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Aquaculture 511 (2019) 634190
Contents lists available at ScienceDirect
journal homepage: www.elsevier.com/locate/aquaculture
Effects of cryopreservation on mitochondrial function and sperm quality in
E. Figueroaa,b, M. Lee-Estevezc, I. Valdebenitod, I. Watanabeh, R.P.S. Oliveirae, J. Romerob,
R.L. Castillof,g, J.G. Faríasc,
Núcleo de Investigación en Producción Alimentaria, Departamento de Ciencias Biológicas y Químicas, Facultad de Recursos Naturales, Universidad Católica de Temuco,
Temuco, Chile
Laboratorio de Biotecnología, Instituto de Nutrición y Tecnología de los Alimentos, Universidad de Chile, Santiago, Chile
Departamento de Ingeniería Química, Facultad de Ingeniería y Ciencias, Universidad de La Frontera, Temuco, Chile
Núcleo de Investigación en Producción Alimentaria, Departamento de Ciencias Agropecuarias y Acuícolas, Facultad de Recursos Naturales, Universidad Católica de
Temuco, Temuco, Chile
Biochemical and Pharmaceutical Technology Department, School of Pharmaceutical Sciences, University of São Paulo, São Paulo, Brazil
Programa de Fisiopatología, Instituto de Ciencias Biomédicas (ICBM), Facultad de Medicina, Universidad de Chile, Av. Salvador 486, Providencia, Santiago 7500922,
Departamento de Medicina Interna Oriente, Facultad de Medicina, Universidad de Chile, Santiago, Chile
Department of Anatomy, Institute of Biomedical Sciences, University of São Paulo, Brazil
Sperm ultrastructure
Mitochondrial function
Antioxidant defences
Fish spermatozoa
Salmo salar
The development of cryopreservation techniques has led to important changes in animal reproductive biotechnology. However, these techniques are associated with cellular and molecular damage, affecting the mitochondrial function and quality of spermatozoa; moreover studies in fish are limited. In this work, the effects of
cryopreservation on ultrastructure, mitochondrial function and antioxidant defences in Atlantic salmon (Salmo
salar) spermatozoa were assessed, along with intracellular calcium (Ca2+)i, mitochondrial DNA sequence and
sperm function (motility and fertilization rate). Significant ultrastructure alterations of the middle piece and
mitochondria were observed in cryopreserved spermatozoa as compared to controls. Oxygen consumption and
ATP were also significantly different in cryopreserved spermatozoa, and in spermatozoa incubated with electron
transport chain (ETC) uncouplers and inhibitors. Mitochondrial membrane potential, motility, fertilization rate
and Ca2+
in cryopreserved spermatozoa displayed significant reductions compared to fresh spermatozoa.
Mitochondrial potential correlated significantly with motility and fertilization rate. A redox imbalance was
observed in frozen spermatozoa due to increased lipid peroxidation and superoxide anion production as compared to fresh spermatozoa. Likewise, increased activity of glutathione peroxidase and total glutathione (GSH/
GSSG), as well as reduced catalase activity, were observed in comparison with fresh spermatozoa. Our results
contribute to a better understanding of cryodamage to mitochondrial functions in fish spermatozoa, and enabled
us to establish potential quality assessment indicators. The data suggest that cryopreservation induces a reduction in overall sperm quality and functionality through disruption of the mitochondrial ultrastructure and
function, leading to energy depletion and increased oxidative stress. This knowledge may also lead to the
identification of a potential biotechnological tool for improving reproductive efficiency in species of commercial
and biological interest.
1. Introduction
The development of sperm cryopreservation techniques has led to
radical changes in human and animal reproductive biotechnology.
However, these techniques cause extensive cellular and molecular damage which has not been studied fully (Figueroa et al., 2016a, 2016b;
Magnotti et al., 2018). Cryodamage generates important structural and
physiological changes: rupture of the plasma membrane, alterations in
mitochondrial membrane potential (ΔΨm), nDNA and mtDNA fragmentation, enzyme inactivation, production of free radicals such as
reactive oxygen and nitrogen species (ROS and RNS), and alterations in
ATP concentration and in the homeostasis of intracellular calcium
Corresponding author at: Universidad de La Frontera, Av. Francisco Salazar 01145, Box 54D, Temuco, Chile.
E-mail address: [email protected] (J.G. Farías).
Received 10 February 2019; Received in revised form 16 May 2019; Accepted 2 June 2019
Available online 06 June 2019
0044-8486/ © 2019 Published by Elsevier B.V.
Aquaculture 511 (2019) 634190
E. Figueroa, et al.
(Ca+2)i (Figueroa et al., 2018b). Few investigations have concentrated
on the effects of cryopreservation on the structural, functional and
genomic stability of the mitochondrion, and how they are related with
the fertilizing capacity of fish spermatozoa in an in vitro model.
These effects may be related with the interruption of oxidative
phosphorylation (OXPHOS) and inactivation of the antioxidant enzymes, changes in mitochondrial integrity and functionality, and
changes affecting mitochondrial ultrastructure, mtDNA and the transcriptome or proteome. These alterations in the mitochondrial dynamic,
ATP production and O2 consumption, have been related with loss of
sperm functionality in mammals and fish. Cartón-García et al. (2013)
reported that the mtDNA genes in Sparus aurata spermatozoa presented
higher levels of damage than nuclear DNA as a result of cryopreservation. Although the relationship between DNA and sperm mitochondrial
function and motility have not been fully explained, some authors indicate that, apart from fragmentation damage to nuclear DNA, the reduction in sperm motility should also be associated with the structure
and metabolic pathways of the mitochondrion (Perchec et al., 1995;
Zilli et al., 2004). Figueroa et al. (2016b) showed experimentally that a
direct correlation exists between the activity of the mitochondrial
membrane potential and the motility and fertilizing capacity of fresh
spermatozoa when vitrified or frozen. This confirms that the mitochondrion is the principal energy source of the spermatozoon, and
that the duration of motility and successful fertilization depend on
mitochondrial functioning.
The mitochondria play a basic role in ATP production through
oxidative phosphorylation. OXPHOS generates an important part of cell
metabolism in ROS production through superoxide (O2%-), hydroxyl
radical (%OH), hydrogen peroxide (H2O2), hypohalous acid (HOX,
where X = Cl−, Br−, I− or SCN−), nitric oxide (NO%) and peroxynitrite
(ONOO−) (Shaliutina-Kolesová et al., 2015; Figueroa et al., 2017).
These molecules are very harmful to cell functioning and contribute to
various diseases associated with alterations to reproductive biology and
fertilizing capacity in animals.
When ROS production exceeds the antioxidant defences in spermatozoa, a state of oxidative stress occurs, producing lipid peroxidation
of the membranes, cell apoptosis and alterations in mitochondrial
function. These alterations have been reported in cryopreserved spermatozoa of Sparus aurata (Cabrita et al., 2005), Morone saxatilis (He and
Woods, 2004), Oncorhynchus mykiss (De Baulny et al., 1997) and recently Salmo salar (Figueroa et al., 2016b); in all cases a significant
reduction is observed in motility and fertilizing capacity, due to the
high sensitivity of these sperm to cold and to the osmotic and oxidative
stress induced during freezing and thawing (Figueroa et al., 2018a,
2018b). Similar alterations have been reported in human and mouse
spermatozoa, with a reduction in sperm viability and motility, increase
in DNA fragmentation, alteration of metabolic pathways and inactivation of enzymes in the seminal plasma and cells (Cabrita et al., 2014;
Figueroa et al., 2015).
To understand the effects of cryopreservation on fish sperm function, it is necessary to explore the relationship between mitochondrial
activity and sperm quality parameters. Oxidative phosphorylation and
the tricarboxylic acids cycle in mitochondria are metabolic pathways
with key roles for motility and fertilizing capacity (Figueroa et al.,
2017; Boryshpolets et al., 2018); however, there is little information
about the effects of freezing/thawing on mitochondria, and the consequences for the physiology of fish spermatozoa. Hence the object of
this work was to evaluate the effects of cryopreservation on mitochondrial function and antioxidant defences as a base model for assessing post-thaw sperm quality in Atlantic salmon (Salmo salar), which
in turn may allow the development of new tools for sperm evaluation
and biotechnological strategies to improve cryopreservation protocols
and reproduction efficiency of valuable fish species for aquaculture
worldwide, such as Atlantic salmon.
2. Materials and methods
2.1. Chemicals and reagents
All chemicals used in this study were purchased from Sigma (St.
Louis MO, USA), unless otherwise indicated. All solutions were prepared using water from a Milli-Q Synthesis System (Millipore, Bedford,
MA, USA). The Mitochondrial Permeability Detection Kit AK-116 (MīTE-Ψ ™, JC-1), the superoxide anion production detection Kit (DHE/
SYTOX® Greed), the Intracellular Calcium Indicator (Fluo-4 AM), the
Extracellular Oxygen Consumption Detection Kit (MitoXpress®-Xtra),
the Luminescent Cell Viability Assay Kit (CellTiter-Glo®), the Total
Glutathione Assay Kit and Catalase Activity Assay Kit (OxiSelect™) and
the Glutathione Peroxidase Activity Assay Kit (GPx, K762-100), were
purchased from Invitrogen (Oregon, USA), Roche Diagnostics GmbH
(Mannheim, Germany), Biomol International LP (Pennsylvania, USA),
Molecular Probes (Oregon, USA), Luxcel Biosciences Ltd. (Cork,
Ireland), Promega Corporation (Madison, USA), Cell Biolabs, INC® (San
Diego, USA) and BioVision® (Milpitas, USA) respectively.
2.2. Broodstock
This investigation was carried out in the Engineering, Biotechnology
and Applied Biochemistry Laboratory (LIBBA) of Universidad de la
Frontera, Temuco, Chile; the Microbial Biomolecule Laboratory,
Universidade de São Paulo, Brazil; the Aquaculture Biotechnology Unit
(BIOACUI) of the School of Aquaculture of Universidad Católica de
Temuco, Chile; and the Biotechnology Laboratory (INTA) of the
University of Chile, Chile. The fifteen male Salmo salar were two to
three years old (sexually mature), with average mass of 8.1 ± 0.4 kg
and total length 83 ± 0.7 cm. They were provided by Hendrix Genetics
Aquaculture S.A. and Aquagen Chile S.A., from a commercial farm in
southern Chile (39° 23′ 17″ S, 71° 40′ 40″ W). During the experimental
period, the broodstock were kept in 3500 L fibreglass tanks with fresh
water (550 L h−1) at 8 °C under a natural photoperiod.
2.3. Gamete collection
Sperm was collected as described by Figueroa et al. (2018a). Briefly,
the ten males were anaesthetized in a 50 L tank with 125 mg L−1 MS222 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 to avoid contamination with faeces, mucus or urine.
Immediately after collection, sperm motility and concentration were
determined using a phase contrast microscope (Carl Zeiss Jena, Jena,
Germany). The sperm motility of the samples was assessed using
Computer-Assisted Sperm Analysis (CASA) as described below.
Spermatozoa concentration was determined with a Neubauer haemocytometer in Cortland non-activating medium (Trus-Cott et al., 1968)
for fish spermatozoa, as described by Figueroa et al. (2016b). Only 15
samples exhibited sufficiently high motility (> 80%) and average
sperm concentration (14 × 109 ± 2.7 spermatozoa ml−1) for use in
this study.
2.4. 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 modified
protocol of Figueroa et al. (2016b). The frozen semen was diluted at
4 °C in Cortland medium, supplemented with 1.3 M dimethyl sulphoxide (DMSO), 0.3 M glucose and 2% bovine serum albumin (BSA) to
establish the cryoprotectant medium. The dilution ratio was 1:3
(semen: cryoprotectant medium). The semen was stored for 7 to 10 min
after dilution in 0.5 ml plastic straws, which were sealed after charging.
Aquaculture 511 (2019) 634190
E. Figueroa, et al.
Subsequently, the straws were cryopreserved with the Freeze Control®
programmable freezing system (Cryologic, Australia) at a freezing rate
of 62.3 °C/min from 4 °C to −120 °C, controlled by CryoGenesis 5.0
software (Cryologic) in liquid nitrogen vapour (N2L), for later transfer
to cryotanks. After 2 months' frozen storage, the straws were removed
from the cryotank and thawed in a thermo-regulated bath at 40 °C for
9 s. The thawed semen was used immediately for sperm function assessment.
fluorescence and luminescence in real time of the mitochondrial metabolism at constant temperature and pH (4 °C and pH 8.0). The oxygen
consumption was expressed in RFU (relative fluorescence units)/
109spermatozoa and the ATP content was expressed in nmol/109
2.6.2. Mitochondrial membrane potential (ΔΨM)
To evaluate mitochondrial activity, changes in the ΔΨM were determined using JC-1 fluorescent cation. JC-1 is a lipophilic dye that is
internalized by all functioning mitochondria, where it fluoresces green.
In highly functional mitochondria, the concentration of JC-1 inside the
mitochondria increases and the stain forms aggregates that fluoresce
red. In brief, 250 μL of sperm sample were centrifuged at 720 g for
5 min. The pellet was re-suspended 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 720 g, the supernatant was
discarded and the sperm pellet re-suspended in 400 μL Cortland extender and immediately analysed by flow cytometry. The analysis in
each trial was replicated three times.
2.5. Mitochondrial and sperm ultrastructure
The protocol of Lim and Le (2013) (modified) was applied for
viewing by Scanning Electron Microscope (SEM) and Transmission
Electron Microscope (TEM). Fixing for SEM was performed in 2.5%
glutaraldehyde in 0.1 M cacodylate buffer (TC) at pH 7.4 for 2 h at 4 °C;
then the samples were washed twice with TC for 10 min and post fixing
was carried out for 2 h with 1% osmium tetroxide. The samples were
dehydrated in graduated solutions of ethanol for 2 h and dried at ambient temperature. They were then dehydrated again in liquid CO2 and
coated with gold at 35–40 nm by cathodic pulverization (EDWARDs,
model S150 Sputter Coater). For TEM the samples were fixed in 2.5%
glutaraldehyde in 0.1 M TC at pH 7.4 for 10–12 h at 4 °C; then the
samples were washed in TC and post fixing was carried out for 2 h in 1%
osmium tetroxide. After four washes in TC, the samples were dehydrated in graduated solutions of ethanol and immersed in propylene
oxide, then embedded in resin at a 1:1 ratio of propylene oxide and
Spurr resin (EMS—Electron Microscopy Sciences; Hatfield, PA, USA) in
a rotatory mixer for 16 h. Ultrafine sections (40–50 nm) were cut by
ultramicrotome (Leica Ultracut UCT—Leica Biosystems; Wetzlar, Germany) and then stained with 2% alcoholic uranyl acetate and lead citrate for evaluation.
2.6.3. Motility
The percentage of motile spermatozoa (%) was determined in a
phase contrast optical microscope (Olympus BX 41, Japan; with ×200
magnification) through CASA. The sperm samples were activated in
Powermilt® (280 mOs Kg−1 and pH 9.0). To prevent the spermatozoa
from adhering to the slide, 0.25% (w/v) of Pluronic (Sigma-Aldrich)
was added to the activator. The spermatozoa were recorded with a SSCG818digital video camera (SONY; www.sony.com) mounted on the
microscope, filming at 25 frames s−1 at 50 Hz. They were analysed
using ImageJ software (JAVA, version 1.49, NIH, Bethesda, MD, USA;
www.virtualdub.org) for processing images and videos. The analysis
was replicated three times in each trial.
2.6. Sperm quality parameters
2.6.4. Fertility
A pool of oocytes from 20 females was used to test the fertility of the
fresh and cryopreserved semen. The procedure described by Figueroa
et al. (2016b) was followed; all the fertility tests were carried out in
quintuplicate with 200 oocytes per replication. The sperm density used
in all the treatments was 1.5 × 107 spermatozoa/oocyte. The eggs were
incubated in open flow at 10 °C. Fertilization was evaluated by observation of the first cleavages (segmentation) after 16 h incubation at
10 °C.
For oxygen consumption and ATP content the following experimental groups were considered: Group 1: sperm suspension + Cortland
(basal); Group 2: sperm suspension + Cortland®; Group 3: sperm
suspension + Cortland® + potassium cyanide or 2,4-dinitrophenol;
Group 4: sperm suspension + Cortland® + Antimycin A and Group 5:
sperm suspension + Cortland® + Rotenone. For Group 1 (basal) sperm
motility was not activated, while for Groups 2, 3, 4 and 5 (treatment)
sperm motility was activated with Powermilt®. The order and number
of groups varied, depending on the type of evaluation performed.
2.6.5. Intracellular calcium (Ca2+)i
We used the intracellular fluorescent probe Fluo-4 AM (non-fluorescent acetoxymethyl ester, Molecular Probes, Eugene, Oregon, USA); this
probe is hydrolysed (loss of acetomethyl group (AM)) by cytosolic esterases, leaving the fluo-4 molecule free and sensitive to Ca2+. We
followed the modified protocol of Guthrie et al. (2011), adjusting the
concentration to 2 × 109 spermatozoa/ml followed by centrifugation at
720 g for 5 min in Cortland medium. The pellet was re-suspended in
200 μL Cortland medium with calcium and/or free of calcium (EGTA
5 mM). Then 1.6 μl of fluo-4 AM (1 μM) was added and this was incubated for 45 min at ambient temperature. The system was calibrated
(Ca2+)i by incorporating ionomycin (1 mM) into the various media.
Spermatozoa positive for fluo-4 emitted green fluorescence (Fig. 8). The
data were expressed as concentration of free calcium (Ca2+)free/1 × 109
spermatozoa as per the modified specifications of Molecular Probes
2.6.1. O2 consumption and ATP content
The MitoXpress®-Xtra kit (Luxcel, Biosciences) was used to measure
extracellular oxygen consumption. The assay was based on the capacity
of O2 to complete the excited state of the MitoXpress probe. When the
O2 in the medium is exhausted, the fluorescence signal in the probe
increases. We also used the CellTiter-Glo® luminescence kit for viable
cells (Promega®); this test is a combination of reactions (CellTiter-Glo®
Buffer and CellTiter-Glo® Substrate) which allows cell lysis and the
generation of luminescence resulting from the thermo-stable properties
of luciferase (Ultra-Glo™ recombinant luciferase). The sperm density
was adjusted to 2 × 109 spermatozoa/mL per treatment, then the
sample was centrifuged at 1800 rpm for 2 min and the supernatant
discarded. The pellet was re-suspended in 150 μL of Cortland® medium.
After each suspension of spermatozoa in which O2 consumption was
assessed, inhibiting agents were added independently: antimycin A
(10 μM) and rotenone (10 μM); and an uncoupling agent: 2,4 dinitrophenol (0.5 mM), incorporating 10 μL of probe (MitoXpress). The
ATP content was assessed independently by adding inhibiting agents
only: antimycin A (10 μM), rotenone (10 μM) potassium cyanide
(10 mM), plus 100 μL of the reagent CellTiter-Glo®. The assessment was
read in a multimodal reader for approx. 3 min, with assessment periods
at 0, 5, 10, 20, 40, 80 and 160 s. The analyses were based on
2.7. Oxidative stress and enzyme activity
To assess LPO and antioxidant enzyme activity the samples were
centrifuged at 1800 rpm for 10 min at 4 °C. The supernatant was carefully collected and discarded. The sperm pellet was re-suspended with
1 mL of lysis buffer (Tris 50 mM, NaCl 100 mM, EDTA 1 mM, EGTA
Aquaculture 511 (2019) 634190
E. Figueroa, et al.
Fig. 1. Sperm ultrastructure of Atlantic salmon (Salmo salar) by scanning electron microscopy (SEM): A, B and C: Fresh sperm (Control), hr: head region, mp:
midpiece, f: fagellum and tr: tail region; D, E and F: Frozen sperm. Grey arrows indicate in D: loss of the terminal portion of the flagellum; E: Lumps in the head; F:
Protuberances and decoupling of the middle piece.
2.5 mM, Tween-20 0.1%, PMSF 100 μg/mL, pH 8.0) at a concentration
of 2 × 109 sperm/mL. Cell disruption was performed using glass beads
(1 mm diameter); the samples were homogenised in a vortex at 40 Hz
for 5 min and incubated in an ice bath for 2 min. Then the samples were
centrifuged at 10,000 rpm for 30 min at 4 °C. The supernatant was divided into aliquots and stored at −80 °C for evaluation of the following
2.7.2. Glutathione (GSH/GSSG)
The OxiSelect™ Total Glutathione Assay Kit (Cell Biolabs, INC®) was
used; this is a quantitative assay to measure the total glutathione content in the samples (GSH/GSSG). A volume of 100 μL of fresh and
cryopreserved samples was adjusted and incubated with 25 μL of glutathione reductase (1×) and 25 μL of nicotinamide adenine dinucleotide phosphate (1× NADPH) for 3 min; then 50 μL of chrome gene (1×)
was added to start the reaction. Glutathione reductase reduces the
oxidized glutathione (GSSG) to reduced glutathione (GSH) in the presence of NADPH. The chrome gene then reacts with the thiol group of
GSH to produce a red compound. The GSH/GSSG content was obtained
from the absorbance compared with the standard curve for glutathione.
The absorbance was determined at 405 nm during 10 min of incubation
with readings at 1 min intervals using a microplate reader. GSH/GSSG
concentration was expressed as μM/mL.
2.7.1. Lipid peroxidation
The thiobarbituric acid reactive substances (TBARS) protocol described by Shaliutina-Kolesová et al. (2015) was used, adapted to assess
lipid peroxidation in fish spermatozoa. A volume of 70 μL of fresh and
cryopreserved samples was adjusted and incubated with 130 μL of
thiobarbituric acid in a thermoregulated bath (98 °C) for 20 min and
then chilled on ice for 5 min. The TBARS values were obtained by absorbance compared with the standard malondialdehyde (MDA) curve
generated by hydrolysis with catalyst 1,1,3,3-tetraethoxypropane acid.
The absorbance was determined at 532 nm using a microplate reader.
The MDA values were expressed as nmol MDA/mL.
2.7.3. Glutathione peroxidase (GPx)
The Glutathione Peroxidase Activity Colorimetric Assay kit
(BioVision®) was used; this is an indirect quantitative assay to measure
GPx activity. GPx reduces the concentration of cumene hydroperoxide
in samples, generating the oxidation of reduced glutathione (GSH) to
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Table 1
Morphometric parameters of fresh and cryopreserved spermatozoa of Atlantic
salmon (Salmo salar).
Fresh sperm
Frozen sperm
Overall Length
Head (h)
38.71 ± 0.02μma
L: 2.1 ± 0.2μma
W: 1.9 ± 0.2μma
Spherical shaped
L: 0.48 ± 0.01μma
W: 0.34 ± 0.03μma
Bell shaped
L: 0.53 ± 0.01μma
W: 0.44 ± 0.02μma
Cylindrical shaped
L: 0.42 ± 0.01μma
W: 0.27 ± 0.02μma
1 Cylindrical and helical
Normal cytoplasmic
(m. crests)
L: 36.1 ± 0.10μma
W: 0.21 ± 0.01μma
Conical shape
9 (peripheral) + 2
36.88 ± 0.02μma
L: 2.4 ± 0.3μma
A: 1.4 ± 0.1μma
Ovoid shaped
0.46 ± 0.02μma
0.31 ± 0.02μma
Bell shaped
0.77 ± 0.02μmb
0.81 ± 0.02μmb
Cylindrical shaped
0.69 ± 0.01μmb
0.45 ± 0.02μmb
1 Amorphous shape
Nucelar Fossa (nf)
Midpiece (mp)
Mitochondria (m)
Flagella (F)
condensed cytoplasmic
(m. crests)
33.8 ± 0.12μma
0.18 ± 0.01μma
Conical shape
9 (peripheral) + 2
L: Length of the structure axis; W: Width of the structure axis. Different letters
presented statistically significant difference (p < .05, n = 35 for the control
and cryopreserved groups).
oxidized glutathione (GSSG). The reduction of GSSG to GSH depends on
NADPH consumption by glutathione reductase (GR). Thus, the diminution in NADPH is proportional to GPx activity. A volume of 10 μL
of fresh and cryopreserved samples was adjusted and incubated with
40 μL of reaction mix (33 μl buffer, 3 μl 40 mM NADPH solution, 22 μl
GR solution; 2 μl GSH solution) for 15 min to exhaust all the GSSG in
the samples. Then 10 μl of cumene hydroperoxide was added to start
the GPx reaction. GPx activity was obtained by the variation in absorbance compared with the standard NADPH curve during 10 min reaction at 25 °C. The absorbance was determined at 340 nm using a microplate reader. GPx activity was expressed as nmol/min/mL.
Fig. 2. Sperm ultrastructure of Atlantic salmon (Salmo salar) by transmission
electron microscopy (TEM): A: Modified sperm comparative model of
Lahnsteiner and Patzner (2008), nu: nucleus, nf: nucelar fossa, pc: proximal
centriole, dc: distal centriole, mp: midpiece, pm: plasmatic membrane, m: mitochondria, rc: ring of cytokeletal filament, f: flagellum, tr: tail region. B and C:
Longitudinal sections of nu, pc, dc and M in cryopreserved sample; D and C:
transversal sections of the mp, m and rc in fresh samples controls.
2.7.4. Catalase (CAT)
The OxiSelect™ Catalase Activity Assay kit (Cell Biolabs, INC®) was
used; this is a quantitative assay to measure CAT activity. The disintegration velocity of hydrogen peroxide (H2O2) by CAT to water and
oxygen is proportional to the concentration of catalase during 1 min of
reaction. The remaining hydrogen peroxide is mixed with a reaction
mix (chrome gene) generating a coupling product called quinoneimine,
which is correlated with the quantity of H2O2 remaining in the reaction
mix. A volume of 20 μL of fresh and cryopreserved samples was adjusted and incubated with 50 μl of H2O2 (12 mM) for 1 min. The reaction was stopped with 50 μL Catalase Quencher® and 5 μl of the suspension was transferred to a free well. Then 250 μL of chrome gene was
added to each well and incubated for 60 min in continuous orbital
movement. The CAT activity was obtained by comparing absorbance
with the standard curve for catalase using the second order polynomial
equation. The absorbance was determined at 520 nm using a microplate
reader. CAT activity was expressed as U/mL.
(2.5 × 106 sperm/mL) was added to 2.5 μL of DHE (5 mM) and 0.7 μL of
SYTOX (5 mM) and incubated for 10 min at 10 °C in darkness. Then it
was centrifuged at 13,000 rpm for 5 min, the supernatant was discarded
and the pellet was re-suspended in 300 μL of Cortland® medium before
detection by flow cytometry. The analysis in each trial was replicated
three times.
2.8. Mitochondrial DNA sequencing and analysis
Mitochondrial DNA (mtDNA) was extracted, sequenced and assembled as described by Lee-Estevez et al. (2017), using the Wizard®
Plus SV Minipreps DNA Purification System (Promega Corp.). DNA Next
Generation Sequencing (NGS) Service was hired from Macrogen Inc.
(Korea) using Illumina HiSeq 2000 platform with 1 Gigabase output.
Data was processed with the NGS QC Toolkit (Patel and Mukesh, 2012)
and a homology search was performed for Salmo salar reads, using a
reported sequence as reference (NCBI Accession number NC_001960.1).
Genome assembly was done using SOAP2 and the resulting sequences
were scanned for SNP (SOAPsnp), insertions/deletions (SOAPindel) and
structural variations (SOAPsv).
2.7.5. Intracellular superoxide (O2−
i )
The DHE (Dihydroethidium)/SYTOX® green kit (Invitrogen®) was
used following the modified protocol of Figueroa et al. (2018a) for fish
spermatozoa. In general, the DHE probe is permeable and reacts with
O2– to form 2-hydroxyethidium, which becomes interspersed in DNA
emitting red fluorescence. SYTOX is a green nucleic acid colorant which
only penetrates cells that present membrane damage, but does not cross
viable cell membranes. A sperm suspension of 250 μL
Aquaculture 511 (2019) 634190
E. Figueroa, et al.
Fig. 3. Sperm ultrastructure of Atlantic salmon
(Salmo salar) by transmission electron microscopy
(TEM): A and B: Longitudinal section of the nu, m
and rc of fresh samples (control), the banded strips
indicate membranes (A), ridges and mitochondrial
matrix (B). C and D: Longitudinal section of nu, m, rc
and pm of cryopreserved samples. Grey arrows indicate morphological alterations and uncoupling of
the mitochondria and loss of the envelope of the
middle piece (C and D) and the white arrow indicates
the mitochondrial crests (D).
minimum of 3 × 106 spermatozoa and 1–2 mg/mL proteins were examined at a reading speed of 31 s for 96 wells. The fluorescence was
measured from a Tunsgten halogen Xenon flash light source through
emission interference filters (485/20 nm) with excitation (528/20 nm),
jointly with a PMT (photomultiplier) detector for greater reading sensitivity. The UV–Visible absorbance was measured from a Xenon flash
light source through a photodiode detector and a monochromator
(200–999 nm).
2.9. Electron microscopy
The samples were observed under a SEM (JSM 6380 LV; JEOL Inc.,
Peabody, MA, USA), at 20 kV, and TEM (JEM-1200EX II; JEOL Inc.,
Peabody, MA, USA), at 60–80 kV, to determine the ultrastructure of
spermatozoa and mitochondria. SEM and TEM images were analysed
with the digital image processing ImageJ software (JAVA, version 1.49,
NIH, Bethesda, MD, USA; www.virtualdub.org) to evaluate morphometric parameters of the ultrastructure of 25 spermatozoa selected at
random from 60 images.
3. Statistical analysis
2.10. Confocal microscopy
The data were expressed as mean ± standard deviation. Normality
and homoscedasticity were analysed with the Prisma® statistics programme, version 6.0. Wilcoxon's t-test (paired samples) was applied to
assess morphometric, functional and enzymatic parameters.
Nonparametric One-Way ANOVA (Kruskal-Wallis test) with Dunn's
multiple comparisons post-test was used to assess the different treatments in the oxygen consumption dynamic and the ATP content.
Spearman's correlation was used to relate the intracellular calcium with
mitochondrial membrane potential, motility and fertilization rates. The
level of significance was set at p < .05.
The sperm samples were observed under a synchronised laser
scanning inverted confocal microscope (Olympus, Fluoview FV1000,
http://www.olympus-lifescience.com). The probe emissions were
evaluated over a wavelength range of 405–635 nm. The samples were
subjected to multiple excitation by Ar laser (488 nm) and He/NeG laser
(543 nm).
2.11. Flow cytometry
FACs Canto II flow cytometer (BD Biosciences; www.bdbiosciences.
com) was used to determine the following variables: mitochondrial
membrane potential (with JC-1), intracellular calcium (fluo-4 AM) and
intracellular superoxide (DHE/SYTOX® green). A minimum of 10,000
spermatozoa were examined in each assay at a flow rate of
100 cells 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 (20 mW) and 633 nm
HeNe lamp (17 mW). Green fluorescence was measured in the FITC
channel (533/30 nm) and red fluorescence in the PE channel (585/
42 nm).
4. Results
4.1. Ultrastructure
Under SEM the fresh spermatozoa of Atlantic salmon (control)
presented a total length of 38.71 ± 0.02 μm, round head (hr) and
wrinkled zones in the plasma membrane, with no acrosomal complex. A
small middle piece (mp) was observed connecting the head with the
flagellar structure (f). (Fig. 1A, B and C). The cryopreserved spermatozoa presented an ovoid head with multiple protuberances in the
plasma membrane and disconnection of the middle piece; the terminal
part of the flagellum was shorter than in the control (36.88 ± 0.02 μm)
but with no statistically significant differences (Fig. 1D, E and F,
Table 1).
The findings of morphometric analysis by SEM of the fresh and
2.12. Microplate reader
A Synergy™ HTX Multi-Mode Microplate Reader (BioTek®) was used. A
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Fig. 4. Dynamics of oxygen consumption (A) and ATP content in non-activated (B) and activated (C) fresh (F) and cryopreserved (T) spermatozoa of Atlantic salmon
(Salmo salar) incubated for a period of 160 s at 4 °C. Different letters presented statistically significant difference (p < .05, n = 12 for each treatment). RFU: Relative
fluorescence unit.
cryopreserved spermatozoa are described in Table 1; statistically significant differences were observed in the length (0.53 μm) and width
(0.44 μm) of middle piece in frozen spermatozoa as compared with the
control group (0.77 μm; 0.81 μm respectively; p < .05).
Statistically significant differences were observed in the length
(0.42 μm) and width (0.27 μm) of the mitochondrion between cryopreserved and native (control) spermatozoa (0.69 μm; 0.45 μm respectively, p < .05). In cryopreserved spermatozoa, we observed loss of
mitochondrial shape, loss of the external mitochondrial membrane and
middle piece, and uncoupling, leading to a significant augmentation of
the morphometric measurements of the middle piece detected with
SEM and TEM in frozen spermatozoa (Table 1; Fig. 2A, B, C, D and E;
Fig. 3A, B, C and D). In fresh spermatozoa, TEM images of mitochondrial ultrastructure showed partial presence of the external and internal
membrane, mitochondrial crests and matrix. In the cryopreserved
samples however, more condensed internal structures were detected by
their high electron density, allowing a partial view of the mitochondrial
crests and matrix (Fig. 3A, B and D).
4.2. Physiology
4.2.1. Mitochondrial function
The cryopreserved spermatozoa incubated for 160 s showed statistically significant difference in initial oxygen consumption rate (0 s) in
basal state (2963 ± 512 RFU/109spz) compared to fresh samples
(4130 ± 344 RFU/109spz; p < .05). Similarly, in cryopreserved
spermatozoa after motility activation oxygen consumption increased at
20s (3444 ± 631 RFU/109spz) and decreased at 160 s incubation
(3107 ± 398 RFU/109spz), showing statistically significant differences
compared to fresh spermatozoa (4740 ± 612 RFU/109spz;
4501 ± 428 RFU/109spz, respectively, p < .05; Fig. 4a).
On the other hand, cryopreserved spermatozoa incubated with
0.5 mM of 2,4-dinitrophenol exhibited increased oxygen consumption
(3446 ± 398 RFU/109spz), but the difference was not significant
compared to activated cryopreserved spermatozoa not incubated with
uncoupling agent (3386 ± 419 RFU/109spz). Conversely, a decrease in
the oxygen consumption rate was observed in spermatozoa incubated
with lectron transport chain inhibitors, such as 10 μM Rotenone
(2682 ± 411 RFU/109spz) and 10 μM Antimycin A (2777 ± 511
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Fig. 6. Dynamics of ATP content in fresh (A) and cryopreserved (B) spermatozoa in Atlantic salmon (Salmo salar) incubated with inhibitory agents for a
period of 160 s at 4 °C. Different letters presented statistically significant difference (p < .05, n = 12 for each treatment).
Fig. 5. Dynamics of oxygen consumption in fresh (A) and cryopreserved (B)
spermatozoa of Atlantic salmon (Salmo salar) incubated with inhibitors and
uncoupling for a period of 160 s at 4 °C. Different letters presented statistically
significant difference (p < .05, n = 12 for each treatment). RFU: Relative
fluorescence unit.
treatments showed statistically significant differences with respect to
spermatozoa in basal condition (p < .05, Fig. 6b). Statistically significant differences were also observed in fresh spermatozoa incubated
with the different inhibitors (p < .05, Fig. 6a).
RFU/109spz), being statistically significant with respect to activated
and basal cryopreserved spermatozoa not incubated with those agents
(3274 ± 431 RFU / 109spz; 3110 ± 399 RFU / 109spz, respectively,
p < .05; Fig. 5b). Similarly, statistically significant differences were
observed in fresh spermatozoa incubated with inhibitors and uncoupling agents (p < .05, Fig. 5a).
With respect to ATP, basal content in cryopreserved spermatozoa
was significantly lower compared to fresh spermatozoa (p < .05,
Fig. 4b) during the incubation period (160 s). After activation, both
fresh and cryopreserved spermatozoa showed a progressive decrease in
ATP content reaching near-zero values at 160 s. Activated cryopreserved spermatozoa displayed statistically lower values of ATP content
at all measuring points, and the decreasing pattern was faster than that
exhibited by fresh spermatozoa (Fig. 4c).
Cryopreserved spermatozoa incubated with 10 mM potassium cyanide showed a significant decrease of ATP content at 5 s post-activation
(1.315 ± 0.6 nmol/109spz) compared to cryopreserved spermatozoa
in basal condition (5.743 ± 0.5 nmol/109spz), activated condition
(3.511 ± 0.8 nmoles/109spz) and those incubated with 10 μM
Antimycin A (3.217 ± 0.6 nmoles / 109spz) and 10 μM of Rotenone
(2.9 ± 0.7 nmoles/109spz) (p < .05; Fig. 6b). Similarly, sperm incubated with inhibitory agents such as 10 μM Antimycin A showed an
increase in ATP concentration compared to 10 μM Rotenone; both
4.2.2. Sperm function
The mitochondrial membrane potential in cryopreserved spermatozoa was significantly lower (61 ± 5.4%) than in the control group
(89 ± 4.7%; p < .05; Fig. 7). Also, a diminution of 63 ± 7.7% was
observed in the motility of cryopreserved spermatozoa as compared to
the control group (97 ± 5.1%; p < .05; Fig. 7) and diminution of
85 ± 7.2% in the fertilization rate of cryopreserved spermatozoa as
compared to the control group (98 ± 5.2%; p < .05; Fig. 7).
Turning to the intracellular calcium concentration, statistically
significant correlations were observed between the (Ca2+)i the mitochondrial membrane potential (r = 0.77), the motility (r = 0.70) and
the fertilization rates (r = 0.75; Fig. 8). Moreover, different Ca2+
fluorescence distribution patterns were observed in the head, middle
piece and flagellum of cryopreserved spermatozoa from those of the
control group (Fig. 8d).
4.2.3. Oxidative stress and enzyme activity
In cryopreserved spermatozoa, a statistically significant increase
was observed in MDA concentration (3.66 ± 0.8 nmol MDA/ml), due
to lipid peroxidation of the cell membrane, as compared to fresh
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Fig. 7. Physiological parameters in cryopreserved spermatozoa of Atlantic salmon (Salmo salar) ( ) and fresh sperm as a
control group (□). Different letters: Indicates statistically
significant differences between the control and cryopreserved
group (p < .05, n = 12).
Fig. 8. (Ca+2)i free ratio in post-thawed spermatozoa of Atlantic salmon (Salmo salar) with (a) mitochondrial m. potential (b) motility and (c) fertility. Confocal
microscopy (d) with Fluo-4 AM: (1) fresh green spermatozoa, distribution of fluorescence in basal bodies of the head and middle piece (indicated in the red box) and
(2) green post-thaw spermatozoa, distribution of fluorescence in the head, middle part and part of the flagellum (p < .05, n = 12). (For interpretation of the
references to colour in this figure legend, the reader is referred to the web version of this article.)
spermatozoa: 1.23 ± 0.5 nmol MDA/ml (p < .05; Fig. 9a). Likewise a
significant increase was detected in the percentage of viable cells expressing levels of DHE (O2−
i ) in frozen samples (24.3 ± 2.8%) as
compared to fresh spermatozoa: 16.5 ± 3.3% (p < .05; Fig. 9b).
Turning to antioxidant enzymatic activity, a statistically significant
increase was observed in GPx activity (12.8 ± 3.4 nmol/min/ml) and
total GSH/GSSG content (0.73 ± 0.3 μM/ml) in cryopreserved spermatozoa as compared to enzymatic activity in fresh spermatozoa
(7.9 ± 1.1 nmol/min/ml; 0.51 ± 0.1 μM/ml respectively; Fig. 9c, d;
p < .05). On the other hand there was a reduction in CAT enzymatic
activity in cryopreserved spermatozoa (0.79 ± 0.1 U/ml), with significant differences as compared to the control group (Fig. 9e;
p < .05).
4.2.4. mtDNA sequence
For each condition (fresh and cryopreserved), mtDNA-enriched
samples were obtained with concentration above 170 ng/uL and high
purity (A260/A280 > 1.8). DNA integrity was confirmed by the appearance of single thick bands after agarose gel electrophoresis. The
assembled genomes and reference sequence were all identical in size
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Fig. 9. Effects of cryopreservation on (a) Lipid peroxidation (LPO), (b) production of superoxide (O2−
SYTOX-/DHE+), (c) activity of glutathione peroxidase (GPX),
(d) Total glutation (GSH/GSSG) and (e) Catalase activity (CAT) in spermatozoa of Atlantic salmon. Different letters presented statistically significant differences
(p < .05, n = 12 for the control and cryopreserved groups).
(16,665 bp) and showed the same gene content and organization. The
comparative analysis of reconstructed mitochondrial genomes did not
show any difference among samples, or between them and the reference
sequence. No insertions, deletions or structural variations were found in
cryopreserved samples compared to fresh samples and the reference
sequence. Two SNP were detected in all samples, at positions
2271A > G and 2272G > A.
needed by the spermatozoon for flagellar movement, compromising
cellular osmoregulation, ion exchange, lipid peroxidation and the enzymatic mechanisms which regulate motility (Cabrita et al., 2010;
Ciereszko et al., 2014; Figueroa et al., 2018a).
The metabolic pathways, which maintain ATP concentration and
flagellar activity in fish spermatozoa depend on oxygen consumption.
In Atlantic salmon spermatozoa, respiration varies around 1 to 4 nmol
O2/min/109sperm and the ATP concentration from 1 to 8 nmol/
109sperm (Vladić et al., 2002; Ingermann et al., 2003; Cosson, 2010).
The results obtained in the dynamics of oxygen consumption and ATP
content in fresh and cryopreserved spermatozoa of Atlantic salmon
presented a gradual diminution of ATP accompanied by an increase in
oxygen consumption in fresh spermatozoa; differences were found in
comparison with cryopreserved spermatozoa, due to the rapid loss of
ATP content and increase in oxygen consumption. Christen et al. (1987)
observed in rainbow trout spermatozoa that when sperm motility was
activated, the oxygen consumption increased and the ATP content diminished rapidly; however the oxygen consumption did not differ significantly from unactivated sperm in the basal state (not activated).
Boryshpolets et al. (2009) and Dzyuba et al. (2014) reported in carp
spermatozoa that an increase in osmolarity and pH of the storage
5. Discussion
According to the morphometric results of this study, the greatest
structural alterations were detected in the middle piece and mitochondrion of Atlantic salmon spermatozoa. This agrees with reports
of spermatozoa of Macrozoarces americanus, Thymallus thymallus,
Micropogonias undulatus and Oncorhynchus mykiss, which describe
morphological changes found in SEM and TEM. Damage is found in the
middle piece of the spermatozoon after thawing, leading to the formation of protuberances or thickening of the plasma membrane as a
result of loss of the dense sheath around the mitochondria (Zhang et al.,
2003; Cabrita et al., 2010). These alterations lead to loss of mitochondrial functionality and a diminution of the energy reserves
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medium, and a decrease in temperature, lead to significant reductions
in O2 consumption and ATP content. These effects would be related
with the cryoprotectant medium used in freezing the spermatozoa
(Figs. 4 and 5). According to Christen et al. (1987) and Perchec et al.
(1995), cellular respiration is not necessary during sperm motility in
salmonids and cyprinids. In this case oxygen consumption by the mitochondrion would be fundamental for the production and maintenance
of ATP reserves for use later during the activation of motility and flagellar movement (Cosson et al., 2015). However, in our results, we
observed that 10s post activation of motility in fresh spermatozoa and
5 s post activation of motility in cryopreserved spermatozoa there is a
small increase in the ATP content which may be related with the increase in ATPase activity, which has not previously been reported for
Atlantic salmon.
When Atlantic salmon spermatozoa were incubated with the uncoupling agent 2,4 dinitrophenol, their oxygen consumption showed
that sperm respiration reaches a higher level than that of spermatozoa
not incubated with the uncoupler. However, there was no difference
between these treatments in basal spermatozoa. These results agree
with studies using uncoupling agents in carp and rainbow trout spermatozoa, in which maximum oxygen consumption was recorded but
with no difference between activated and non-activated spermatozoa
(Christen et al., 1987; Boryshpolets et al., 2009). Terner and Korsh
(1963) reported that fish sperm cells present a low respiration rate in
basal conditions and during the motility period when compared to
mammal spermatozoa. However, the variation in oxygen consumption
in fish spermatozoa is regulated to some degree by the proportion of
ADP and/or intracellular ATP, which inhibit or activate respiration
(Dzyuba et al., 2014; Cosson et al., 2015). Spermatozoa incubated with
antimycin A and rotenone presented a reduced oxygen consumption
rate caused by inhibition of I and III complexes of the electron transport
chain. This reduction in the oxygen consumption rate may be related
with the intracellular ATP levels, and also a physiological response to
the blocking of the electron transport chain complexes. In cryopreservation, a reduction was observed in oxygen consumption caused by
alterations to the mitochondrial function or the low physiological response, of the mitochondrion.
Freezing fish spermatozoa alters their oxygen consumption, affecting ATP production and storage in the mitochondrion due to the low
functioning of the mitochondrial dynamic. According to Billard and
Cosson (1990) and Ingermann (2007), > 70% of total ATPase activity is
directly coupled to cell movement and flagellar activity potential
(50–90 Hz frequency), which consumes between 35 and 60% of the
total free energy available from ATP hydrolysis (Perchec et al., 1995;
Cosson et al., 2000); however, this energy availability changes when
the spermatozoa are subjected to cryopreservation processes, affecting
the motility and mitochondrial membrane potential by around 60% and
related with a low mitochondrial dynamic.
In our results, when fresh and cryopreserved spermatozoa of
Atlantic salmon were incubated with potassium cyanide, a reduction in
the ATP content was observed in the first 10 s after activation of motility; however, this reduction was significant in frozen spermatozoa,
where the content was close to 1 nmol/109sperm, compared with
5 nmol/109sperm in basal spermatozoa (Fig. 6). The use of various
compounds which interfere with the respiratory chain (cyanide) or an
agent to uncouple the respiratory chain from ATP synthase (CCCP) or to
block ATP synthase (oligomycin) has been studied in order to understand the dynamic of the ATP content in the spermatozoa of salmonids
and cyprinids (Perchec et al., 1995; Boryshpolets et al., 2009; Cosson
et al., 2015). For example, Boryshpolets et al. (2009) and Cosson (2010)
showed that when motility is activated in rainbow trout and carp
spermatozoa, the ATP concentration diminishes rapidly because ATP
synthesis in the mitochondrion is insufficient to maintain the rate of
dynein ATPase hydrolysis. Furthermore, when inhibitor agents of the
electron transport chain were used, a significant reduction in the ATP
content was observed during motility activation (Vladić et al., 2002).
The mitochondrial function has been considered a key factor in
sperm functioning, particularly in salmonid spermatozoa in which mitochondrial energy activity is of great importance for maintaining
motility and fertility (Cabrita et al., 2010; Figueroa et al., 2016b). According to Cabrita et al. (2014) and Figueroa et al. (2017), the mitochondria in cryopreserved spermatozoa of Dicentrarchus labrax, Acipenser ruthenus, Cyprinus carpio, L., Oncorhynchus mykiss, Salvelinus
fontinalis and Sparus aurata present high sensitivity to cryopreservation.
For example, the frozen spermatozoa of trout and Atlantic salmon
present 40–50% of the mitochondrial membrane potential. This agrees
with data obtained on the mitochondrial membrane potential (ΔΨM) of
61% in cryopreserved Atlantic salmon spermatozoa; moreover, the
motility and fertilizing capacity were also reduced after cryopreservation (Fig. 7). This relationship is similar to that reported by Figueroa
et al. (2015, 2016b) showing positive correlation between mitochondrial membrane potential and fertilization rate in spermatozoa of
Onchorynchus mykiss and Salmo salar, and supports the idea that cryopreservation reduces sperm motility and fertilization capacity, possibly
by disrupting the mitochondrial function.
There is little information about the effects of cryopreservation on
the intracellular calcium concentration (Ca2+)i in fish spermatozoa.
According to Morisawa et al. (1983) and Cosson (2016) the increase in
(Ca2+)i is necessary for the initiation of sperm motility in fish. In salmonids, (Ca2+)i presents a range from 30 nM for basal spermatozoa to
180 nM for motile spermatozoa (Alavi and Cosson, 2006), which agrees
with data obtained in fresh and frozen Atlantic salmon spermatozoa.
However a significant diminution in basal (Ca2+)i was observed in
cryopreserved spermatozoa, which correlated with a diminution in
mitochondrial membrane potential, motility and fertilizing capacity in
spermatozoa post thawing (Fig. 8a, b, c). Okuno and Morisawa (1989)
and Takei Gen et al. (2012) suggest that in spermatozoa of Oncorhynchus mykiss and Oncorhynchus keta, the alteration in sperm motility
is caused by a reduction in (Ca2+)i due to the effects of osmolality
exercised by the dilution media during in vitro storage or freezing,
hence altering the phosphorylation of proteins like PKA (protein kinase
A) and dynein in the axoneme of the flagellum.
The Ca2+
signal detected by the fluo-4 AM probe in fresh Atlantic
salmon spermatozoa was located in the head close to the basal bodies
(nuclear fossa, proximal and distal centriole) and in the middle piece of
the spermatozoon. Ho and Suarez (2001) report that in the nuclear
wrapping and the neck region of bull spermatozoa there is a second IP3
(Inositol trisphosphate) messenger for transducing cell signals to mobilise the stored Ca2+. Similar structures have been observed in the
plasma membrane, basal bodies and mitochondrion in rainbow trout,
agreeing with the observations obtained by confocal microscope in
fresh Atlantic salmon spermatozoa. However, these fluorescence patterns change in frozen spermatozoa, with Ca2+
distributed in much of
the head, middle piece and flagellum (Fig. 8d). This observation agrees
with Takei Gen et al. (2012), that when salmonid spermatozoa suffer
thermal and osmotic shock during storage and cryopreservation, alterations are caused in the calcium distribution patterns which affect
sperm motility and fertilizing capacity.
In cryopreservation the exposure of the cell to thermal shock and
variations in the atmospheric oxygen concentration during freezing
leads to increased lipid peroxidation of the sperm plasma membrane,
due to the generation of ROS (Chatterjee et al., 2001; Li et al., 2010).
According to Lahnsteiner et al. (2011), Martínez-Páramo et al. (2013)
and Shaliutina-Kolesová et al. (2015), an increase in lipid peroxidation
has been detected in the frozen spermatozoa of Dicentrarchus labrax,
Acipenser ruthenus, Cyprinus carpio, L., Oncorhynchus mykiss and Salvelinus fontinalis. This agrees with the data obtained in cryopreserved
Atlantic salmon spermatozoa and differs from observations in fresh
spermatozoa. This effect would be caused by high levels of polyunsaturated fatty acids (PUFAs) contained in the plasma membrane,
which is susceptible to oxidative damage – principally by the superoxide anion (O2−), hydrogen peroxide (H2O2) and nitric oxide (NO%)
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(Trenzado et al., 2006; Figueroa et al., 2018a).
Similar results were observed in the antioxidant enzyme activity in
frozen Atlantic salmon spermatozoa, which presented significantly increased GPx activity and total glutathione content (GSH/GSSG), while a
diminution in CAT activity was observed. According to Kun et al.
(2010), Lahnsteiner et al. (2011) and Liu et al. (2015), the reduction of
antioxidant capacity may be caused by saturation of enzyme activity
due to significant reduction of intracellular ROS, and to the low endogenous antioxidant capacity to control lipid peroxidation and plasma
membrane disruption during cryopreservation. These results suggest
that the molecules remain sufficiently mobile at low temperature, allowing cell ageing reactions to occur. This agrees with the results obtained in the cryopreservation of spermatozoa of Oncorhynchus mykiss,
Salvelinus fontinalis, Pagrus major, Cyprinus carpio L. and Clarias gariepinus (Hisahi et al., 1984; Steyn and Van Vuren, 1987; Kun et al.,
2010; Lahnsteiner et al., 2011). Sperm protection depends on antioxidants and antioxidant enzymes found in the seminal plasma and the
spermatozoon that prevent oxidative damage (Li et al., 2010; Shaliutina
et al., 2013; Dzyuba et al., 2014; Figueroa et al., 2018a).
Complete sequencing was carried out for Salmo salar mtDNA in
fresh and cryopreserved semen samples. Comparative analysis indicates
that cryopreservation does not induce alterations in the sequence and
structure of mitochondrial genome in Salmo salar spermatozoa, hence
disruption of mitochondrial function through this mechanism is unlikely.
In conclusion, the model of structural, functional and genomic
analysis in mitochondria of frozen Atlantic salmon spermatozoa enabled us to identify alterations in the breadth, length and structural
shape of the middle piece, along with reductions in sperm functionality
expressed as decreased mitochondrial membrane potential, motility
and, more importantly, fertilizing capacity, which correlated positively
with ΔΨM. The O2 consumption rate and ATP content were also affected
by cryopreservation in a similar way to uncoupling agents and electron
transport chain inhibitors, altogether supporting our hypothesis that
cryopreservation reduces sperm motility and fertility by disrupting
mitochondrial function. Moreover, a pro-oxidant state was evidenced in
cryopreserved spermatozoa, as per increased of O2– production and
LPO, and reduced activity of antioxidant enzyme catalase, which also
suggests that cryopreservation causes an imbalance in the mitochondrial REDOX state of the spermtaozoa. Collectively, these results support the bases for the study, which was intended to gain understanding
of the effects of cryopreservation on the mitochondria as a base model
for the evaluation of sperm quality; and to seek future biotechnological
applications associated with the quality of gametes and reproduction of
fish of interest for aquaculture.
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This work was supported by the Fund for the Promotion of Scientific
and Technological Development of CONICYT, FONDECYT/
and N° 1171129) and CONICYT National Doctorate Scholarship (N°
21150246) Chile. Special thanks to the companies AquaGen Chile S.A.
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