechanus), spotted sea trout (Cynoscion nebulo-
sus) and red drum (Sciaenops ocellatus) (Table 1).
However, they obtained good results for motility and mem-
brane integrity using other mixtures of permeable and non-
permeable compounds (e.g. 10% DMSO + 30%
EG + 0.25 M trehalose).
Seminal plasma has recently been used successfully as
a cryoprotectant for the vitrification of salmonid semen.
Merino et al. (2011, 2012) used Cortlandâ solu-
tion + 1% BSA and substituted the permeable cryopro-
tectant with 40% seminal plasma for vitrifying the
semen of rainbow trout, obtaining >80% motility, >82%
plasma membrane integrity and 55% mitochondrial
membrane potential. Figueroa et al. (2013, 2015a) used
Cortlandâ solution + 10% DMSO + 2% BSA + 0.13M
sucrose + 30, 40 or 50% seminal plasma for vitrifying
the semen of sex-reversed rainbow trout and Atlantic
salmon, respectively. The authors obtained better results
for motility, plasma membrane integrity, mitochondrial
membrane potential and fertilisation when 50% seminal
plasma was added to the cryoprotectant. In general,
marine fish produce semen with a higher viscosity and
lower volume than freshwater fish. However, use of the
total portion of seminal plasma and a fraction of
extracted and purified proteins should be considered and
tested as cryoprotectants for the cryopreservation of
semen from marine fish species.
Perspectives
Research into conservation of the semen of marine fish has
intensified in recent years. However, most of this work
deals only with technical questions, modifying diluents and
cryoprotectants and reporting the results on sperm motility
and fertility. Some works present a deeper attempt to deter-
mine the effects on cell physiology and morphology of
these chemical substances and of cryopreservation by slow
freezing and vitrification. In future, cellular and molecular
techniques will have to be used to characterise cryodamage
(Figueroa et al. 2014). Techniques to verify plasma mem-
brane integrity (e.g. flow cytometry (SYBR-14/PI) and opti-
cal microscopy (eosin), DNA fragmentation (e.g. TUNEL)
and to test mitochondrial integrity by analysis of the mito-
chondrial membrane potential (e.g. epifluorescence micro-
scopy and flow cytometry (Rhodamine and JC-1) are
recommended by Figueroa et al. (2014). Other technology
like CASA has been used to assess motility patterns in cry-
opreserved spermatozoa, as well as to evaluate the effects of
different cryoprotectants and to characterise different sub-
populations of spermatozoa in semen samples. It has been
8 © 2016 Wiley Publishing Asia Pty Ltd
also used to assess the effect of cryopreservation on sperm
structure (Billard & Zhang 2001; Cosson et al. 2008;
Cosson 2010).
The addition of new substances to the diluents and new
cryoprotectants is also recommended to obtain progress in
these techniques. The application of substances already
tried and tested in freshwater species may produce good
results in marine species, for example, supplementing dilu-
ents with antioxidants and adding organic acids, and the
use of macromolecular seminal plasma as a non-permeable
cryoprotectant in cryopreservation and vitrification.
Acknowledgement
The authors are sincerely thankful by support provided by
FONDECYT No. 1151315 (JGF), FONDECYT MEC No.
80140066 (IV), FONDEF D10I1064 (IV), CONICYT
Scholarships for PhD in Chile (EF) and DIUFRO Grant
DI15-2018 (JGF).
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