[2016]JFB ResearchJF

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Journal of Fish Biology (2016)
doi:10.1111/jfb.13052, available online at wileyonlinelibrary.com
Cryopreservation of Atlantic salmon Salmo salar sperm:
effects on sperm physiology
E. F*†, I. V*, O. M‡, A. U*,
J. R‡ J. G. F†§
*School of Aquaculture, Catholic University of Temuco, Temuco, Chile, †Departamento de
Ingeniería Química, Facultad de Ingeniería y Ciencias, Universidad de La Frontera, Temuco,
Chile and ‡BIOREN – Center for Biotechnology in Reproduction, La Frontera University,
Temuco, Chile
(Received 6 November 2014, Accepted 28 April 2016)
The objective of this study was to determine the effect of freezing on the function in Atlantic
salmon Salmo salar spermatozoa. The semen was frozen in Cortland’s medium+1.3M dimethyl
sulphoxide +0.3M glucose +2% bovine serum albumin (nal concentration) in a ratio of 1:3
(semen:cryoprotectant) as the treatment (T) and fresh semen as the control (F). Straws of 0·5ml of
sperm suspension were frozen in 4 cm of N2L. They were thawed in a thermoregulated bath (40∘C).
After thawing, the percentage of spermatozoa with fragmented DNA [transferase dUTP (deoxyuridine
triphosphate) nick-end labelling (TUNEL)], plasma membrane integrity (SYBR-14/PI) and mito-
chondrial membrane potential (ΔΨMMit, JC-1) were evaluated by ow cytometry and motility was
evaluated by optical microscope under stroboscopic light. The fertilization rates of the control and
treatment semen were tested at a sperm density of 1·5×107spermatozoa oocyte−1, by observation of
the rst cleavages after 16 h incubation at 10∘C. In the cryopreserved semen (T), the mean±.. DNA
fragmentation was 4·8±2·5%; plasma membrane integrity 75·2±6·3%; mitochondrial membrane
potential 51·7±3·6%; motility 58·5±5·3%; sperm velocity (VCL)61·2±17·4μms
−1; average-path
velocity (VAP)50·1±17·3μms
−1; straight-line velocity (VSL)59·1±18·4μms
−1; fertilization rate
81·6±1·9%. There were signicant differences in the plasma membrane integrity, mitochondrial
membrane potential, motility and VCL compared with the controls (P<0·05). Also the mitochondrial
membrane potential correlated with motility, fertilization rate, curved-line velocity (VCL)andVSL
(r=0·75; r=0·59; r=0·77 and r=0·79, respectively; P<0·05); and the fertilization rate correlated
with VCL and VSL (r=0·59 and r=0·55, respectively).
© 2016 The Fisheries Society of the British Isles
Key words: fertility; sh; sperm function; spermatozoa freezing.
INTRODUCTION
Sperm cryopreservation in shes is now an alternative for storing germplasm from
brood stock. This allows new, genetically improved lines to be protected and facilitates
reproduction of species of commercial interest, as well as those in danger of extinction
or genotypes of biological interest (Dziewulska et al., 2011; Cartón-García et al., 2013;
Ciereszko et al., 2014).
§Author to whom correspondence should be addressed at present address: Av. Francisco Salazar 01145,
Temuco, Chile. Tel.: 56 45 2325472-2592189; email: jorge.farias@ufrontera.cl
1
© 2016 The Fisheries Society of the British Isles

2E. FIGUEROA ET AL.
Atlantic salmon Salmo salar L. 1758 is one of the three most important salmonid
species in global aquaculture. In the wild, this species spawns during autumn and win-
ter, but by manipulating temperature and photoperiod, spawning can be induced all
year-round with minimal apparent effects on gamete quality. Biotechnologies such as
cryopreservation can also be applied allowing good quality gametes to be obtained all
year round (Cabrita et al., 2010; Aas et al., 2011).
Cryopreservation of spermatozoa has been reported in various salmonid species, but
with variable results on fertilization rates (Scott & Baynes, 1980; Scheerer & Thor-
gaard, 1989; Billard, 1992; Lahnsteiner et al., 1996a,b; Cabrita et al., 2001; Robles
et al., 2003; Jodun et al., 2007; Merino et al., 2011a). This could be due to a lack of
standardisation of freezing and thawing processes, causing damage to sperm structure
and physiology, such as plasma membrane integrity, nuclear and mitochondrial DNA
integrity, mitochondrial membrane potential and sperm ultrastructure (Pérez-Cerezales
et al., 2010). Damage of this kind could affect the cell viability, motility and fertilizing
capacity of the spermatozoa (Cabrita et al., 2001, 2005; Gallego et al., 2013; Figueroa
et al., 2015a).
The effects of osmotic stress, combined with the formation of ice crystals and the
toxicity of some cryoprotectants, generates alterations in sperm physiology due to
an increase in reactive oxygen species (ROS). It has been shown that ROS cause
alterations in the plasma membrane and breaks in the nuclear and mitochondrial
DNA chains in mammalian and piscine spermatozoa (Slupphaug et al., 2003; Aitken
et al., 1996; Koppers et al., 2008; Thomson et al., 2009), generating alterations in the
metabolic paths related with motility and viability, as well as other structural lesions
in the cell; leading to a reduction in the fertilization rate and probable alterations in
the development of the progeny (Lahnsteiner et al., 1996a,b; Ogier de Baulny et al.,
1997; Cabrita et al., 1998, 2005; Drokin et al., 1998; Babiak et al., 2002; Dziewulska
et al., 2011; Figueroa et al., 2015b).
Semen freezing protocols have been optimised and standardised in some sh species,
but they are being revised continuously to reduce negative effects on the fertilizing
capacity of the spermatozoa (Robles et al., 2009; Figueroa et al., 2016). The ef-
ciency of cryopreservation protocols has been evaluated recently by characterising
post-thawing motility patterns using computer-assisted semen analysis (CASA). This
technique provides objective, basic information on semen quality derived from inter
alia agellar movement, considering the percentage of motile spermatozoa, duration
of motility, velocity, acceleration, progressive movement, amplitude of agellar wave
and motility pattern (Fauvel et al., 2010).
Other types of cryodamage can be assessed at cell and molecule level, such
as plasma membrane integrity and uidity (SYBR-14/PI), mitochondrial activity
(JC-1/rhodamine), DNA integrity [transferase dUTP (deoxyuridine triphosphate)nick-
end labelling (TUNEL)]–single-cell electrophoresis (COMET)] using ow cytom-
etry or electrophoresis. These variables allow the quality of the thawed semen to
be evaluated, as expressed by the motility and fertilization rate. The importance
of studying sperm function in cryopreserved spermatozoa arises from its rela-
tionship with motility, fertilizing capacity and the quality of the offspring (Fahy,
1986; Carpenter & Crowe, 1988; Álvarez & Storey, 1992; Gao et al., 1997; Labbé
et al., 2001; Pérez-Cerezales et al., 2010; Merino et al., 2011b). There are, how-
ever, relatively few studies of the effects of freezing on the sperm physiology
of S.salar.
© 2016 The Fisheries Society of the British Isles, Journal of Fish Biology 2016, doi:10.1111/jfb.13052

CRYOPRESERVATION OF S. SALAR SPERM 3
MATERIALS AND METHODS
All chemicals used in this study were purchased from Sigma (www.sigmaaldrich.com), unless
otherwise indicated. All solutions were prepared using water from a Milli-Q Synthesis System
(www.merckmillipore.com). The live –dead sperm viability kit (SYBR-14/PI; Thermo-Fisher;
www.thermosher.com/cl/es/home/brands/invitrogen.html), the situ cell-death detection kit
(TUNEL; Roche Diagnostics GmbH; www.roche.com) and the mitochondrial permeability
detection kit AK-116 [M¯
ıT-E-Ψ, JC-1; Biomol International LP (www.enzolifesciences.com/
biomol/)] test kits were also used.
BROODSTOCK
This research was conducted at the Center for Biotechnology in Aquaculture (BIOACUI),
School of Aquaculture, Catholic University of Temuco, Chile, at the Engineering, Biotechnol-
ogy and Applied Biochemistry Laboratory (LIBBA) and at the Center for Biotechnology in
Reproduction (CEBIOR), La Frontera University, Chile. The 20 male S. salar were 2 –3 years
old (sexually mature), with mean ±.. mass of 8·6±0·7kg and total length (LT) 84·0±0·8cm.
They were provided by a commercial farm (Troutlodge Chile; www.troutlodge.com). During
the experimental period, the broodstock were kept in 3500 l breglass tanks with fresh water
(550 l h−1)at8
∘C under a natural photoperiod.
COLLECTION OF GAMETES
Sperm collection was performed as described by Ciereszko et al. (2014) with some modica-
tions. Briey, the 10 males were anaesthetized in a 50-l tank with 125mg l−1MS-222 for 10 min.
The urogenital pore was dried and semen collected by gentle abdominal massage, directly into a
graduated, sterile, dry, disposable plastic container, maintained at a temperature of 4∘C, taking
care not to contaminate them with faeces, mucus or urine.
Immediately after collection, sperm motility and concentration were determined using
a phase-contrast microscope (Carl Zeiss; www.zeiss.com). Prior to motility analysis, the
samples were diluted 1:3 (semen: medium) in Cortland non-activating medium (Trus-Cott
et al., 1968). Sperm motility (pre-diluted sperm) was activated with Powermilt (Universidad
Católica de Temuco, Chile) and evaluated by subjective microscopic examination using a
phase contrast microscope (Carl Zeiss) at ×400 magnication. Sperm motility was assessed
as described by Cosson (2008). Spermatozoa concentration was determined with a Neubauer
haemocytometer on Cortland’s culture medium for sh spermatozoa, described by Figueroa
et al. (2013, 2015b). Only 15 samples exhibited sufciently high motility (>80%) and aver-
age sperm concentration (mean ±.. 14·0×109±2·7×109spermatozoa ml−1)tobeused
in this study.
FREEZING AND THAWING
Two experimental groups were formed: group (1) fresh sperm (F) and group (2) frozen sperm
(T). The semen was frozen by the modied protocol of Lahnsteiner et al. (2011). Frozen semen
was diluted at 4∘C in Cortland’s medium as described by Figueroa et al. (2013, 2015b), sup-
plemented with 1·3 M dimethyl sulphoxide (DMSO), 0·3 M of glucose and 2 % bovine serum
albumin (BSA) to establish the cryoprotectant medium (nal concentration). The dilution was
1:3 (semen:cryoprotectant medium). For 7– 10 min after dilution, the semen was stored in 0·5ml
plastic straws. Subsequently, the straws were frozen in liquid nitrogen vapour in a styrofoam
box. The straws were deposited in a horizontal tray oating 2 cm above the surface of the
nitrogen; they were frozen for 10 min and subsequently immersed in liquid nitrogen for later
transfer to cryotanks. After 2 months of frozen storage, the straws were removed from the
cryotank and thawed in a thermo-regulated bath at 35∘C for 9 s, as per the modied pro-
cedure described by Ciereszko et al. (2014). The sperm function of the thawed semen was
evaluated immediately.
© 2016 The Fisheries Society of the British Isles, Journal of Fish Biology 2016, doi:10.1111/jfb.13052

4E. FIGUEROA ET AL.
SPERM EVALUATION
DNA fragmentation
To assess DNA fragmentation, the TUNEL protocol was followed according to instructions
provided by the manufacturer, adapted to spermatozoa by Cabrita et al. (2011). The suspension
at a concentration 3 ×106spermatozoa ml−1waswashedinPBSfor5minat720g. The pellet
was xed with 2% (w/v) paraformaldehyde in phosphate buffered saline (PBS) pH7·4for1h
at room temperature. Next, the suspension was washed in PBS for 5min at 320g, the pellet was
resuspended in 100 μlof0·1% (v/v) Triton X-100 prepared in 0·1% (w/v) sodium citrate for 2
min at 4∘C and then washed in PBS for 5 min at 320g. The pellet was resuspended in 50 μlof
TUNEL reaction mixture (TdT and uorescein-dUTP) and incubated for 60 min at 37∘Cina
humidied atmosphere in the dark. Next, the spermatozoa were stained with 5 μl of propidium
iodide (1·5 mM), resuspended in PBS and washed for 5 min at 320g. Then the pellet was resus-
pended in 400 μl PBS. After washing, the label at damaged DNA sites was analysed directly
by ow cytometry and confocal microscopy. TUNEL positive spermatozoa (with fragmented
DNA) were stained green. The analysis was replicated three times in each trial.
Cytoplasmic membrane integrity
The viability of the spermatozoa and the integrity of the cytoplasmic membrane were
determined by SYBR-14/PI according to the modied procedure described by Cabrita
et al. (2009). For this purpose, 4 ×106spermatozoa ml−1were resuspended in 250 μl
PBS +1·25 μl SYBR-14 +1·25 μl propidium iodide (PI). After exposure of the spermatozoa
to this solution for 6 min at 10∘C, 250 μl of PBS were again added for analysis of the sper-
matozoa by ow cytometry and confocal microscopy. SYBR-14-positive spermatozoa, with
cytoplasm membrane integrity, are stained green. The analysis was replicated three times
in each trial.
Mitochondrial membrane potential
To evaluate mitochondrial activity, changes in the mitochondrial membrane potential (ΔΨM)
were determined using a uorescent cation JC-1, following the modied procedure described
by Asturiano et al. (2006). JC-1 is a lipophilic dye that is internalized by all functioning mito-
chondria, where it uoresces green. In highly functional mitochondria, the concentration of
JC-1 inside the mitochondria increases and the stain forms aggregates that uoresce red. In
brief, 250 μl of sperm sample were centrifuged at 720gfor 5 min. The pellet was resuspended in
250 μl JC-1 solution (3 mM JC-1 in DMSO) and incubated for 15 min at 10∘C in the dark.
After this, the cell suspension was centrifuged for 5 min at 720g, the supernatant was dis-
carded and the sperm pellet resuspended in 400 μl Cortland’s extender and immediately anal-
ysed by ow cytometry and confocal microscopy. The analysis in each trial was replicated
three times.
Motility
The method used was a modied protocol of Cosson (2004) and Li et al. (2012) for optical
microscope with Exposure Scope stroboscopic light (FROV, Vodnany, Czech Republic) to deter-
mine motility patterns (velocity) through CASA. The percentage of motile spermatozoa and the
spermatozoa average velocity (μms
−1) were determined at ×200 magnication in a phase con-
trast Olympus BX 41 microscope (Olympus; www.olympus.com) after activation of motility.
The samples were diluted in Powermilt (280 mOs kg−1and pH 9·0). To prevent the spermatozoa
from adhering to the slide, 0·25% (w/v) of Pluronic was added to the activator. The spermatozoa
were recorded with a SSC-G818digital video camera (SONY; www.sony.com) mounted on the
microscope, lming at 25 frames s−1at 50 Hz. They were analysed using ImageJ CASA software
(www.virtualdub.org) for processing images and videos. The following sperm motility variables
were evaluated: curved line velocity (VCL,μms
−1), average path velocity (VAP,μms
−1), straight
line velocity (VSL,μms
−1), linearity (LIN, %), wobble (WOB , %) and beat cross frequency (FBC,
Hz). The analysis was replicated three times in each trial.
© 2016 The Fisheries Society of the British Isles, Journal of Fish Biology 2016, doi:10.1111/jfb.13052

CRYOPRESERVATION OF S. SALAR SPERM 5
Fertility
A pool of oocytes from 20 females was used for testing the fertility of the fresh and cryopre-
served semen. The procedure described by Figueroa et al. (2013) was followed; all the fertility
tests were carried out ve times with 200 oocytes per replication. The sperm density used in all
the treatments was 1·5×107spermatozoa oocyte−1. The eggs were incubated in open ow at
10∘C. Fertilization was evaluated by observation of the rst cleavages (segmentation) after 16 h
incubation at 10∘C(Efferet al., 2014).
FLOW CYTOMETRY
A FACs Canto II ow cytometer (BD Biosciences; www.bdbiosciences.com) was used
to determine the following variables: sperm-membrane integrity (with SYBR-14/PI),
mitochondrial-membrane potential (with JC-1) and DNA fragmentation (by TUNEL). A
minimum of 10 000 spermatozoa were examined in each assay at a ow rate of 100cells s−1.
The spermatozoon probe was gated using 90∘and forward-angle light scatter to exclude debris
and aggregates. The excitation wavelength was 488 nm solid state laser (20mW) and 633 nm
HeNe lamp (17 mW). Green uorescence was measured in the FITC channel (533/30 nm) and
red uorescence in the Pe channel (585/42 nm).
CONFOCAL MICROSCOPY
The sperm samples were observed under a Fluoview FV1000 synchronised laser-scanning
inverted confocal microscope (Olympus). The probe emissions were evaluated over a wave-
length range of 405–635 nm. The samples were subjected to multiple excitation by Ar laser
(488 nm) and He –NeG laser (543 nm).
STATISTICAL ANALYSIS
Data were expressed as percentages (mean ±..) and analysed using Prisma 6.0
(www.graphpad.com). A Wilcoxon matched-pair t-test was used to assess rates of DNA
fragmentation, viability–plasma-membrane integrity, mitochondrial-membrane integrity,
motility and fertility. Spearman correlation was used to relate the mitochondrial-membrane
potential with the motility and fertilization rates. The level of signicance was set at P<0·05.
RESULTS
There was 2·4±1·8% DNA fragmentation in fresh semen, statistically simi-
lar to frozen semen (4·8±2·5%; PF–T>0·05; Fig. 1). The plasma-membrane
integrity of the frozen semen was 75·2±6·3%, which was signicantly different
from that of the fresh control semen (90·5±3·9%; PF–T<0·05; Fig. 1). The high-
est mitochondrial-membrane potential was found in fresh semen (91·1±3·7%),
which was signicantly different from that of the frozen semen (51·7±3·6%;
PF–T<0·05, Fig. 1). There was a statistically signicant positive correlation between
mitochondrial-membrane potential and motility, VCL and VSL [Fig. 2(a)–(d)].
The motility for frozen semen was 58·5±5·3%, representing a statistically sig-
nicant difference from fresh semen (91·3±6·2%; PF–T<0·05; Fig. 1). Likewise
the VCL,VAP and VSL for fresh semen presented values of 104·4±25·6, 78·1±24·2
and 102·4±26·0μms
−1, were signicantly different from frozen semen [61·2±17·1,
50·1±17·3 and 59·1±18·4μms
−1, respectively; PF–T<0·05; Fig. 3(b)]. The BCF
(4·2±2·1Hz), LIN (83·2±5·6%) and WOB (94·6±2·7%) patterns on the other
hand presented no differences between frozen semen and the control [3·8±2·3Hz;
77·5±6·5 and 98·4±1·5%; PF–T>00·05; Fig. 3(a), (b)].
© 2016 The Fisheries Society of the British Isles, Journal of Fish Biology 2016, doi:10.1111/jfb.13052

6E. FIGUEROA ET AL.
100
*
*
*
80
60
Rate (%)
40
20
0DNA
fragmentation
Cytoplasmic-
mebrane integrity
Mitochondrial-
membrane potential
Sperm function
MotilityFertility
F. 1. Survival rate (%) of Salmo salar spermatozoa ( ) after freezing in Cortland’s medium+1·3 M dimethyl
sulphohoxide +0·3 M glucose +2% bovine serum albumin and ( ) fresh sperm used as a control. *, signif-
icantly different rates (P<0·05, n=10).
The fertilization rate for fresh semen was 98·3±3·3%, representing no statistically
signicant difference from frozen semen (90·4±3·8%; PF–T<0·05; Fig. 1). The fer-
tilization rate presented a statistically signicant correlation with the motility variables
VCL and VSL [Fig. 2(e), (f)].
CONFOCAL MICROSCOPY
Confocal microscope images of the spermatozoa after freezing–thawing and staining
are shown in Fig. 4.
DISCUSSION
Cryopreservation of spermatozoa has been reported for various salmonid species
(Scott & Baynes, 1980; Scheerer & Thorgaard, 1989; Billard, 1992; Lahnsteiner
et al., 1996b), including Oncorhynchus mykiss (Walbaum), brown trout Salmo trutta
L. 1758, brook trout Salvelinus fontinalis (Mitchill 1814), Artic charr Salvelinus
alpinus (L. 1758) and S. salar (Lahnsteiner et al., 1996a; Cabrita et al., 1998, 2001;
Labbé et al., 2001; Babiak et al., 2002; Horváth et al., 2008; Martínez-Páramo et al.,
2009; Dziewulska et al., 2011; Ciereszko et al., 2014; Figueroa et al., 2015b). Several
authors, however, have reported variable fertilization rates due to alterations in sperm
physiology that reduce motility and functional parameters after thawing.
The freezing protocol serves to reduce the formation of large, extracellular ice crys-
tals, thus avoiding damage to the cytoplasm-membrane integrity of the spermatozoa.
It has a signicant effect, however, on the mitochondrial-membrane potential through
the possible formation of intracellular ice crystals or osmotic alterations to which the
cells may be subjected.
The lipophilic compound JC-1 has been used to evaluate the depolarisation of the
mitochondrial membrane in the spermatozoa of several species of mammals and shes.
© 2016 The Fisheries Society of the British Isles, Journal of Fish Biology 2016, doi:10.1111/jfb.13052

CRYOPRESERVATION OF S. SALAR SPERM 7
100(a) (b)
(c) (d)
(e) (f)
80
60
Motility (%)
Fertility (%)
40
20
0
150
100
50
0
150
100
50
0
Mitochondrial-membrane potential (%) Mitochondrial-membrane potential (%)
020406080 100
100
90
80
70
60
100
90
80
70
60
050
VCL (ms–1)
VCL (ms–1)
Fertility (%)
Fertility (%) VSL (ms–1)
VSL (ms–1)
100 150 0 50 100 150
100
90
80
70
60
020406080 100
F. 2. Relationship between the mitochondrial-membrane potential and (a) motility (y=1·044x+11·660;
r=0·75, P<0·001, n=10), (b) fertility (y=0·275x+8·240; r=0·59, P<0·05, n=10), (c) curved-line
velocity (VCL;y=1·131x+16·800; r=0·77, P<0·05, n=10), (d) straight-line velocity (VSL;
y=1·118x+10·600; r=0·79, P<0·05, n=10) and the relationship between fertility and (e) VCL
(y=0·178x+6·970; r=0·59, P<0·05, n=12) and (f) VSL (y=0·211x+6·321; r=0·55, P<0·05, n=12).
The use of this technique on the mitochondria of S. salar spermatozoa, however, has
been little reported and could be considered a test of the effectiveness of the freezing
protocol (Labbé et al., 2001; Aitken et al., 1996; Merino et al., 2011a,b). In the current
experiments, there was a positive correlation between the mitochondrial-membrane
potential and the fertilization rate, which agrees with the ndings of Figueroa et al.
(2013, 2015b) in cryopreserved spermatozoa of O. mykiss and S salar.
Many authors have reported low sperm motility after thawing in salmonids (Lahn-
steiner et al., 1996b;Gwoet al., 1999; Cabrita et al., 2001), which agrees with the
observations of this work. This may be associated with damage to the membrane poten-
tial or direct damage to the DNA caused by fragmentation of nuclear or mitochondrial
© 2016 The Fisheries Society of the British Isles, Journal of Fish Biology 2016, doi:10.1111/jfb.13052

8E. FIGUEROA ET AL.
100
(a)
(b)
80
*
*
**
60
40
20
0
0
50
100
150 10
8
6
4
Frequency (Hz)
Velocity (m s–1) Rate (%)
2
Motility variables
0
MOTLIN
VCL VAPVSL FBC
WOD
F. 3. (a) Mean +.. rate of motility variables (MOT, motility; LIN, linearity; WOB , wobble) of Salmo salar
sperm ( ) after freezing in Cortland’s medium+1·3 M dimethyl sulphoxide +0·3 M glucose +2% bovine
serum albumin and ( ) fresh semen. (b) Mean +.. velocity and frequency for: VCL, curved-line velocity;
VAP, velocity average path; VSL, velocity straight line; FBC , beat cross frequency. *, statistically signicant
difference (P<0·05, n=10).
DNA. Furthermore, the mitochondria depend on certain proteins of the non-encoding
genome (Kurland & Andersson, 2000). ROS may also damage mitochondrial DNA and
induce lipid peroxidation in the membrane, which may cause transition alterations in
the permeability of the aperture of the mitochondrial pore (Bucak et al., 2015; Figueroa
et al., 2015a). This may lead to release of cytochrome cand the induction of cell apop-
tosis (Wang et al., 2013).
In salmonids, the motility and swimming speed of the spermatozoa diminish rapidly
after thawing (Muchlisin, 2005; Dziewulska et al., 2011; Figueroa et al., 2016). In
other sh families, however, such as Cyprinidae and Sparidae, the percentage of sperm
motility after thawing presents no differences compared to fresh semen (Horváth et al.,
2003; Liu et al., 2007). This sensitivity of salmonid spermatozoa to freezing normally
results in no more than 5 to 20% motile spermatozoa post-thawing (Ohta et al., 1995;
Glogowski et al., 1996, 1997; Lahnsteiner et al., 1996b;Gwoet al., 1999; Mansour
et al., 2006), with occasional results as high as 40–60% (Conget et al., 1996; Cabrita
et al., 2001; Bozkurt et al., 2005; Tekin et al., 2007), as in this study.
The activation and duration of motility requires a large amount of ATP (Cosson et al.,
1991, 1995), which is provided by the mitochondria in most shes. Damage to the mito-
chondria will therefore have a negative effect on motility (Dreanno et al., 1999), which
© 2016 The Fisheries Society of the British Isles, Journal of Fish Biology 2016, doi:10.1111/jfb.13052

CRYOPRESERVATION OF S. SALAR SPERM 9
(a) (b) (c)
F. 4. Confocal microscopy of Salmo salar spermatozoa. (a) TUNEL [transferase dUTP (deoxyuridine triphos-
phate) nick-end labelling] staining, yellow-green spermatozoa with fragmented DNA, red spermatozoa with
intact DNA. (b) SYBR-14/PI staining: ( ) green spermatozoa with intact cytoplasm membrane, ( )red
spermatozoa with disrupted cytoplasm membrane. (c) JC-1 staining: ( ) red spermatozoa with an intact
mitochondrial membrane.
agrees with the current results. The results of this study indicate a positive correlation
of the mitochondrial-membrane potential with motility (r=0·75) and the variables VCL
and VSC (r=0·77 and 0·79, respectively), agreeing with the results of Figueroa et al.
(2013, 2015b). This may also be related to the ndings of Christen et al. (1987), who
indicated that low oxidative capacity of the mitochondria appeared to be one of the
crucial parameters limiting the duration of sperm motility in salmonids, due to the lack
of ATP. This may therefore be the limiting factor in increasing the motility (motility
patterns) and fertilizing capacity of the spermatozoa (Gallego et al., 2013).
When they assessed the motility patterns of S. salar spermatozoa by CASA after
thawing, Dziewulska et al. (2011) found no signicant differences compared to fresh
semen in the linearity percentage, which agrees with results found here for LIN in frozen
(83%) and fresh (77%) semen. Linearity and velocity are related with fertilizing capac-
ity. Lahnsteiner et al. (1996a,b, 2000) reported a close relationship of the velocity of
fresh and frozen semen with the fertilization rate in salmonids, including O. mykiss; this
is comparable with results found here, where the fertilization rate presented a positive
correlation with VCL (r=0·77) and VSL (r=0·79) in fresh and frozen semen [Fig. 2(b)].
Furthermore, Gage et al. (2004) and Dziewulska et al. (2011) mention that in S. salar
spermatozoa, the highest velocities determine fertilizing capacity.
The effect of cryopreservation on DNA fragmentation is low (4·8%), although it
sometimes reaches signicant levels (Drokin et al., 1998; Zilli et al., 2003); it is
approximately 20 times less than the damage caused to cytoplasm-membrane and
mitochondrial integrity (Labbé et al., 2001). DNA damage is therefore only a minor
component of the damage caused in cryopreserved cells (Ogier de Baulny et al.,
1997; Labbé et al., 2001). It has been shown that DNA fragmentation increases as the
spawning season progresses and also increases with freezing (Pérez-Cerezales et al.,
2010). DNA damage may cause alterations at various stages of embryo development
(abnormal cleavage patterns in segmentation) and these may lead to signicant mor-
tality rates in salmonid embryos and hatched larvae. There is a strong correlation
between DNA damage and the appearance of mutagenic alterations. Spermatozoa
with genetic material damaged by freezing, however, do present fertilizing capacity
(Twigg et al., 1998) and the oocytes have the capacity to repair this damage partially
© 2016 The Fisheries Society of the British Isles, Journal of Fish Biology 2016, doi:10.1111/jfb.13052

10 E. FIGUEROA ET AL.
(Pérez-Cerezales et al., 2010). According to Cabrita et al. (2005), DNA fragmentation
is greater in salmonids than in other species, but in S. salar the results of the freezing
protocol produce a high level of DNA integrity after thawing, with a range of 3–5%
fragmented DNA for both fresh and frozen semen.
According to Dziewulska et al. (2011) the best cryoprotectant for maintaining
cytoplasm-membrane integrity, mitochondrial function and motility patterns is DMSO
supplemented with antioxidants and cytoplasm-membrane stabilisers, such as BSA.
In salmonids, this maintains the lipid composition of the plasma membrane and
increases the stability of the spermatozoa (Baynes & Scott, 1987; Lahnsteiner et al.,
1996a; Cabrita et al., 2001; Bergeron & Manjunath, 2006; Mirzoyan et al., 2006).
Furthermore, the incorporation of sugars like sucrose and glucose into the cryopreser-
vation medium has been used effectively for freezing (Mansour et al., 2006; Tekin
et al., 2007) spermatozoa of O. mykiss, asp Leuciscus aspius (L. 1758) and S. salar
(Lahnsteiner et al., 1996b; Babiak et al., 1998; Lahnsteiner, 2000; Kusuda et al.,
2005; Mansour et al., 2006). The carbohydrates used in sperm freezing function as an
additional solvent; thus, they are able to reduce and stabilise the osmotic pressure of
the medium caused by permeable cryoprotectants such as DMSO and methanol. For
example, it has been suggested that rafnose plays the role of a membrane stabiliser
and dehydrating agent (Wakayama et al., 1998; Koshimoto & Mazur, 2002).
The results show that the fertilizing capacity of cryopreserved S. salar spermatozoa
may be similar to that of fresh semen and the same may occur with DNA fragmentation
and some motility patterns. This method needs to be improved, however, to increase
the amount of semen stored in containers, to meet the volume requirements of com-
panies for their production process. This method may have important applications in
the conservation and handling of biological and genetic material of great commercial
value and may also allow the establishment of a germplasm bank for aquatic species
of interest. The high fertility (90%) of the cryopreserved semen indicates that the sper-
matozoa of this species may be frozen with the addition of DMSO +glucose +BSA to
the freezing medium, thus reducing cryodamage to sperm physiology. More tests need
to be done, however, to improve these results with larger storage volumes.
The authors are sincerely grateful for support provided by FONDECYT N∘1151315 (J.G.F.),
FONDECYT MEC N∘80140066 (I.V.) and FONDEF D10I1064 (I.V.), CONICYT Scholarships
for PhD in Chile (E.F. and O.M.) and DIUFRO Grant DI15-2018 (J.G.F.) and the company
Troutlodge Chile, which provided the gametes used in the biotests.
References
Aas, Ø., Einum, S., Klemetsen, A. & Skurdal, J. (2011). Atlantic Salmon Ecology. Oxford:
Wiley-Blackwell.
Aitken, R.J., Buckingham, D.W., Harkiss, D., Paterson, M., Fisher, H., & Irvine, D.S. (1996).
The extragenomic action of progesterone on humanspermatozoa is inuenced by redox
regulated changes in tyro-sine phosphorylation during capacitation. Molecular and Cel-
lular Endocrinology 117, 83–93.
Álvarez, J. G. & Storey, B. T. (1992). Evidence for increased lipid peroxidative damage and loss
of superoxide dismutase activity as a mode for sublethal cryodamage to human sperm
during cryopreservation. Journal of Andrology 13, 232 –241.
Asturiano, J., Marco-Jiménez, F., Pérez, L., Balasch, S., Garzón, D., Peñaranda, D., Vicente,
J., Viudes-de-Castro, M. & Jover, M. (2006). Effects of HCG as spermiation inducer on
European eel semen quality. Theriogenology 66, 4.
© 2016 The Fisheries Society of the British Isles, Journal of Fish Biology 2016, doi:10.1111/jfb.13052

CRYOPRESERVATION OF S. SALAR SPERM 11
Babiak, I., Glogowski, J., Kujawa, R., Kucharczyk, D. & Mamcarz, A. (1998). Cryopreservation
of sperm from asp Aspius aspius.Progressive Fish-Culturist 60, 146–148.
Babiak, I., Glogowski, J., Dobosz, S., Kuzminski, H. & Goryczko, K. (2002). Semen from
rainbow trout produced using cryopreserved spermatozoa is more suitable for cryopreser-
vation. Journal of Fish Biology 60, 561 –570.
Baynes, S. M. & Scott, A. P. (1987). Cryopreservation of rainbow trout spermatozoa: the inu-
ence of sperm quality, egg quality and extender composition on post-thaw fertility. Aqua-
culture 66, 53– 67.
Bergeron, A. & Manjunath, P. (2006). New insights towards understanding the mechanism of
sperm protection by egg yolk and milk. Molecular Reproduction and Development 73,
1338–1344.
Billard, R. (1992). Reproduction in rainbow trout: sex differentiation, dynamics of gametogen-
esis, biology and preservation of gametes. Aquaculture 100, 263–298.
Bozkurt, Y., Seçer, S., Tekin, N. & Akçay, E. (2005). Cryopreservation of rainbow trout
(Oncorhynchus mykiss) and mirror carp (Cyprinus carpio) sperm with glucose based
extender. Süleyman Demirel Üniversitesi 1, 21–25.
Bucak, M. N., Ataman, M. B., Ba¸spınar, N., Uysal, O., Ta¸spınar, M., Bilgili, A., Öztürk,
C., Güngör, S., Inanc, M. E. & Akal, E. (2015). Lycopene and resveratrol improve
post-thaw bull sperm parameters: sperm motility, mitochondrial activity and DNA
integrity. Andrologia 47, 545–552.
Cabrita, E., Alvarez, R., Anel, L., Rana, K. & Herráez, M. P. (1998). Sublethal damage during
cryopreservation of rainbow trout sperm. Cryobiology 37, 245–253.
Cabrita, E., Anel, L. & Herráez, M. P. (2001). Effect of external cryoprotectants as membrane
stabilizers on cryopreserved rainbow trout sperm. Theriogenology 56, 623 – 635.
Cabrita, E., Robles, V., Cuñado, S., Wallace, J. C., Sarasquete, C. & Herráez, M. P. (2005).
Evaluation of gilthead sea bream, Sparus aurata, sperm quality after cryopreservation in
5 ml macrotubes. Cryobiology 50, 273–284.
Cabrita, E., Engrola, S., Conceição, L. E. C., Pousão-Ferreira, P. & Dinis, M. T. (2009). Success-
ful cryopreservation of sperm from sex-reversed dusky grouper, Epinephelus marginatus.
Aquaculture 287, 152– 157.
Cabrita, E., Sarasquete, C., Martinez-Páramo, S., Robles, V., Beirão, J., Pérez-Cerezales, S. &
Herráez, M. P. (2010). Cryopreservation of sh sperm: applications and perspectives.
Journal of Applied Ichthyology 26, 623–635.
Cabrita, E., Ma, S., Diogo, P., Martinez-Paramo, S., Sarasquete, C. & Dinis, M. T. (2011). The
inuence of certain aminoacids and vitamins on post-thaw sh sperm motility, viability
and DNA fragmentation. Animal Reproduction Science 125, 189–195.
Carpenter, J. F. & Crowe, J. H. (1988). The mechanism of cryoprotection of proteins by solutes.
Cryobiology 25, 244–255.
Cartón-García, F., Riesco, M., Cabrita, E., Herráez, M. & Robles, V. (2013). Quantication
of lesions in nuclear and mitochondrial genes of Sparus aurata cryopreserved sperm.
Aquaculture 402– 403, 106–112.
Christen, R., Gatti, J. L. & Billard, R. (1987). Trout sperm motility: the transient movement of
trout sperm is related to changes in the concentration of ATP following the activation of
the agellar movement. European Journal of Biochemistry 166, 667–671.
Ciereszko, A., Dietrich, G. J., Nynca, J., Dobosz, S. & Zalewski, T. (2014). Cryopreserva-
tion of rainbow trout semen using a glucose-methanol extender. Aquaculture 420 – 421,
275–281.
Conget, P., Fernández, M., Herrera, G. & Minguell, J. J. (1996). Cryopreservation of rainbow
trout (Oncorhynchus mykiss) spermatozoa using programmable freezing. Aquaculture
43, 319–329.
Cosson, J. (2004). The ionic and osmotic factors controlling motility of sh spermatozoa. Aqua-
culture International 12, 69– 85.
Cosson, J. (2008). The motility apparatus of sh spermatozoa. In Fish Spermatology (Alavi, S.
M. H., Cosson, J. J., Coward, K. & Raee, G., eds), pp. 281– 316. Oxford: Alpha Science
Ltd.
Cosson, M. P., Cosson, J. & Billard, R. (1991). cAMP dependence of movement initiation
in intact and demembraned trout spermatozoa. In Proceedings of Fourth International
© 2016 The Fisheries Society of the British Isles, Journal of Fish Biology 2016, doi:10.1111/jfb.13052

12 E. FIGUEROA ET AL.
Symposium on Reproductive Physiology of Fish (Scott, A. P., Sumpter, J. P., Kime, D. E.
& Rolfe, M. S., eds), pp. 262–264. Shefeld: University of Shefled.
Cosson, M. P., André, F., Billard, R. & Cosson, J. (1995). cAMP/ATP relationship in the activa-
tion of trout sperm motility: their interaction in membrane-deprived models and in live
spermatozoa. Cell Motility and the Cytoskeleton 31, 159– 176.
Dreanno, C., Seguin, F., Cosson, J., Suquet, M. & Billard, R. (1999). Metabolism of tur-
bot (Scophthalmus maximus) spermatozoa: relationship between motility, intracellular
nucleotide content, mitochondrial respiration. Molecular Reproduction and Development
53, 230–243.
Drokin, S., Stein, H. & Bartscherer, H. (1998). Effect of cryopreservation on the ne structure
of spermatozoa of rainbow trout (Oncorhynchus mykiss) and brown trout (Salmo trutta
cf. fario). Cryobiology 37, 263– 270.
Dziewulska, K., Rzemieniecki, A., Czerniawski, R. & Domagała, J. (2011). Post-thawed motil-
ity and fertility from Atlantic salmon (Salmo salar L.) sperm frozen with four cryodilu-
ents in straws or pellets. Theriogenology 76, 300–311.
Effer, B. R., Sánchez, R. R., Ubilla, A. M., Figueroa, E. V. & Valdebenito, I. I. (2014). Morpho-
metric of blastomeres in Salmo salar.Zygote 22, 470–475.
Fahy, G. M. (1986). The relevance of cryoprotectant “Toxicity” to cryobiology. Cryobiology
23, 1–13.
Fauvel, C., Suquet, M. & Cosson, J. (2010). Evaluation of sh sperm quality. Journal of Applied
Ichthyology 26, 636– 643.
Figueroa, E., Risopatrón, J., Sánchez, R., Isachenko, E., Merino, O., Valdebenito, I. &
Isachenko, V. (2013). Sperm vitrication of sex-reversed rainbow trout (Oncorhynchus
mykiss): effect of seminal plasma on physiological parameters. Aquaculture 372– 375,
119–126.
Figueroa, E., Valdebenito, I., Zepeda, A. B., Figueroa, C. A., Dumorne, K., Castillo, R. L. &
Farias, J. G. (2015a). Effects of cryopreservation on mitochondria of sh spermatozoa.
Reviews in Aquaculture. doi: 10.1111/raq.12105/abstract
Figueroa, E., Merino, O., Risopatrón, J., Isachenko, V., Sánchez, R., Effer, B., Isachenko, E.,
Farias, J. G. & Valdebenito, I. (2015b). Effect of seminal plasma on Atlantic salmon
(Salmo salar) sperm vitrication. Theriogenology 83, 238–245.
Figueroa, E., Valdebenito, I. & Farias, J. G. (2016). Technologies used in the study of sperm
function in cryopreserved sh spermatozoa. Aquaculture Research 47, 1691 –1705. doi:
10.1111/are.12630
Gage, M. J. G., Macfarlane, C. P., Yeates, S., Ward, R. G., Searle, J. B. & Parker, G. A. (2004).
Spermatozoa traits and sperm competition in Atlantic salmon: relative sperm velocity is
the primary determinant of fertilization success. Current Biology 14, 44–47.
Gallego, V., Pérez, L., Asturianoa, J. F. & Yoshida, M. (2013). Relationship between spermato-
zoa motility parameters, sperm/egg ratio and fertilization and hatching rates in puffersh
(Takifugu niphobles). Aquaculture 416 –417, 238–243.
Gao, D., Mazur, P. & Critser, J. (1997). Fundamental cryobiology of mammalian spermatozoa.
In Reproductive Tissue Banking Scientic Principles (Karow, A. M. & Critser, J. K., eds),
pp. 263–328. New York, NY: Academic Press.
Glogowski, J., Babiak, I., Goryczko, K. & Dobosz, S. (1996). Activity of aspartate amino-
transferase and acid phosphatase in cryopreserved tout sperm. Reproduction Nutrition
Development 8, 1179– 1184.
Glogowski, J., Babiak, I., Goryczko, K., Dobosz, S. & Kuüminski, H. (1997). Properties and
cryopreservation of Danube salmon (Hucho hucho) milt. Archives of Polish Fisheries 5,
235–239.
Gwo, J. C., Ohta, H., Okuzawa, K. & Wu, H. C. (1999). Cryopreservation of sperm from the
endangered Formosan landlocked salmon (Oncorhynchus masou formosanus). Theri-
ogenology 51, 569 –582.
Horváth, A., Miskolczi, S. Z. & Urbanyi, B. (2003). Cryopreservation of common carp. Aquatic
Living Resources 16, 457– 460.
Horváth, Á., Wayman, W. R., Dean, J. C., Urbányi, B., Tiersch, T. R., Mims, S. D., Johnson, D.
& Jenkins, J. A. (2008). Viability and fertilizing capacity of cryopreserved sperm from
three North-American acipenseriform species: a retrospective study. Journal of Applied
Ichthyology 24, 443– 449.
© 2016 The Fisheries Society of the British Isles, Journal of Fish Biology 2016, doi:10.1111/jfb.13052

CRYOPRESERVATION OF S. SALAR SPERM 13
Jodun, W. A., King, K., Farrell, P. & Wayman, W. (2007). Methanol and egg yolk as cryopro-
tectants for Atlantic salmon spermatozoa. North American Journal of Aquaculture 69,
36–40.
Koppers, A. J., De Iuliis, G. N., Finnie, J. M., McLaughlin, E. A. & Aitken, R. J. (2008). Sig-
nicance of mitochondrial reactive oxygen species in the generation of oxidative stress
in spermatozoa. Journal of Clinical Endocrinology & Metabolism 93, 3199– 3207.
Koshimoto, C. & Mazur, P. (2002). The effect of the osmolality of sugar-containing media,
the type of sugar and the mass and molar concentration of sugar on the survival of
frozen–warmed mouse sperm. Cryobiology 45, 80 –90.
Kurland, C. G. & Andersson, S. G. (2000). Origin and evolution of the mitochondrial proteome.
Microbiology and Molecular Biology Reviews 64, 786– 820.
Kusuda, S., Koide, N., Kawamula, H., Teranishi, T., Nakajima, J.-I., Yamaha, E., Arai, K. &
Ohta, H. (2005). Cryopreservation diluents for spermatozoa of Sakhalin taimen Hucho
perryi.Fisheries Science 71, 293–298.
Labbé, C., Martoriati, A., Devaux, A. & Maisse, G. (2001). Effect of sperm cryopreservation on
sperm DNA stability and progeny development in rainbow trout. Molecular Reproduction
and Development 60, 397–404.
Lahnsteiner, F. (2000). Semen cryopreservation in the Salmonidae and in the northern pike.
Aquaculture Research 31, 245 – 258.
Lahnsteiner, F., Berger, B., Weismann, T. & Patzner, R. (1996a). Changes in morphology, phys-
iology, metabolism and fertilization capacity of rainbow trout semen following cryop-
reservation. Progressive Fish-Culturist 58, 149–159.
Lahnsteiner, F., Berger, B., Weismann, T. & Patzner, R. (1996b). The inuence of various cry-
oprotectants on semen quality of the rainbow trout (Oncorhynchus mykiss) before and
after cryopreservation. Journal of Applied Ichthyology 12, 99–106.
Lahnsteiner, F., Mansour, N. & Kunz, F. (2011). The effect of antioxidants on the quality of
cryopreserved semen in two salmonid sh, the brook trout (Salvelinus fontinalis)andthe
rainbow trout (Oncorhynchus mykiss). Theriogenology 76, 882 – 890.
Li, P., Li, Z. H., Hulak, M., Rodina, M. & Linhart, O. (2012). Regulation of spermatozoa motility
in response to cations in Russian sturgeon Acipenser gueldenstaedtii.Theriogenology 78,
102–109.
Liu, Q. H., Li, J., Xio, Z. Z., Ding, F. H., Yu, D. D. & Xu, X. Z. (2007). Use of computer-assisted
sperm analysis (CASA) to evaluate the quality of cryopreserved sperm in red seabream
(Pagus major). Aquaculture 263, 20– 25.
Mansour, N., Richardson, G. F. & McNiven, M. A. (2006). Effect of extender composition and
freezing rate on post-thaw motility and fertility of Arctic char, Salvelinus alpinus (L.),
spermatozoa. Aquaculture Research 37, 862 – 868.
Merino, O., Risopatrón, J., Sánchez, R., Isachenko, E., Figueroa, E., Valdebenito, I. &
Isachenko, V. (2011a). Fish (Oncorhynchus mykiss) spermatozoa cryoprotectant-free vit-
rication: stability of mitochondrion as criterion of effectiveness. Animal Reproduction
Science 124, 125–131.
Merino, O., Sánchez, R., Risopatrón, J., Isachenko, E., Katkov, I. I., Figueroa, E., Valdeben-
ito, I., Mallmann, P. & Isachenko, V. (2011b). Cryoprotectant-free vitrication of sh
(Oncorhynchus mykiss) spermatozoa: rst report. Andrologia 44(Suppl. 1), 390–395.
Mirzoyan, A. V., Nebesikhina, N. A., Voynova, N. V. & Chistyakov, V. A. (2006). Preliminary
results on ascorbic acid and lysine suppression of clastogenic effect of deep-frozen sperm
of Russian sturgeon (Acipenser gueldenstaedti). International Journal of Refrigeration
29, 374–378.
Muchlisin, Z. A. (2005). Current status of extenders and cryoprotectants on sh spermatozoa
cryopreservation. Biodiversitas 6, 12– 15.
Ogier De Baulny, B., Le Vern, Y., Kerboeuf, D. & Maisse, G. (1997). Flow cytometric evaluation
of mitochondrial activity and membrane integrity in fresh and cryopreserved rainbow
trout (Oncorhynchus mykiss) spermatozoa. Cryobiology 34, 2.
Ohta, H., Shimma, H. & Hirose, K. (1995). Relationship between fertility and motility of cryop-
reserved spermatozoa of the amago salmon Oncorhynchus masou ishikawae.Fisheries
Science 61, 886–887.
© 2016 The Fisheries Society of the British Isles, Journal of Fish Biology 2016, doi:10.1111/jfb.13052

14 E. FIGUEROA ET AL.
Pérez-Cerezales, S., Martínez-Páramo, S., Beirão, J. & Herráez, M. P. (2010). Evaluation of
DNA damage as a quality marker for rainbow trout sperm cryopreservation and use of
LDL as cryoprotectant. Theriogenology 74, 282–289.
Robles, V., Cabrita, E., Cuñado, S. & Herraéz, M. P. (2003). Sperm cryopreservation of
sex-reversed trout (Oncorhynchus mykiss): parameters that affect its ability for freezing.
Aquaculture 224, 203– 212.
Robles, V., Cabrita, E. & Herraez, M. P. (2009). Germplasm cryobanking in zebrash and other
aquarium model species. Zebrash 6, 281– 293.
Scheerer, P. & Thorgaard, G. (1989). Improved fertilization by cryopreserved rainbow trout
semen treated with theophylline. The Progressive Fish-Culturist 51, 179–182.
Scott, A. P. & Baynes, S. M. (1980). A review of the biology, handling and storage of salmonid
spermatozoa. Journal of Fish Biology 17, 707–739.
Slupphaug, G., Kavli, B. & Krokan, H. E. (2003). The interacting pathways for prevention and
repair of oxidative DNA damage. Mutation Research 531, 231–251.
Tekin, N., Secer, S., Akcay, E., Bozkurt, Y. & Kayam, S. (2007). Effects of glycerol additions
on post-thaw fertility of frozen trout sperm, with an emphasis on interaction between
extender and cryoprotectant. Journal of Applied Ichthyology 23, 60–63.
Thomson, L. K., Fleming, S. D., Aitken, R. J., De Iuliis, G. N., Zieschang, J. A. & Clark, A. M.
(2009). Cryopreservation-induced human sperm DNA damage is predominantly medi-
ated by oxidative stress rather than apoptosis. Human Reproduction 24, 2061 – 2070.
Trus-Cott, B., Idler, D., Hoyle, R. & Freeman, H. (1968). Sub-zero preservation of Atlantic
salmon sperm. Journal of the Fisheries Research Board of Canada 25, 363–372.
Twigg, J. P., Irving, D. S. & Aitken, R. J. (1998). Oxidative damage to DNA in human spermato-
zoa does not preclude pronucleus formation at intracytoplasmic sperm injection. Human
Reproduction 13, 1864–1871.
Wakayama, T., Whittinhgam, D. G. & Yanagimachi, R. (1998). Production of normal offspring
from mouse oocytes injected with spermatozoa cryopreserved with or without cryopro-
tection. Journal of Reproduction and Fertility 112, 11–17.
Wang, B., Van Veldhoven, P., Brees, C., Rubio, N., Nordgren, M., Apanasets, O., Kunze,
M., Baes, M., Agostinis, P. & Fransen, M. (2013). Mitochondria are targets for
peroxisome-derived oxidative stress in cultured mammalian cells. Free Radical Biology
and Medicine 65, 882–894.
Zilli, L., Schiavone, R., Zonno, V., Storelli, C. & Vilella, S. (2003). Evaluation of DNA damage
in Dicentrarchus labrax sperm following cryopreservation. Cryobiology 47, 227–235.
Electronic Reference
Martínez-Páramo, S., Martínez-Pastor, F., Martínez-Rodríguez, G., Herraez, P. & Cabrita,
E. (2009). Antioxidant status in fresh and cryopreserved sperm from gilthead sea
bream (Sparus aurata). Paper presented at the Second International Workshop on
the Biology of Fish Gametes (Valencia, Spain, September 9–11, 2009). Available at
http://digital.csic.es/bitstream/10261/52276/4/Cryopreserved_sperm.pdf/
© 2016 The Fisheries Society of the British Isles, Journal of Fish Biology 2016, doi:10.1111/jfb.13052