ORIGINAL RESEARCH

published: 08 July 2020

doi: 10.3389/fphys.2020.00772

High Temperature, pH, and Hypoxia

Cause Oxidative Stress and Impair
the Spermatic Performance of the
Amazon Fish Colossoma
macropomum
Jonatas S. Castro1,2*, Susana Braz-Mota2, Derek F. Campos 2, Samara S. Souza2 and
Adalberto L. Val1,2
1
Aquaculture Graduate Program, Nilton Lins University, Manaus, Brazil, 2Laboratory of Ecophysiology and Molecular
Evolution, National Institute of Amazonian Research, Manaus, Brazil

The control of abiotic parameters is fundamental for fish survival, growth and reproduction.
These factors have a direct effect on sperm quality. Thus, this study evaluated the effect
of different temperatures (29, 31, 33, and 35°C), pHs (4 and 8), and hypoxia (1 mgO2 L−1)
Edited by:
Alexssandro Geferson Becker, on sperm motility of Colossoma macropomum (tambaqui). The results indicated a longer
Universidade Federal do Paraná, duration of sperm motility at 29°C (50.1 ± 2.70 s) that progressively decreased when
Setor Palotina, Brazil
exposed to 35°C (31.2 ± 1.31 s) and hypoxia at pH 4 (27.4 ± 1.42 s) and pH 8
Reviewed by:
Michael L. Fine,
(30.44 ± 1.66 s; p < 0.05), respectively. Sperm oxygen consumption increased in hypoxia
Virginia Commonwealth University, at both pH (pH 4 = 61.22; pH 8 = 54.74 pmol s−1). There was an increase in the activity
United States of glutathione-S-transferase (GST) and superoxide dismutase (SOD), as well as in lipid
Charlene Menezes,
Federal University of Santa Maria, Brazil peroxidation levels (LPO) and DNA damage in sperm exposed to higher temperatures
*Correspondence: and hypoxia. The pH 4 and pH 8 under normoxia did not affect the quality of C.
Jonatas S. Castro macropomum sperm. These results suggest that water warming and acidification,
[email protected]
consequences of climate changes, significantly affect the reproduction of C. macropomum,
Specialty section: reducing the quality of spermatozoids during fertilization.
This article was submitted to
Aquatic Physiology, Keywords: aquaculture, antioxidant enzymes, climate change, comet assay, fish breeding, motility, tropical fish
a section of the journal
Frontiers in Physiology

Received: 26 September 2019 INTRODUCTION

Accepted: 11 June 2020
Published: 08 July 2020 Climate changes are universal and are causing unprecedented environmental and life damage.
Citation: Among the changes are higher temperatures, increases in precipitation rates, extreme drought,
Castro JS, Braz-Mota S, Campos DF, and changes in the habitat of terrestrial and aquatic species, which can result in reallocation
Souza SS and Val AL (2020) High and adaptation to survival (Isaak et al., 2012). Freshwater species face particular challenges
Temperature, pH, and Hypoxia Cause within this scenario, worsened by increased pollution and over-exploitation of commercial
Oxidative Stress and Impair the
fisheries, leading to declining fish populations (Wenger et al., 2011), that is, elimination of
Spermatic Performance of the
Amazon Fish Colossoma
sensitive species, through direct and indirect effects on the biology of these organisms (Jeppesen
macropomum. et al., 2010), as well as on sperm quality and viability (Jenkins et al., 2018). Thereby, in order
Front. Physiol. 11:772. to minimize the impacts caused by fishing and climate change in natural environments,
doi: 10.3389/fphys.2020.00772 aquaculture is becoming increasingly important for the maintenance of fish stocks and for

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Castro et al. Spermic Performance of the Amazonian Fish

animal protein production. Therefore, targeted efforts to assess November and the species presents annual and total spawning.
how fish in confined environments will be affected by the Mature individuals of C. macropomum have high fecundity, releasing
climate change in terms of reproductive efficiency are needed. more than one million oocytes. The fertilization is external and
Dissolved oxygen (DO), pH, and temperature are important the species does not show parental care (Araujo-Lima and Goulding,
factors affecting survival, growth, and reproduction of fish 1998; Vieira et al., 1999). At the beginning of the flood season,
(Caldwell and Hinshaw, 1994). In the Amazon region, these individuals start their upstream migration into whitewater
factors are influenced by diurnal variations of DO, seasonal rivers, and when the gonads are fully developed they spawn
flooding, thermal stratification, aquatic plants, and anthropogenic along the grassy leaves that are being inundated with rising water.
effects, such as eutrophication and global warming (Richards After spawning, the fish enter the floodplain areas where they
et al., 2009). The control of these factors is pivotal for the feed mainly on fruits (Goulding and Carvalho, 1982;
reproductive success of fish that have external fertilization Araujo-Lima and Goulding, 1998).
(Dong et al., 2011), since sperm activity is only triggered when This species is of great interest for researchers and producers
it comes into contact with water (Alavi and Cosson, 2006). due to its acclimatization facility, rapid growth, and great social
Thus, sperm motility is strongly influenced by the activation and economic importance for the central region of the Amazon
medium (Dadras et al., 2016). (Mendonça et al., 2009). Because it is a tropical species,
Sperm motility is a key factor determining semen quality C. macropomum is expected to be especially vulnerable to
being directly related to fertilization success, and is potentially climate change, specifically to warming, because it has a narrower
used as a biomarker of environmental quality (Gallego et al., thermal sensitivity range compared to subtropical and temperate
2018). Negative effect of hypoxia, temperature, and pH on the species and tends to live closer to its thermal limits (Lapointe
spermatic quality of Porichthys notatus, Sparus aurata et al., 2018). Therefore, even small temperature increases can
(Lahnsteiner and Caberlotto, 2012), and Genypterus blacodes lead to declining performance of individuals and populations
(Dumorné et al., 2018) have been documented. The duration (Tewksbury et al., 2008).
of sperm motility of farmedfish species is approximately 1 min; Considering that homeostasis of the sperm of C. macropomum
for this reason, accurate assessments using rapid and sensitive is extremely important for the aquaculture industry,
methods are needed to increase the efficiency of artificial understanding how environmental parameters dependent on
fertilization (Kime et al., 2001; Rurangwa et al., 2004). climate change affect sperm quality is critical in improving
The motility and fertilization capacity of sperm are dependent the methods and manipulations required for successful in vitro
on energy stored in the form of ATP, which in turn is strongly fertilization. Thus, the present study aims to investigate in
influenced by the oxygen concentration (Bencic et al., 1999). vitro effects of temperature, pH and hypoxia on motility and
However, molecular oxygen, although necessary, can induce antioxidant responses of the sperm of the Amazonian fish
the production of reactive oxygen species (ROS), causing C. macropomum.
oxidative stress. During periods of environmental stress to
which sperm are subjected at the time of activation, several
antioxidant enzymes act to neutralize ROS (Dadras et al., 2016). MATERIALS AND METHODS
The antioxidant capacity of semen can vary among fish species
(Slowinska et al., 2013) and the formation of ROS can cause Semen Collection
different types of damage, in particular lipid peroxidation of Males of C. macropomum (n = 3, 60.4 ± 0.36 cm, 4.13 ± 0.05 kg)
membranes, due to the abundance of polyunsaturated fatty were selected from 600 m2 nurseries at the Center for Technology,
acids, and DNA damage, with consequent loss of motility and Training and Production in Aquaculture (CTTPA; Balbina,
decreased fertilization (Aramli and Kalbassi, 2016). Presidente Figueiredo, Amazonas – 1°55′54.4"S; 59°24′39.1"W).
The tambaqui, Colossoma macropomum (Cuvier, 1818), is a The fish were randomly selected for a light abdominal massage
migratory tropical fish species native from the Amazon and and in cranium-caudal direction, and those who released semen
Orinoco basins (Araujo-Lima and Goulding, 1998), belonging were chosen. The fish were recovered from this initial handling
to family Serrasalmidae (Mirade, 2010). The spawning of tambaqui in 1000 L tanks with oxygen 5.54 ± 0.22 mg L−1, temperature
coincides with the beginning of the flood, occurring in October/ 29.2 ± 0.07°C, and pH 7.73 ± 0.28 for a period of 6 h.
Subsequently, spermiation was induced by intraperitoneal
Abbreviations: ADAPTA, Adaptations of aquatic biota of the Amazon; ATP, injection of crude extract of carp pituitary hormone (EBHC).
Adenosine triphosphate; BHT, Butylate hydroxytoluene; BSA, Bovine serum albumin; The procedure was performed by injecting two boluses of
BTS, Beltsville thawing solution; CAPES, Coordination for the improvement of EBHC. The first bolus was 0.5 mg/kg EBHC, and after an
higher education personnel; CDNB, 1-Chloro-2,4-dinitrobenzene; CHP, Cumene
hydroperoxide; CNPq, Brazilian National Research Council; CTTPA, Center for
interval of 12 h, a second bolus of 1.0 mg/kg EBHC was
Technology Training and Production in Aquaculture; DDT, Dichloro-diphenyl- injected. Six hours after injection of second hormone bolus,
trichlorethane; DMSO, Dimethyl sulfoxide; EBHC, Crude extract of carp pituitary the semen was collected in graduated Falcon tubes, avoiding
hormone; EDTA, Ethylene diamine tetra-acetic acid; ROS, Reactive oxygen species; contact with water, blood, feces, or urine, thus preventing
FAPEAM, Amazonas State Research Foundation; FOX, Ferrous oxidation/xylenol
sperm activation. From each animal, 4 ml of semen was collected
orange; GST, Glutathione S transferase; HCL, Hydrochloric acid; INPA, National
Institute for Research of the Amazon; LEEM, Laboratory of Ecophysiology and and immediately diluted 1:10 (50 μl semen: extender 450 μl)
Molecular Evolution; LPO, Lipoperoxidation; NaOH, Sodium hydroxide; SEM, in Beltsville Thawing Solution (BTS-MINITUB®) and refrigerated
Standard error of mean; SOD, Superoxide Dismutase. at 4°C to be transported to the laboratory Ecophysiology and

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Castro et al. Spermic Performance of the Amazonian Fish

Molecular Evolution (LEEM) of the Brazilian National Institute (CDNB) as the substrate. GST activity was calculated as nmol
for Research of the Amazon (INPA). All procedures and of conjugate CDNB·min−1·mg−1 protein, using the molar extinction
experimental manipulations used in this study were performed coefficient 9.6 mM·cm−1. SOD activity was quantified from the
according to the Brazilian Guidelines for Animal Care and inhibition of the reduction rate of cytochrome c influenced by
were approved by the Ethics Committee for the use of Animals the xanthine/xanthine oxidase system at 550 nm (Flohé and
of INPA, protocol number 004/2018. Otting, 1984) and is expressed as U min−1·mg of protein−1. CAT
activity was determined following the method of Beutler (1975)
Activation and is expressed as μmol H2O2·min−1·mg−1 protein. The inhibition
For activation of sperm motility, the following treatments were rate of H2O2 decomposition was monitored by decreasing the
tested: temperature (29, 31, 33, and 35°C), pH (4 and 8), and absorbance at 240 nm. LPO levels were measured by the ferrous
interaction between pH and hypoxia (1 mgO2·L−1) in controlled oxidation/xylenol orange (FOX) method as described by Jiang
activator solution (distilled water-0 mOsm kg−1). The different et al. (1991) which consists of the oxidation of Fe+2 to Fe+3
temperatures were maintained using thermostats HOPAR model hydroperoxide in an acid medium and subsequent formation of
(Aquarium Heater H-606). The pHs 4 and 8 were achieved by Fe3+/xylenol orange in the presence of the stabilizer butylate
adding HCl 1% and NaOH 1%, respectively. The pH values were hydroxytoluene (BHT). The formation of this complex was
then monitored using a digital pH meter (Ohaus, Starter 3100). quantified by increased absorption at 560 nm and is expressed
Hypoxia was adjusted with the addition of gaseous nitrogen until in μM cumene hydroperoxide (CHP)·mg−1 of sperm protein. Total
the concentration of 1 mgO2 L−1. The sperm were activated in sperm protein was determined spectrophotometrically at 595 nm,
the proportion 2:20 (v:v), in a closed environment with controlled according to the colorimetric assay described by Bradford (1976)
temperature (23°C). Immediately after activation, sperm were using bovine serum albumin (BSA) as standard. All assays were
observed under an optical microscope (Leica DM500; 40x), by performed with a SpectraMax M2 spectrophotometer, Molecular
the same observer to avoid the subject bias of the analyzes. The Devices Inc., Sunnyvale, CA, USA.
motility time was monitored with the aid of a timer (s) being
evaluated from the start of movement until 100% sperm immobility. Genotoxic Damage
To identify the percent of sperm motility, a scale from 0 to To verify the DNA integrity of C. macropomum sperm cells,
100% was used, according to Cosson et al. (2008), where: 1 = 0–5%, unicellular gel electrophoresis (comet assay) was used,
2 = 5–25%, 3 = 25–50%, 4 = 50–75%, 5 = 75–100%. For each according to Cabrita et al. (2005). After experimental
treatment, nine replicates were performed in triplicate. exposures, 5 μl of semen was added to 300 μl of RPMI.
Previously, the experimental units were covered with 0.5%
agarose and all procedures were completed in triplicate.
Oxygen Consumption of Sperm After agarose drying, the slides were placed in the lysis
Oxygen consumption of sperm was monitored using a solution (2.5 M NaCl, 100 mM EDTA, 10 mM Tris, pH
high-resolution respirometer (Oxygraph-2 K, Oroboros Instruments) 10–10.5, 1% Triton X-100, and 10% DMSO) for 90 min at
equipped with a Peltier thermostat and an electromagnetic stirrer. 4°C. The slides were then submerged in alkaline sodium
The treatments (temperature, hypoxia, and pH) were controlled hydroxide and EDTA buffer (300 mM NaOH and 1 mM
within two 2 ml closed Oxygraph chambers. With the aid of EDTA, pH > 13) for 20 min. The slides were electrophoresed
injection cannulas, 100 μl of semen were added, immediately for 20 min at 25 V, 300 mA, and 4°C. Subsequently, the
activated inside the chambers. Oxygen consumption was recorded slides were neutralized (0.4 M Tris, pH 7.5 at 4°C) and
immediately after activation until cessation of sperm respiration. stained with silver nitrate solution (5% sodium carbonate,
Recordings of new samples were performed at intervals of 5 min 0.1% ammonium nitrate, 0.1% silver, 0.25% tungstosilicic
between exposures, to the complete stabilization of the equipment. acid, and 0.15% formaldehyde) for 15 min at 37°C. A total
Data are expressed as pmol·s−1·cm−3. of 100 spermatozoids were randomly quantified per slide,
using an optical microscope (Leica DM205) with a
Biochemical Analysis magnification of 100x. Damage was classified according the
For analysis of antioxidant enzymes, 200 μl of semen from each size of the tail that represents the degree of fragmentation
fish was activated, with five replicates in triplicate for each of the DNA. DNA fragments of damaged nucleus migrate
treatment and after motility cessation, it was immediately immersed when exposed to an electrical field (electrophoresis). Fragments
in liquid nitrogen. The semen samples were homogenized in a of different sizes migrate at different speeds, forming the
buffer solution (Tris base 200 mM, ethylene diamine tetraacetic typical figure of a comet. Thus, during the electrophoretic
acid (EDTA) 1 mM, dichloro-diphenyl-trichlorethane (DDT) run, the more intense the breaks in the DNA molecule, the
1 mM, sucrose 500 mM, KCl 150 mM, and pH 7.6) and centrifuged smaller the fragments generated and the greater the extent
at 9,000 g for 10 min at 4°C. The supernatant was used for of migration and so the comet tail (Singh et al., 1988; Collins
analysis of the activity of glutathione S-transferase enzyme (GST), et al. 2008). The cells were divided into five categories,
superoxide dismutase (SOD) and catalase (CAT), and for according to tail size, ranging from 0 to 4, i.e., class 0
determination of lipid peroxidation (LPO) levels. GST activity was corresponds to intact DNA, without tail; class 1, low damage
determined as described by Keen et al. (1976), considering index; class 2, intermediate damage; class 3, high damage;
absorbance changes at 340 nm using 1-chloro-2,4-dinitrobenzene and class 4, extreme damage.

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Castro et al. Spermic Performance of the Amazonian Fish

Statistical Analysis
Data are expressed as mean ± SEM. To compare the different
treatments, an ANOVA one way, followed by Tukey’s post-hoc
test when differences (p < 0.05) between means were detected.
Data that did not meet the premises of linear models were
log transformed. All analysis was performed using the R-3.5.1
software (R Core Team, 2018), with additional car package
(Fox and Weisberg, 2011).

RESULTS
Sperm Motility
The duration of C. macropomum sperm motility was 50.1 ± 2.7 s
at 29°C and significantly reduced (p < 0.05) at 33°C (33.66 ± 2.58 s)
FIGURE 1 | In vitro effect of temperature on time (s) and percentage mobile
and 35°C (31.22 ± 1.31 s; Figure 1). The percentage of mobile (%) of sperm of Colossoma macropomum. Data are presented as
cells showed no differences at 29 and 31°C, but decreased mean ± SEM. Lowercase letters indicate difference in motility time between
significantly at 33 and 35°C (Figure 1). Exposure to pH 4 and treatments. Uppercase letters indicate differences in motility rate between
pH 8 in hypoxia caused a significant reduction in motility time treatments (p < 0.05).
and percentage of mobile sperm (p < 0.05; Figure 2). Motility
time and percentage of mobile sperm showed no differences when
exposed to pH 4 and pH 8 in normoxia (p > 0.05; Figure 2).

Oxygen Consumption of Sperm

The oxygen consumption of sperm was not altered with increasing
temperatures (p > 0.05; Figure 3A) or under different pHs
within the same treatment (Figure 3B). However, exposure to
hypoxia resulted in an increase in oxygen consumption at
both pH 4 (61.22 pmol s−1) and pH 8 (54.74 pmol s−1) compared
to normoxia at pH 4 (14.20 pmol s−1) and pH 8 (10.59 pmol s−1;
p < 0.05; Figure 3B).

Biochemical Analysis
Activities of GST, SOD, and CAT and levels of LPO of C.
macropomum sperm exposed to different temperatures are shown FIGURE 2 | In vitro effect of pH and hypoxia (Hy) on time (s) and percentage
in Figure 4. GST activity was increased at 35°C, differing from mobile (%) of sperm of C. macropomum. Data are presented as
sperm exposed to 33°C (p < 0.05), and similar to other treatments mean ± SEM. Lowercase letters indicate differences in motility time between
Hy and normoxia (No). Uppercase letters indicate differences in motility rate
(p > 0.05; Figure 4A). SOD activity was significantly increased
between Hy and No (p < 0.05).
at 35°C compared to all other treatments (p < 0.05; Figure 4B).
CAT did not differ in any of the treatments (p > 0.05; Figure 4C).
Exposure to 35°C caused an increase in LPO levels compared at 29 and 31°C (Figure 6). At 29 and 31°C, there was a
to cells exposed to 29 and 33°C, while for sperm exposed to prevalence of intact DNA with about 71 and 66%, respectively.
33°C LPO levels were reduced compared to other groups (p < 0.05; At the highest temperature, 35°C, approximately 60% of the
Figure 4D). Low oxygen concentration exposure during sperm sperm cells had extreme damage, class 4 (Figure 6). Activated
activation caused an increase of GST and SOD activities spermatozoa under normoxia showed the highest rate of intact
(Figures 5A,B). CAT activity at pH 8 in normoxia was equal cells (pH 4 = 43%, pH 8 = 57%), but low oxygen activation
to pH 4 normoxia (p > 0.05) and showed a similar not significant medium on both pHs caused an increased classes 2, 3, and
increase of sperm exposed to hypoxia at both pHs (p < 0.05; 4 damages, considered intermediate, high, and extreme,
Figure 5C). Increased enzymatic activities in hypoxic environments respectively (Figure 6).
contributed to increased LPO levels (Figure 5D). Normoxic
environments at pHs 4 and 8 did not affect the activities of
SOD and GST, as well as the LPO levels (p > 0.05; Figure 5). DISCUSSION
Genotoxic Damages Temperature Effects
Genetic damage to sperm of C. macropomum increased at Sperm motility, a critical semen characteristic, depends on activation
higher temperatures (33 and 35°C) compared to sperm activated media (Williot et al., 2000) that strongly influence the fertilization

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Castro et al. Spermic Performance of the Amazonian Fish

A B

FIGURE 3 | Oxygen consumption (pmol⋅s−1) of C. macropomum sperm activated at different in vitro temperatures (A) and at pH 4 and pH 8 in No (5.5 mgO2/L)
and Hy (1 mgO2/L). (B) Lowercase letters indicate differences between treatments.

A B

C
D

FIGURE 4 | In vitro effect of temperature on Glutathione-S-transferase (GST), (A) superoxide dismutase (SOD), (B) catalase (CAT), (C) and lipoperoxidation (LPO)
(D) levels in sperm of C. macropomum. Lowercase letters indicate differences between treatments.

in teleosts (Bera et al., 2016). In the present study, sperm exposure and Dadras et al. (2016) for Cyprinus carpio, which showed that
to increased temperature (33 and 35°C) caused a reduction on higher temperatures caused lower percentage of mobile sperm.
time and percentage of mobile sperm (Figure 1) without altering According to Purchase et al. (2010), fish sperm has limited
the oxygen consumption (Figure 3A). These findings corroborate energetic resources; thus, temperature increase causes a motility
the report of Lahnsteiner and Caberlotto (2012) for S. aurata decrease due to the acceleration of the metabolic processes.

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Castro et al. Spermic Performance of the Amazonian Fish

A B

C
D

FIGURE 5 | In vitro effect of pH in No (5.5 mgO2/L) and Hy (1 mgO2/L) on GST, (A) SOD, (B) CAT, and (C) LPO, (D) in sperm of C. macropomum. Lowercase
letters indicate differences between treatments.

A B

FIGURE 6 | Frequency of DNA damage of sperm of C. macropomum exposed to different temperatures (A) and exposed to pH 4 and pH 8 in No and Hy
(B) in vitro.

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Castro et al. Spermic Performance of the Amazonian Fish

Low temperatures reduce the intensity of flagellar movement identifying that the lipid peroxidation in these cells is
significantly, prolonging motile duration of spermatozoids (Alavi temperature sensitive. Lipids are probably used as a source
and Cosson, 2006). In the present study, the high temperature of energy by sperm to increase motility duration (Baeza
accelerated the swimming speed of C. macropomum sperm cells, et al., 2015) that plays an important role in fertilization
increasing the metabolic consumption of the cells and decreasing capacity (Gholami et al., 2011). The lipid peroxidation of
the motility time and the percentage of mobile cells. Activated these cells is associated with deterioration of membrane
sperm at low temperatures consume metabolic energies more function, decreased fluidity, and increased non-specific
slowly, increasing the duration of these cells, corroborating the permeability to ions and other transmembrane transport
report of Alavi and Cosson (2006), who found that low temperatures processes (Wong-Ekkabut et al., 2007).
reduce the intensity of flagellar movement, significantly prolonging Although C. macropomum sperm cells were activated at
the mobile duration of sperm. the high temperature evaluated in this experiment, they
As a result, at lower temperatures, the consumption of showed a significant increase in tail damage (class 4). The
ATP is reduced, which is directly related to the longer evaluation of fish sperm DNA damage is important for fish
motility time (Lahnsteiner and Caberlotto, 2012). Dzyuba farming, since the loss of genetic information or the appearance
et al. (2019), studying the tropical species Oreochromis of larval deformities is detrimental to future generations
niloticus, showed that higher temperatures induce an increase (Cabrita et al., 2005; Lewis and Galloway, 2009). Due to
in sperm motility velocity, which is accompanied by a the reduced motility time of freshwater fish spermatozoid,
reduction in motility time. The reduction in time and the activation environment is critical for the successful
percentage of mobile sperm at the higher temperatures delivery of paternal DNA to the egg (Gazo et al., 2015).
observed in the present study may be related to increased However, oxidative stress is highly detrimental to DNA
energy demands, which was not compensated for, since integrity, since this damage is not limited to the direct effect
oxygen consumption remained constant at all temperatures. on the fragmentation of chromatin; it can also alter gene
Therefore, our results suggest that semen from tambaqui expression or induce epigenetic deregulation (Chervona and
has limited energy supply to deal with increased temperature, Costa, 2012). The extreme DNA damage of C. macropomum
suggesting high vulnerability of this species under sperm caused by activation at high temperatures is similar
warming scenarios. to the results found by Lacaze et al. (2011) in assessing
Another factor that may explain the lower percentage of DNA damage of the sperm of Gammarus fossarum. The
mobile sperm observed at the higher temperature is related authors explain that the increase in damage is associated
to the oxidative damages caused by ROS production, as with an overall stress caused by increased temperature and
observed in the present study. The exposure of sperm cells decreased oxygen concentration. Our results corroborate the
at 35°C resulted in an increase in the activity of antioxidant findings of De Andrade et al. (2004), who found an increase
enzymes (SOD and GST; Figure 4); however, such increase in DNA damage of Dreissena polymorpha and Netuma sp.
was not enough to avoid damage to macromolecules, since exposed to 37°C. Despite increased DNA damage in various
there was an increase in lipoperoxidation and DNA damage species of fish exposed to various stressors, Pérez-Cerezales
(Figures 4, 6A). Oxidative stress can induce metabolic et al. (2010) reported that DNA damaged spermatozoids
disorders that reduce sperm motility. As suggested by Aramli are capable of performing fertilization and that the oocyte
and Kalbassi (2016), oxidative stress causes disturbances in can repair this damage to some extent in Oncorhynchus
the sperm metabolism, which results in decreased mykiss. These authors stated that when the DNA fragmentation
sperm motility. rate is high, the oocyte repair capacity is insufficient, and
The oxidative damage observed in the present study infertility rate increases considerably.
suggests a inefficiency in the neutralization of ROS by the
antioxidant defenses, since even with the increase of Effects of pH and Hypoxia
antioxidant enzyme activity, such as SOD, sperm presented In the present study, activation media pHs (pH 4 and pH 8)
high levels of lipoperoxidation and DNA damage. Aramli did not influence the sperm quality of C. macropomum. This
and Kalbassi (2016) also showed that the increase of the may be related to the adaptation of this species to large pH
antioxidant defenses in the species Acipenser persicus was range that drives resilience of sperm cell to pH changes and
accompanied by the increase of oxidative damages. Fish so, possibly, increasing reproductive success. Aride et al.
sperm have characteristics that make them susceptible to (2004) reported that in confined environments C. macropomum
damage related to oxidative stress, considering the high is able to tolerate large variations in pH, which was confirmed
amount of polyunsaturated fatty acids of sperm membrane by Wood et al. (2018). Cosson (2004) showed that the pH
(Shaliutina et al., 2013). of activating medium usually affects motility, but in small
Studies have shown that temperature increases potentially proportions. In addition, the external pH can reduce or
induce oxidative stress in different fish tissues (Wiens et al., prolong motility, but it is not the main environmental factor
2017), including sperm cells, as presented in this paper affecting initial motility (Cosson et al., 2008). The results
with the increase in antioxidant enzymes, contributing to found in this study are similar to the findings by
the increase in lipid peroxidation levels, as shown by PerchecPoupard et al. (1997), who showed reduced pH impact
Dadras et al. (2016) when evaluating the sperm of C. carpio, on carp sperm motility, indicating that fish that inhabit

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Castro et al. Spermic Performance of the Amazonian Fish

tropical/subtropical environments are possibly more resistant shown that hypoxia induces oxidative stress in different fish
to pH variation. However, for species that inhabit temperate tissues (Mustafa et al., 2011). The results of our study are
environments such as Samotrutta, variations in pH affect similar to those of Bera et al. (2016), evaluating the activity
sperm motility (Dziewulska and Domagała, 2013). of antioxidant enzymes in the testes and ovary of C. carpio
Hypoxia exposure affected sperm quality of C. macropomum, in hypoxic environments. The authors reported an increase
suggesting that hypoxia, a common environmental condition in CAT and SOD activities compared to tissues under
in tropical waters and aquaculture, is highly detrimental to normoxia. Other authors report the relationship between
the reproduction of this tropical species. The results observed loss of sperm quality and oxidative damage in fish sperm
in the present study are in agreement with Wu et al. (2003), under different stress conditions (Dadras et al., 2016; Wiens
which suggested that hypoxia caused reduction of sperm et al., 2017). Shaliutina et al. (2013) also showed a relationship
motility in carp and other reproductive dysfunctions, such between loss of sperm quality and oxidative stress in Acipenser
as the retardation of gametogenesis. Thomas et al. (2007) gueldenstaedtii and Acipenser bae. Taken together, these results
evaluated the formation of sperm cells of Micropogonias suggest that tambaqui reproduction is especially sensitive
undulates and found that sperm production, gametogenesis to disturbances caused by exposure to hypoxia.
and all stages of spermatogenesis were impaired in
hypoxic environments.
The decrease in the time and percentage of mobile sperm CONCLUSIONS
of C. macropomum under hypoxic conditions was accompanied
by an increase in oxygen consumption (Figure 3B). At low The elevation of temperature and the decrease in DO, factors
oxygen, sperm cells present an increase in the rate of flagellar associated among others to climate change, will negatively
motility, not measured in the present study, which resulted affect sperm quality of C. macropomum. No pH effects
in decreased motility time due to the rapid consumption of on spermatozoids of C. macropomum were observed. This
ATP. As a result, oxygen consumption increased. Fitzpatrick study provides basic information for future research on
et al. (2009) showed that sperm velocity of P. notatus exposed sperm quality of tropical fish in confined environments.
to hypoxia was positively related to oxygen consumption. However, further investigations are needed to describe the
Both species breed in hypoxic environments. During the mechanisms involved in metabolism and sperm motility in
breeding period, males of P. notatus are trapped in small C. macropomum under warm hypoxic environments. In
pools facing drastic hypoxia, similar to C. macropomum in general, the present results suggest that C. macropomum
their natural environments. It is known that the mitochondria sperm are vulnerable to warm hypoxic waters, conditions
of fish sperm supply ATP demand aerobically (Dzyuba et al., associated with global warming, and may impair the
2017). According to Cosson (2010), as the ATP reserve is reproduction of the species.
depleted along the spermatozoid activity, there is a gradual
decrease of the flagellum movement, settling off the movement
of the cell. In aquaculture, fish are commonly kept under
artificial conditions, exposed to various adverse conditions, DATA AVAILABILITY STATEMENT
which may contribute to the establishment of hypoxic
The datasets generated for this study are available on request
environments (Rurangwa et al., 2004). Therefore, sperm
to the corresponding author.
exposed in these environments consume more quickly ATP
reserves, directly affecting the duration of motility and the
efficiency of fertilization. In addition, recent studies suggest
that changes in sperm quality may be related to changes at ETHICS STATEMENT
the molecular level, such as changes in gene expression or
epigenetic factors. Wang et al. (2016) verified the occurrence The animal study was reviewed and approved by Brazilian
of epigenetic changes in the sperm methyloma of Oryzias Guidelines for Animal Care and were approved by the Ethics
melastigma exposed to hypoxia, as well as differential expression Committee for the use of Animals of INPA, protocol number
of genes and proteins involved in spermatogenesis and gene 004/2018.
silencing, which was associated with a reduction in sperm
motility, even in generations that never had been exposed
to hypoxic conditions. AUTHOR CONTRIBUTIONS
We also observed that exposure to hypoxia caused an
increase in antioxidant activity (SOD, CAT, and GST; AV and JC designed the work. JC performed the experiment,
Figure 5), which suggests an increased capacity to prevent collected data, analyzed and drafted the original version of
ROS effects. However, despite the higher activity of antioxidant the manuscript. SS, SB-M, and DC helped with laboratory
enzymes under hypoxia condition, there was an increase in analyzes, discussed and reviewed the manuscript and AV
lipoperoxidation levels and DNA damage. Thus, the oxidative supervised this work, obtained financial support, discussed and
damages observed in the present study may be related to revised the manuscript. All authors contributed to the article
loss of sperm quality under hypoxic conditions. It has been and approved the submitted version.

Frontiers in Physiology | www.frontiersin.org 8 July 2020 | Volume 11 | Article 772

Castro et al. Spermic Performance of the Amazonian Fish

FUNDING ACKNOWLEDGMENTS
The Brazilian National Research Council (CNPq, 465540/2014-7), JC, SB-M, and DC were recipients of post-grad fellowships
Amazonas State Research Foundation (FAPEAM, 0621187/2017) from CAPES (finance code 001); SS was recipients of
and Brazilian Coordination for the Improvement of Higher post-grad fellowships from FAPEAM; AV is the recipient
Education Personnel (CAPES) (88887.136340/2017-00) support of a research fellowship from CNPq (303930/2014-4).
the INCT ADAPTA. The CAPES – Finance Code 001 granted We would also like to thank the collaborators at the Balbina
the fellowships. fish farm.

De Andrade, V. M., De Freitas, T. R. O., and Da Silva, J. (2004). Comet assay

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Richards, J. G., Farrell, A. P., and Brauner, C. J. (2009). Fish physiology: Hypoxia.
1st Edn. Vol. 27. London, UK: Academic Press. Copyright © 2020 Castro, Braz-Mota, Campos, Souza and Val. This is an open-access
Rurangwa, E., Kime, D. E., Ollevier, F., and Nash, J. P. (2004). The measurement article distributed under the terms of the Creative Commons Attribution License
of sperm motility and factors affecting sperm quality in cultured fish. (CC BY). The use, distribution or reproduction in other forums is permitted,
Aquaculture 234, 1–28. doi: 10.1016/j.aquaculture.2003.12.006 provided the original author(s) and the copyright owner(s) are credited and that
Shaliutina, A., Hulak, M., Gazo, I., Linhartova, P., and Linhart, O. (2013). the original publication in this journal is cited, in accordance with accepted academic
Effect of short-term storage on quality parameters, DNA integrity, and practice. No use, distribution or reproduction is permitted which does not comply
oxidative stress in Russian (Acipenser gueldenstaedtii) and Siberian (Acipenser with these terms.

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