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Aquaculture Reports 29 (2023) 101477
Available online 20 January 2023
2352-5134/© 2023 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
Cryostorage of white cachama (Piaractus orinoquensis) sperm: Effects on
cellular, biochemical and ultrastructural parameters
Víctor Mauricio Medina-Robles
a
,
*
, Leydy Yasmin Sandoval-Vargas
a
,
b
,
Roger Oswaldo Su´
arez-Martínez
c
, Edwin G´
omez-Ramírez
d
, Diana Nataly Guaje-Ramírez
a
,
Pablo Emilio Cruz-Casallas
a
a
Research Group on Aquatic Organism Reproduction and Toxicology. Institute of Aquaculture, Faculty of Agricultural Sciences and Natural Resources, Universidad de los
Llanos, Villavicencio, Colombia
b
Nucleus of Research in Food Production, Faculty of Natural Resources, Catholic University of Temuco, Temuco, Chile
c
Animal Science Research Group - Faculty of Agricultural Sciences, University of Applied and Environmental Sciences (UDCA), Bogot´
a D.C., Colombia
d
Grupo de Ecotoxicología, Evoluci´
on, Medio Ambiente y Conservaci´
on, Facultad de Ciencias B´
asicas y Aplicadas, Universidad Militar Nueva Granada, Cajic´
a, Colombia
ARTICLE INFO
Keywords:
Cryostorage time
Cryostorage damage
Sperm cryopreservation
Sperm ultrastructure
Post-thaw sperm quality
ABSTRACT
To date, little attention has been paid to identifying the effects of long-term cryopreservation on sperm quality
for Piaractus orinoquensis. The object of this study was therefore to evaluate the effect of long-term cryopreser-
vation (24 h, 1, 6 and 12 months) on sperm motility, viability, DNA integrity, ATP content, total antioxidant
capacity (TAC), morphology and sperm ultrastructure in this species. Milt samples from six males were cry-
opreserved in a medium containing nal concentrations of 7.5% Me
2
SO, 4.1% glucose and 9.0% egg yolk. The
samples were frozen in liquid nitrogen (LN) vapor and stored in LN for periods of 24 h and 1, 6 and 12 months.
After thawing, both the motility rate and the viability decreased signicantly compared with fresh sperm;
however, these parameters did not differ among the four cryopreservation times. The DNA integrity and ATP
content decreased signicantly after 6 months of cryopreservation. There were no signicant differences in TAC
values between fresh and cryopreserved sperm. The total sperm abnormalities in cryopreserved samples were
about 5-fold higher than in fresh sperm; short tail was the most common defect occurring after cryostorage. The
ultrastructural analysis reveals that P. orinoquensis spermatozoa consist of an ovoid head without acrosome, a
cylindrical mid-piece, and a single agellum. The nuclear fossa is located at the base of the nucleus and contains
the centriolar complex. There are 1–2 ring-shaped mitochondria located in the mid-piece. The agellum shows a
9 +2 organization of microtubules in the axoneme. Post-thaw spermatozoa presented damage such as swelling
and rupture of the plasma membrane, mitochondrial damage, loss of the electron-dense chromatin of the nu-
cleus, and degeneration in the middle region.
1. Introduction
White cachama (Piaractus orinoquensis) is a neotropical sh,
belonging to the order of Characiformes, that has recently been studied
by molecular analysis and classied as a new species of the Serra-
salmidae shes restricted to the Orinoco basin (Escobar et al., 2019).
According to these authors, P. orinoquensis differs from congeners such
as Piaractus brachypomus distributed in the Amazon basin. Therefore, it
is reasonable to assume that previous studies performed in the Orinoco
region were carried out in P. orinoquensis and not in P. brachypomus
(Navarro et al., 2004; Ramírez-Merlano et al., 2011; Su´
arez et al., 2019).
This is the rst study on sperm quality and cryopreservation of Piaractus
orinoquensis using the new classication.
Piaractus orinoquensis is the main native species of economic
importance in Colombian aquaculture production (Valencia, 2019).
Despite the increasing interest in expanding production, bottlenecks
exist such as water quality and availability. In addition, wild populations
are decreasing as a result of overshing for the commercial market
(Gonz´
alez et al., 2019). In this context, special attention should be paid
to avoiding decline, or some degree of risk of extinction, in this species,
as has been reported for at least 81 of the 1500 sh species distributed in
Colombia (Mojica et al., 2012; Reis et al., 2016). Sperm
* Correspondence to: Institute of Aquaculture, Faculty of Agricultural Sciences and Natural Resources, Universidad de los Llanos, Villavicencio, Colombia.
E-mail address: vmmedinarobles@unillanos.edu.co (V.M. Medina-Robles).
Contents lists available at ScienceDirect
Aquaculture Reports
journal homepage: www.elsevier.com/locate/aqrep
https://doi.org/10.1016/j.aqrep.2023.101477
Received 24 June 2022; Received in revised form 5 January 2023; Accepted 18 January 2023
Aquaculture Reports 29 (2023) 101477
2
cryopreservation may enable the establishment of a genetic repository
for the conservation of this species; moreover, the development of a
cryopreservation technique would also be useful to support aquaculture
production. In this case, the use of cryopreserved sperm could facilitate
broodstock management and improve genetic heterogeneity. The con-
servation of biological material (cells, tissues, organs) is possible by
storage of the samples in cryoprotective agents and liquid nitrogen
(–196 ◦C). This temperature should arrest all metabolic processes and
therefore keep the biological material theoretically viable for long pe-
riods of time (Mazur, 1984; ¨
Ozkavukcu and Erdeml, 2002). Nonetheless,
some studies have shown that factors such as oxidative stress, osmotic
stress or mechanical stress related to the cryopreservation process affect
sperm motility (Medina-Robles, 2021; Park et al., 2022), reduce
viability, and alter mitochondrial integrity and normal function (Fig-
ueroa et al., 2016; Sandoval-Vargas et al., 2021), as well as affecting the
normal structure of spermatozoa (Lahnsteiner et al., 1996; Borges et al.,
2020).
Furthermore, the exact duration of cryostorage of the biological
material could be controversial, since sh spermatozoa have been
routinely stored in liquid nitrogen for a few hours (Babiak et al., 2001;
Labbe et al., 2001; Christensen and Tiersch, 2005; Huang et al., 2009;
Nomura et al., 2018), days (Miliorini et al., 2011; Felizardo et al., 2010;
Liu et al., 2018) or months (Tanaka et al., 2002; Ding et al., 2011; Lim
and Le, 2013; Figueroa et al., 2016). To our knowledge, only two studies
have reported a cryostorage time of years, for periods not exceeding 7
years (Chen et al., 2010; Fabbrocini et al., 2015; Park et al., 2022). In
addition, it should be noted that most cryopreservation methodologies
have been developed according to the results evaluated within days of
cryostorage only. So far, the effects of cryopreservation on sperm quality
over time have been scarcely studied (Kurokura et al., 1984; Steyn and
Van Vuren, 1987; Chen et al., 2010). Nevertheless, it seems that cryo-
preservation time plays a crucial role in cryodamage, with motility,
fertility and hatching rate the parameters most affected (Kurokura et al.,
1984; Chen et al., 2010).
Besides cryopreservation time, other factors such as cryoprotectants,
freezing/thawing rates, packaging systems, membrane stabilizers, etc.
are also responsible for the impairment of sperm quality; the assessment
of post-thaw sperm quality therefore plays an important role in the
validation of a cryopreservation protocol. Sperm motility has been his-
torically recognized as the best indicator of sperm quality, since it de-
pends on several aspects of the cell such as the physiological state of the
mitochondria (Chauvign´
e et al., 2015; Figueroa et al., 2016, 2019), ATP
production (Perchec et al., 1995) and plasma membrane integrity (Alavi
et al., 2019) which ultimately determine the fertilizing capacity (Rur-
angwa et al., 2004). Nevertheless, for a better understanding of damage
mechanisms, it is important to carry out specic analyses, such as
viability, mitochondrial membrane potential, DNA integrity (Figueroa
et al., 2015; Merino et al., 2017) and ATP content (Burness et al., 2005;
Cabrita et al., 2005a; Kommisrud et al., 2020).
ROS scavengers, including enzymatic and non-enzymatic ROS-
neutralizing components, play an important role in protecting sperma-
tozoa against damage by free radicals (Lahnsteiner and Mansour, 2010;
Dzyuba et al., 2016). Therefore, assessment of antioxidant systems,
including total antioxidant capacity (TAC) can be also useful as addi-
tional quality markers of sh spermatozoa (Kusano and Ferrari, 2008).
Several studies have described damage to sperm structure due to
cryopreservation (Lahnsteiner et al., 1996; Figueroa et al., 2019; Borges
et al., 2020). Depending on its severity, ultrastructural damage can lead
to signicant decreases in post-thaw motility (Lahnsteiner et al., 1996).
Neither TAC nor sperm ultrastructure has been evaluated in fresh or
cryopreserved sperm of P. orinoquensis. In view of the above, the object
of this study was to evaluate the effect of long-term cryopreservation
(24 h, 1, 6 and 12 months) on sperm motility, viability, DNA integrity,
ATP content, total antioxidant capacity, morphology and sperm ultra-
structure in P. orinoquensis.
2. Materials and methods
2.1. Ethics statement
All procedures were performed in compliance with the recommen-
dations given in the Guide for the Care and Use of Laboratory Animals
(National Research Council, 2010) and were previously approved by the
Bioethics Committee of Universidad de los Llanos.
2.2. Fish handling and milt collection
Piaractus orinoquensis were obtained from broodstock born and
raised at Llanos Aquaculture Institute facilities (4◦04′30′′ N 73◦35′07),
Meta, Colombia. The facilities are located at an average altitude above
sea level of 420 m, with an average ambient temperature of 26 ◦C,
relative humidity of 75% and annual precipitation of 4050 mm (http://
www.dhime.ideam.gov.co/atencionciudadano/). Fish were raised in
247–497 m
2
earth ponds in polyculture with coporo (Prochilodus mariae)
and yamú (Brycon amazonicus). The water exchange was 1% per day, the
temperature 25.80 ±2.12 ◦C, dissolved oxygen 5.53 ±1.60 mg/L and
pH 6.35 ±0.81.
Sexually mature males (3 years old, 4.2 ±0.2 kg of body weight)
were selected in the ponds (according to the release of milt by stripping)
during the spawning season (April to June). Subsequently, the males
were transferred to circular concrete ponds of 7 m
3
equipped with
aeration, daily water exchange of 5% and with a natural photoperiod
(12:12 h light: dark) and temperature. Spermation was stimulated by the
injection of a single dose of carp pituitary extract (CPE: 4 mg/kg body
weight) administered intramuscularly behind the dorsal n, approxi-
mately 18 h before milt collection (Ramírez-Merlano et al., 2011). Prior
to milt collection, the sh were anesthetized by immersion for 3–5 min
in a 300 ppm 2-phenoxyethanol solution (Sigma Co, St Louis, Missouri).
Then, the abdomen and urogenital papilla were dried with a clean paper
towel to avoid contamination with blood, water, urine or feces. Finally,
milt was collected in sterile volumetric glass tubes by applying gentle
abdominal pressure. Immediately after collection, the seminal volume
was recorded and each sample was evaluated under a light microscope
(Nikon-Eclipse E-400, Japan) at 400 ×magnication to verify sperm
motility. Only samples without contaminants and with motility above
90% were used for cryopreservation.
2.3. Fresh sperm quality analyses
The percentage and duration of sperm motility were determined
using an optical microscope (Eclipse E400) at 400 ×magnication.
Briey, 20 µL of milt were placed on a concave glass slide (1.0–1.2 mm
deep, Micro Slides Premiere, China) and activated with 180 µL of 1%
sodium bicarbonate (NaHCO
3
, 238 mOsm/kg) (Medina-Robles et al.,
2021). The motility was estimated in all cases by the same observer and
was expressed in percentage of cells actively moving in a forward di-
rection. The duration of motility was established by means of a stop-
watch immediately after the addition of NaHCO
3
until the total
cessation of sperm movements, and was expressed in seconds (s). pH was
determined in raw milt by means of universal pH indicator strips
(MColorpHastTM /Merck/Germany) with a measurement range be-
tween 0 and 14 units. Briey, the indicator was immersed in the sample
for 5 s and immediately read. Sperm concentration was calculated by
counting spermatozoa in a Neubauer hemocytometer (Bright Line, Optik
Labor, Friedrichshofen, Germany) after dilution of 1 µL of fresh milt in
1200 µL of 0.9% NaCl. The hemocytometer was kept in a humid atmo-
sphere for 10 min and then observed under a light microscope at 400 ×
magnication (Medina-Robles et al., 2021).
2.4. Cryopreservation procedure and experimental design
Milt samples from six males were individually diluted at a ratio of 1:4
V.M. Medina-Robles et al.
Aquaculture Reports 29 (2023) 101477
3
(v/v) in a medium containing 10% dimethyl sulfoxide (Me
2
SO) (Sigma
Chemical Co., St. Louis, MO, USA), 5.5% (w/v) glucose and 12% (v/v)
fresh hen egg yolk resulting in nal concentrations of 7.5% Me
2
SO, 4.1%
glucose and 9% egg yolk. (Navarro et al., 2004; Ramírez-Merlano et al.,
2011). Subsequently, the diluted milt was loaded in 5 mL macrotubes
(280 ×5 mm, Minitüb, Abfüll-und Labortechnik GmbH, Tiefenbach,
Germany) previously labeled. The macrotubes were sealed at both ends
using small glass beads (Minitüb GmbH, Tiefenbach, Germany) and
placed for approximately 10 min on a cylindrical PVC and aluminum
support (Ramírez-Merlano et al., 2011). Immediately thereafter, the
support was placed in a dry shipper (Taylor Wharton CX100, USA) for
freezing, at a rate of 28.6 ◦C/min from 28 to −100 ◦C on average
(adapted from Medina-Robles et al., 2007). After 30 min, the macro-
tubes were transferred to a cryogenic tank (Taylor-Wharton HC 35,
Theodore, AL, USA) at −196 ◦C for storage for different periods of time
(24 h, 1, 6 and 12 months).
On conclusion of the experimental period, the macrotubes were
thawed in a water bath at 35 ◦C for 90 s (Ramírez-Merlano et al., 2011)
and the quality of sperm samples was analyzed as described in the
following section.
2.5. Effect of cryostorage on sperm quality
2.5.1. Viability
Viability was assessed in triplicate for each male using Carboxy-
uorescein Diacetate – CFDA and propidium iodide-(PI) uorescent
staining as per Lahnsteiner et al. (2011), with some modications.
Briey, 2 µL of fresh or cryopreserved milt were suspended in 198 µL of
seminal plasma of the same species (obtained immediately after semen
collection by centrifugation at 14,000 x g for 5 min). To this suspension,
5 µL of Carboxyuorescein Diacetate - CFDA (20 µM) were added and
the mixture was incubated at room temperature for 5 min. Subsequently,
5 µL of propidium iodide-PI (7.3 µM) were added and the sample was
incubated again at room temperature for 5 min in dark conditions
(modied from Harrison and Vickers, 1990). Immediately thereafter, 5
µL of the dilution were placed on a glass slide, covered with a coverslip
and assessed under epiuorescence microscope (Leica DM 2000, Ger-
many) equipped with an excitation lter at 450–490 nm and an emission
lter at 516–610 nm. One hundred spermatozoa from each slide were
examined at 400 ×magnication. Cells exhibiting green uorescence
were considered intact, indicating that CFDA remained within the cell
cytoplasm. In contrast, cells with red or greenish-red uorescence were
considered damaged, as the intact membrane is not permeable to PI
(Gheller et al., 2019). The results are expressed as the percentage of
viable spermatozoa.
2.5.2. DNA integrity
DNA integrity was assessed by using the uorescent probe acridine
orange according to Perry et al. (2019), after slight modications.
Briey, 2 µL of fresh or cryopreserved milt were suspended in 198 µL of
seminal plasma as described in the previous Section (2.5.1). Then, 45 µL
of the diluted milt were mixed with 50 µL of TNE solution (0.01 M
Tris-HCl; 0.15 M NaCl; 0.001 M EDTA; pH 7.2) in a reaction tube. After
30 s, the suspension was mixed with 200 µL of Triton X-100, and after a
further 30 s, 5 µL of 2% acridine orange solution (Sigma, USA) in
deionized water was added. Cells were observed after 5 min under an
epiuorescence microscope, as was done for viability. Special care was
taken to perform the assessment in no more than one minute and in dark
conditions. Spermatozoa with green uorescence were considered to
have intact DNA, while those with red or orange uorescence were
considered to have DNA damage. For each slide, one hundred sperma-
tozoa were observed. The results are expressed as a percentage of
spermatozoa with intact DNA. The procedure was carried out in tripli-
cate for each male.
2.5.3. ATP content
Sperm ATP content was determined using the ATP Bioluminescence
Assay Kit (HS II, Roche Diagnostics GmbH, Germany) as previously
described by Boryshpolets et al. (2009), with some modications. For
fresh milt, 20 µL of the sample were mixed with 180 µL of seminal
plasma of the same species, plus 200 µL of cell lysis reagent and then
mixed on a vortex for 30 s. After incubation for 3 min at 100 ◦C the
samples were centrifuged at 10,000g for 60 s at 4 ◦C. The supernatant
was recovered and frozen at −20 ◦C until evaluation. For cryopreserved
milt, 40 µL of immediately thawed milt was mixed with160 µL of 10%
Me
2
SO and 200 µL of cell lysis reagent. The procedures of incubation,
supernatant recovery and storage were the same as those used for fresh
milt. The ATP content was veried in both seminal plasma and in Me
2
SO
to avoid false positives. Luminescence was read using a FLx 800 multi-
functional microplate (BioTek Instruments, Inc., Winooski, VT, USA).
ATP content was expressed as nM ATP/10
8
spermatozoa. Sperm samples
of each male were assessed in triplicate.
2.5.4. Total Antioxidant Capacity (TAC)
The total antioxidant capacity was determined using the Total
Antioxidant Capacity Assay Commercial Kit (Sigma-Aldrich Catalog
Number: MAK187, USA) following the manufacturer’s protocol. Ali-
quots of 250 µL of fresh or post-thawed milt were centrifuged (Hermle
Z326K, Germany) at 4 ◦C at 14,000 or 18,000g for 5 min, respectively.
The supernatant was recovered and frozen at −20 ◦C until evaluation.
The ability of the supernatant to reduce the ABTS or 2,2′- azino-bis[3-
ethylbenzthiazoline]−6-sulfonic acid was measured at 405 nm in a
microplate reader (BioRad Model 680 California, USA). The TAC values
of the samples were expressed as an equivalence of the mmol concen-
tration of a 6-Hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid
(Trolox) solution. The Trolox standard curve for the assay was prepared
in a range of 0.00–0.42 mM. Each male was assessed in triplicate.
2.5.5. Sperm morphology
The sperm morphology analysis was performed using the method
previously described by Maria et al. (2010). Briey, 1 µL of fresh or
cryopreserved milt was xed in 999 µL of formol-citrate solution [35%
formaldehyde (4%) and sodium citrate (2.9%)]. Then, 15 µL of the xed
sample were placed on a glass slide to prepare a smear with 5 µL of 3%
rose Bengal. Subsequently, the smear was examined under an optical
microscope (Nikon-Eclipse E-400, Japan) at 1000 ×magnication. One
hundred spermatozoa from each slide were counted and classied as
normal or abnormal according to methodology adapted from Maria et al.
(2010) and Miliorini et al. (2011). Briey, the damage was classied as
macrocephaly, degenerated head, separated head, degenerated
mid-piece, loose tail, bent tail, broken tail and short tail. The results
were expressed in percentages.
2.5.6. Scanning and transmission electron microscopy
First, 25 µL of fresh or cryopreserved milt (at each storage time) were
xed with 475 µL 2% glutaraldehyde in 0.2 M phosphate buffer. Next,
the samples were post-xed in 4% osmium tetroxide for 2 h at room
temperature (~ 24 ◦C). Then the samples were dehydrated in an
increasing ethanol series (50%, 70%, 90% and 100%), 10 min for each
concentration, followed by 100% ethanol for 15 min, and nally
immersed in 100% acetone for 15 min
For TEM, the samples were pre-inltrated in different solutions of
100% acetone and epoxy resin (2:1; 1:1, respectively), for 1 h each,
followed by immersion in pure epoxy resin for 2 h and then polymerized
in pure epoxy resin for 12 h at 70 ◦C. Semi-thin Section (1 µm) were cut
with a rotary microtome (Slee Cut 4060, Germany) and stained with
toluidine blue. Subsequently, ultra-thin sections of 0.13 µm were cut
with an ultramicrotome Leica UC7 (Leica Microsystems, Vienna) and
mounted on copper grids. The sections were stained with 2% methanol
and uranyl acetate solution for contrast. Finally, a second contrast was
performed with 0.4% lead citrate. The contrasted sections were
V.M. Medina-Robles et al.
Aquaculture Reports 29 (2023) 101477
4
analyzed in a transmission electron microscope (JEOL JEM-1400 Plus,
USA) equipped with a GATAN model camera, ORIUS brand. Micro-
graphs were taken and analyzed with GATAN DigitalMicrograph
1.80.70 software at 20,000 ×magnication.
For SEM, the samples were dried with liquid carbon dioxide using a
K850 Critical Point Dryer (Quorum, UK) and coated with gold between 9
and 12 nm at 10 KV for 120 s in a metallizer (SPT- 20, Coxem, South
Korea). The samples were examined using scanning electron microscopy
(Coxem, EM 30AX plus, South Korea), taken at 5000 magnications (x
5.0 K) and analyzed with the ImageJ software (https://imagej.net/Fiji).
2.6. Statistical analysis
The normal distribution of the data and the homogeneity of variance
were tested by the Kolmogorov-Simirnov and Bartlett tests, respectively.
The motility rate, motility duration, pH, viability, DNA integrity, ATP
content and TAC were subjected to analysis of variance (one-way
ANOVA) followed by the Tukey post hoc test to determine differences
among cryopreservation times. For all analyses, signicance levels of
95% (P <0.05) were assumed. Data were described as mean ±standard
deviation of the mean (SD), and the number of sperm mitochondria by
absolute frequency (Mode - Mo). Statistical analyses were performed
using R packages (4.0.1) and graphs were constructed with GraphPad
Prism 8.0 for Windows (GraphPad Software Inc.).
3. Results
3.1. Fresh milt quality parameters
Milt volume for the six P. orinoquensis males was 9.95 ±2.45 mL,
with a seminal pH of 8.5 ±0.5 and sperm concentration of 7.75 ±4.74
×10
6
spermatozoa/µL. Sperm motility in fresh milt was 94.67 ±2.42%
with a duration of 50.83 ±5.45 s
3.2. Effect of cryostorage on sperm quality
3.2.1. Sperm motility
After cryopreservation, sperm motility showed a signicant drop to
values between 52.5 ±6.45 and 60.0 ±8.94%, with no signicant
differences between storage times. Motility duration showed a tendency
to increase in post-thaw samples, but only samples cryopreserved for 24
h presented a signicant increase compared to fresh sperm. No
signicant differences in motility duration were recorded between the
four cryostorage times (Table 1).
3.2.2. Sperm viability
Sperm viability in cryopreserved samples was signicantly affected
by the freezing-thawing process when compared with fresh sperm.
However, this parameter did not differ signicantly among the four
cryostorage times, with values ranging from 60.17 ±8.97–66.0 ±
6.26% (Table 1).
3.2.3. DNA integrity
Although the sperm assessed after 24 h and 1 month of cryostorage
showed a reduction in DNA integrity (90.0 ±0.0%), they did not
signicantly differ from fresh samples (99.50 ±0.83%). In contrast,
cryostorage for 6 and 12 months led to a drastic and signicant decrease
in DNA integrity (Table 1).
3.2.4. ATP content
Fresh samples showed an ATP content of 4.06 ±0.30 nM×10
8
spermatozoa, similar to the results found after cryostorage for 24 h and 1
month (4.25 ±0.48% and 4.56 ±1.35 nM×10
8
spermatozoa, respec-
tively). In contrast, the ATP content signicantly decreased to 2.28 ±
0.28 and 2.31 ±0.15 nM×10
8
spermatozoa in the samples stored in
liquid nitrogen for 6 and 12 months, respectively (Table 1).
3.2.5. Total Antioxidant Capacity (TAC)
The lowest Total Antioxidant Capacity (0.38 ±0.16 of Trolox
equivalent units) was recorded in fresh sperm. Nevertheless, there were
no signicant differences between fresh and cryopreserved samples. It is
also important to note that no signicant differences were detected
between the four cryostorage times (Table 1).
3.2.6. Sperm morphology
The lowest percentage of sperm abnormalities was found in fresh
samples (2.5 ±0.83%), with signicant differences compared to sam-
ples cryopreserved during 1, 6 and 12 months. In cryopreserved samples
the sperm damage increased signicantly over time, consequently, the
percentage sperm of abnormalities increased from 5.83 ±1.94% at 24 h
to 14.33 ±2.73% at 12 months, showing a 5-fold increase when
compared with fresh sperm (Fig. 1).
In terms of abnormality type, separated head was the most frequent
defect in fresh spermatozoa (1.16 ±0.40%), while the highest fre-
quency recorded in cryopreserved spermatozoa was short tail (5.16
±2.78%) after 6 months of cryostorage. In fact, short tail increased in all
Table 1
Piaractus orinoquensis sperm motility, motility duration, pH, viability, DNA
integrity, ATP concentration and total antioxidant capacity (TAC) detected in
fresh milt and milt cryostored for 24 h, and 1, 6 and 12 months.
Variable Fresh
milt
Cryopreservation time
24 h 1 month 6 months 12
months
Motility (%) 94.67 ±
2.42
a
60.0 ±
8.94
b
54.0 ±
8.94
b
53.33 ±
8.16
b
52.50 ±
6.45
b
Motility duration
(s)
50.83 ±
5.45
b
66.67 ±
11.0
a
55.83 ±
7.85
ab
55.17 ±
10.52
ab
54.20 ±
5.89
ab
pH 8.50 ±
0.50
a
7.0 ±
0.0
b
7.0 ±
0.0
b
7.0 ±
0.0
b
7.0 ±
0.0
b
Viability (%) 99.33 ±
0.81
a
66.0 ±
6.26
b
61.67 ±
11.62
b
60.17 ±
8.97
b
60.50 ±
7.12
b
ADN integrity (%) 99.50 ±
0.83
a
90.0 ±
0.0
a
90.0 ±
0.0
a
68.83 ±
4.26
b
41.17 ±
13.03
c
ATP (nM×10
8
spermatozoa)
4.06 ±
0.30
a
4.25 ±
0.48
a
4.56 ±
1.35
a
2.28 ±
0.28
b
2.31 ±
0.15
b
TAC (mM of
Trolox)
0.38 ±
0.16
a
1.31 ±
0.68
a
1.31 ±
0.74
a
1.27 ±
0.75
a
1.35 ±
0.57
a
Values are shown as means ±standard deviation (mean ±SD) (n =6). Values
followed by different letters on the same row are signicantly different (P <
0.05).
Fig. 1. Total sperm abnormalities found in fresh and cryopreserved Piaractus
orinoquensis sperm at 24 h, 1, 6 and 12 months. Bars represent the mean
±standard deviation (mean ±SD). Different letters indicate signicant differ-
ences (P <0.05). h: hours, m: months.
V.M. Medina-Robles et al.
Aquaculture Reports 29 (2023) 101477
5
four cryostorage periods.
The cryostorage period of 12 months was signicantly harmful, since
there was an increase in the percentage of spermatozoa with degen-
erated mid-piece, bent tail, broken tail and short tail (Table 2).
3.2.7. Sperm ultrastructure
Spermatozoa of P. orinoquensis have an ovoid head without acro-
some, a mid-piece and a single agellum. The mature sperm has a mean
total length of 23.62 ±0.83 µm. Fresh sperm, as well as those cry-
opreserved for 24 h and for 1 and 6 months, exhibited the following
ultrastructural characteristics: the sperm head showed a nucleus with
abundant electron-dense material, homogeneously distributed, covered
by a nuclear envelope formed by two membranes. At the center of the
nuclear base, the nuclear envelope forms the nuclear fossa into which is
inserted the centriolar complex comprising the proximal and distal
centrioles, oriented perpendicularly to each other. The mid-piece of the
spermatozoa is cylindrical and is surrounded by a cytoplasmic channel.
The mid-piece of fresh spermatozoa contains 2 ring-shaped mitochon-
dria surrounding the agellum (Fig. 2). In cryopreserved samples there
were two mitochondria at 24 h and 1 month and one mitochondrion at 6
and 12 months. The mitochondrial matrix is electron-dense with well-
dened cristae. Measurements of fresh spermatozoa are shown in
Table 3. The agellum is inserted centrally at the base of the nucleus; it
has a length of 19.93 ±0.41 µm and is surrounded by a cell membrane
that forms two lateral ns. The agellum shows a 9 +2 organization of
microtubules in the axoneme, which is apparently continuous and pre-
sents no intra-tubular differences.
The electron micrographs of spermatozoa cryopreserved for 12
months revealed the following ultrastructural abnormalities: i) swelling
and rupture of the plasma membrane; ii) loss of mitochondria; iii)
destruction of mitochondrial cristae; iv) loss of the electron-dense
chromatin of the nucleus; and v) degeneration in the middle region of
the agellum with uncoupling or loss of the agellum in some cases
(Fig. 2). The main structural damages at the head level in spermatozoa of
P. orinoquensis cryopreserved for 12 months are shown in Fig. 3.
4. Discussion
Cryopreservation is a very useful practice for storing all types of
genetic material for long periods of time at very low temperatures
(−196 ◦C) without changing their properties. Nevertheless, there is little
information reported on cryostorage time as a variable in studies of sh
sperm. In this study, the effect of long-term (12 months) cryopreserva-
tion on post-thaw sperm quality of P. orinoquensis spermatozoa is re-
ported for the rst time. Sperm motility is directly related to fertilization
capacity in sh (Gallego et al., 2013); the cryopreservation process
causes cellular damage, and therefore decreases levels of this parameter
(Robles et al., 2003). In our study, post-thaw motility decreased to
percentages between 52.50 ±6.45 and 60.0 ±8.94, with the highest
value in samples thawed after 24 h of cryostorage; nevertheless, no
signicant differences were found in the motility rate after the different
cryostorage periods. Similarly, Chen et al. (2010) reported no differ-
ences in the rst year of cryostorage of sperm of seabream (Pagrus
major); however, they found a signicant decrease after 48 months of
cryostorage. In giant grouper (Epinephelus lanceolatus) there were also no
signicant differences after 1, 3 and 4 years of cryostorage, but there
was a drastic decrease in sperm motility after 5 years (Park et al., 2022).
The motility rates obtained in this study are comparable to those
recorded in other South American Characiformes such as tambaqui
(Colossoma macropomum) (Varela et al., 2012; Medina-Robles et al.,
2019) and Piracanjuba (Brycon orbignyanus) (Perry et al., 2019).
Post-thaw sperm quality depends on the cryopreservation protocol.
Herranz-Jusdado et al. (2019) evaluated two different protocols for
cryopreservation of eel sperm, one based on methanol and the second on
Me
2
SO. Interestingly, they found that the methanol-based protocol
yielded the best post-thaw sperm quality values and did not alter the
methylation level of sperm DNA. In addition, the post-thaw sperm
quality may also depend on the biological characteristics of natural
habitat, testis structure, spermatogenesis, sperm morphology, and sperm
physiology (Yang and Tiersch, 2009; Torres et al., 2016). These factors
could explain the high motility rate (over 70%) reported in some other
studies (Nascimento et al., 2010; de Mello et al., 2017).
Fresh milt showed an alkaline pH (8.5 ±0.5), as reported by Su´
arez
et al. (2019) in white cachama under the old classication of Piaractus
brachypomus, and within the range (8.0–8.6) reported for another
Characidae known in Brazil as pirapitinga-do-sul (Brycon opalinus)
(Orf˜
ao et al., 2011). For post-thawed milt, the pH was neutral (7.0) with
no signicant differences between the four cryostorage times. The dif-
ference in pH between fresh and cryopreserved samples may be
explained in part by the pH of the cryopreservation media, which was
7.5. In addition, Me
2
SO is a known scavenger of free radicals (Jin et al.,
2012); it is able to transfer hydrogens (H) from its methyl groups to the
hydroxyl radical (
•
OH), generating methanesulnate, methyl radical
and nally formaldehyde as reaction products (Lee et al., 2004; Abou
et al., 2017), leading to a decrease in the pH of the solutions.
The different mechanisms by which cryopreservation induces loss of
sperm viability are osmotic, mechanical and oxidative damage (Cabrita
et al., 2001; Li et al., 2010; Sandoval-Vargas et al., 2021). In this study,
fresh sperm showed viability close to 100% just prior to cryopreserva-
tion, which decreased to around 60% in all cryopreserved samples,
indicating that after 24 h further storage for one year had limited effect
on sperm viability. The values found in our study are higher than those
reported by Navarro et al. (2004) (0 – 29.4%) for spermatozoa of
P. brachypomus cryopreserved in a different cryoprotective solution. Our
ndings are also higher than those reported for tambaqui, where the best
viability was found with dimethylformamide at 8% (52.9 ±5.4%); the
Table 2
Piaractus orinoquensis sperm abnormalities found in fresh milt and milt cryostored for 24 h, and 1, 6 and 12 months.
Cryopreservation time Sperm abnormality (%)
Macrocephaly* Separated head* Degenerated head* Degenerated mid-
piece
Loose tail* Bent tail Broken tail Short tail
Fresh sperm 0.0 ±0.0 1.16 ±0.40 0.0 ±0.0 0.16 ±0.40
a
0.66
±1.20
0.16
±0.40
a
0.0 ±0.0
a
0.33 ±0.81
a
24 h 0.0 ±0.0 1.33 ±1.21 0.16 ±0.40 0.16 ±0.40
a
0.66
±0.81
1.33
±1.36
a
0.0 ±0.0
a
2.16
±0.98
ab
1 month 0.16 ±0.40 0.33 ±0.81 0.66 ±0.81 0.16 ±0.40
a
1.50
±0.54
0.50
±0.83
a
0.50
±0.54
a
3.33
±2.06
bc
6 months 2.50 ±3.27 1.16 ±1.16 0.66 ±0.81 0.66 ±0.81
a
1.0 ±0.89 1.0 ±1.26
a
0.0 ±0.0
a
5.16 ±2.78
c
12 months 0.33 ±0.51 1.50 ±1.04 0.0 ±0.0 2.0 ±0.89
b
1.33
±1.03
4.0 ±1.78
b
2.50
±1.04
b
2.66 ±0.51
c
Values are expressed as means ±standard deviation (mean ±SD) (n =100 spermatozoa/treatment)
a,b,c
Different superscript letters within the same column indicate signicant differences, (P˂0.05).
* Sperm abnormality with no statistical difference between treatments (P˃0.05).
V.M. Medina-Robles et al.
Aquaculture Reports 29 (2023) 101477
6
Fig. 2. Transmission electron micrograph (TEM) of fresh
and cryopreserved spermatozoa of Piaractus orinoquensis. A-
B: Fresh spermatozoa showing normal structures. C-D:
Spermatozoa cryopreserved for 24 h showing: * loss of
plasma membrane continuity; * * shortening of agellum. E-
F: Spermatozoa cryopreserved for one month showing:
* cytoplasmic channel contraction. G-H; Spermatozoa cry-
opreserved for six months showing * chromatin dispersion
and cytoplasmic vesicles in agellum (circled). I-J: Sper-
matozoa cryopreserved for 12 months showing agellum
loss and decrease in the number of mitochondria with
damage to mitochondrial cristae and uncoupling of the
midpiece (circled); continuity loss and increased thickness of
the plasma membrane (red arrow); less electrodense chro-
matin (white arrow). n: nucleus; f: agellum; m: mitochon-
dria; cr: chromatin; nm: nuclear membrane; pc: proximal
centriole; dc: distal centriole; mp: mid-piece; cc: cytoplasmic
channel; cm central microtubules; pm: peripheral microtu-
bules; f (9 +2): cross section of agellum showing 9 +2
axonemal structure.
V.M. Medina-Robles et al.
Aquaculture Reports 29 (2023) 101477
7
use of 10% Me
2
SO in the same study reduced the viability to less than
25% (Varela et al., 2012). Such differences between ndings may be
related to different factors such as the species-specic toxicity of the
cryoprotectants (Santana et al., 2020), freezing rate (Balamurugan and
Munuswamy, 2017), lipid membrane composition (Bai et al., 2019), milt
dilution (Marco-Jim´
enez et al., 2006) and even their interaction
(Gait´
an-Espitia et al., 2013).
Several studies have demonstrated that cryopreservation increases
DNA damage in sh spermatozoa (Labbe et al., 2001; Zilli et al., 2003;
Cabrita et al., 2005b; P´
erez-Cerezales et al., 2009; Varela et al., 2012). In
our study, sperm from P. orinoquensis cryostored for both 24 h and 1
month presented no negative effects on DNA integrity. However, cryo-
genic storage for 6 and 12 months affected DNA quality signicantly,
resulting in a loss of integrity of approximately 31% and 58%, respec-
tively. Some previous studies have shown that short periods of cryo-
storage (one week and two months) do not affect signicantly the sperm
DNA integrity of grey mullet (Mugil cephalus) (Balamurugan et al., 2019)
and Atlantic salmon (Salmo salar) (Figueroa et al., 2016). On the other
hand, human sperm stored for more than 15 weeks in liquid nitrogen
showed a statistically signicant increase in
α
-tubulin detection (Des-
rosiers et al., 2006). Therefore, DNA damage and other sperm injuries
are probably associated with the length of storage time in liquid nitro-
gen. For example, the quality of giant grouper sperm cryostored for 1–5
years differed signicantly from that of fresh sperm; and although there
were no signicant differences in the percentage of tail DNA during the
cryostorage time, the tail length was greatly increased after 5 years of
cryopreservation (Park et al., 2022).
In human sperm, DNA damage appears to be related to the excess of
reactive oxygen species (ROS) (Agarwal et al., 2017), since ROS affect
DNA bases, especially guanine, leading to oxidative products such as
8-hydroxy-20’deoxyguanosine (8-OHdG) and single- or double-stranded
breaks (Agarwal et al., 2014). Nevertheless, this effect still seems to be
unclear in sh spermatozoa. It is important to highlight that cryopres-
ervation induces oxidative stress in sh spermatozoa (Li et al., 2010;
Sandoval-Vargas et al., 2020, 2021); however, some studies have not
found oxidized bases in samples with fragmented DNA, suggesting that
mechanisms other than oxidative stress could be responsible for DNA
fragmentation during freezing (P´
erez-Cerezales et al., 2009, 2011). In
addition, Cabrita et al. (2005b) note that the differences in the detection
of DNA damage among species could be explained by differences in the
chromatin packaging and the histone/protamine rate of the
spermatozoa.
Stored ATP has been considered to be the primary source of energy to
support sperm motility (Cosson, 2013). Both storage and cryopreser-
vation processes are responsible for a decrease in ATP content (Aramli
et al., 2013; Boryshpolets et al., 2009; Figueroa et al., 2019). In our
study, the intracellular ATP content decreased signicantly at months 6
and 12 of cryostorage. Figueroa et al. (2019) also reported signicantly
lower basal ATP contents for cryopreserved Atlantic salmon (Salmo
salar) spermatozoa compared to fresh samples. Likewise, the intracel-
lular ATP content in Persian sturgeon (Acipenser persicus) spermatozoa
decreased signicantly from 6.57 to values around 2.5 nmol ATP/10
8
spermatozoa after six days of storage (Aramli et al., 2013).
The decrease of intracellular ATP contents could be associated with:
i) ATP consumption caused by sperm cell volume changes and ATP
hydrolysis in damaged cells during the freezing process (Boryshpolets
et al., 2009); ii) ROS production; or iii) a combination of these factors.
Studies in sperm from other animal species have revealed that the
decrease of ATP is associated with the presence of ROS, which produce
inactivation of mitochondrial ATP synthesis (Zhu et al., 2019); or with
activation of the nuclear enzyme poly(ADP-ribose) polymerase-1 (Ara-
mli, 2014). In this context, it may be deduced i) that the metabolic
process of sh sperm did not stop completely at temperatures of
−196 ◦C as was previously concluded by Chen et al. (2010); and it may
be speculated ii) that the decrease of ATP and DNA integrity after 6 and
12 months could be related to mitochondrial damage mediated by ROS,
since ROS generated during cryopreservation may be more damaging to
mitochondrial membranes than other cellular membranes (Whelehan
et al., 2022). The latter could explain the stable viability reported in our
study. It has also been reported that mitochondrial dynamics regulate
DNA damage and genome instability (Cao et al., 2022); nevertheless, to
evaluate this hypothesis, future studies must be conducted including
complementary tests such as mitochondrial membrane potential and
oxidative stress indices.
Fish milt has an antioxidant system with enzymatic (superoxide
dismutase, catalase, glutathione peroxidase, glutathione reductase,
methionine reductase) and non-enzymatic components (ascorbic acid,
uric acid, tocopherol, β-carotenes, selenium, zinc) that act as ROS
scavengers, protecting the cellular structure (Lahnsteiner and Mansour,
2010; Lahnsteiner et al., 2010). The antioxidant activity of these com-
ponents can be evaluated individually. However, the method can be
tedious and time-consuming due to the large variety of antioxidants and
the possible interactions between them (Słowi´
nska et al., 2013; Ni et al.,
2021). Therefore, an alternative way is to measure the antioxidant
Table 3
Measurements of fresh Piaractus orinoquensis spermatozoa.
Values are means ±standard deviation (mean ±SD).
Structure Dimensions (µm)
Head width 1.56 ±0.02
Head length 2.16 ±0.05
Mid-piece width 1.15 ±0.03
Mid-piece length 1.56 ±0.05
Flagellum width 0.28 ±0.008
Flagellum length 19.93 ±0.41
Total length 23.62 ±0.83
Fig. 3. Scanning Electron Microscope images of Piaractus orinoquensis spermatozoa. A) fresh spermatozoa (10.0 KV; SP=9.0; WD=12.4; x 5.0 K). B: Spermatozoa
cryostored for 12 months (10.0 KV; SP=12.0; WD=12.3; x 5.0 K). Spermatozoa with cryo-damage at head level (white arrows). KV: voltage acceleration; SP: spot
size; WD: working distance; K: 1000.
V.M. Medina-Robles et al.
Aquaculture Reports 29 (2023) 101477
8
capacity of all antioxidants present in the sample using a test known as
Total Antioxidant Capacity (TAC) (Kusano and Ferrari, 2008).
Previous studies have stated that protection against oxidative stress
during cryopreservation is insufcient, since the mature spermatozoa
contain a low volume of cytoplasm (which is a rich source of antioxi-
dants). In addition, dilution of the milt in the cryopreservation medium
leads to a reduction in antioxidant concentration (Cabrita et al., 2011;
Martínez-P´
aramo et al., 2012). Nevertheless, in the present study, we
did not nd any decrease in the TAC values between fresh and cry-
opreserved sperm. On the contrary, there was a slight increase in TAC in
cryopreserved samples, which may have protected the plasma mem-
brane against cryodamage, allowing the stability of this parameter and
of post-thaw motility after the different cryostorage periods.
The TAC value for fresh sperm of P. orinoquensis was 0.38 ±0.16 mM
Trolox. This result is within the range of 13 species reported by Słow-
i´
nska et al. (2013) using a similar method to our study. Clearly, those
authors reported great variation between species, with the lowest values
in rainbow trout (0.008 mM of Trolox) and the highest in Eurasian perch
(1.909 mM of Trolox).
With regard to sperm morphology, the percentage of abnormal
spermatozoa in the present study increased signicantly over the cryo-
storage period. Thus, the main negative effects were observed at months
6 and 12 of cryostorage (12.1 ±0.4% and 14.3 ±1.1%, respectively).
However, these percentages of abnormalities are lower than those
detected in spermatozoa of amazon catsh, Leiarius marmoratus (64.50
±12.46%) (Borges et al., 2020), piracanjuba, Brycon orbignyanus
(45.60 ±4.15%) (Galo et al., 2011), common carp (19.4%) (Linhares
et al., 2015), South American silver catsh, Rhamdia quelen (78.52
±0.95%) (da Costa et al., 2019), pejerrey, Odontesthes bonariensis (60%)
(G´
arriz and Miranda, 2013) and tambaqui (61.3 ±8.1%), (Medi-
na-Robles et al., 2019). The difference between studies may be related to
species-specic cryopreservation processes, the cryoprotective capacity
of the media used, or even the reproductive season. Sperm damage
during freezing has been attributed to different causes, such as: osmotic
and oxidative stress (Li et al., 2010; Sandoval-Vargas et al., 2021);
biophysical processes, such as crystallization of intracellular and
extracellular water; or possibly iatrogenic alterations caused during
sample handling (Meryman, 2007; Wolfe and Bryant, 2001; Benson
et al., 2012; Medina-Robles et al., 2019). A previous study in South
American silver catsh showed a high positive correlation between the
percentage of normal spermatozoa and motility (da Costa et al., 2019).
Although no such association was determined in the present study, the
increase in sperm abnormalities in cryopreserved sperm apparently did
not inuence post-thaw motility, which remained signicantly stable.
Some authors have classied sh sperm abnormalities as primary
and secondary, based on the classication applied in mammals (Galo
et al., 2011; Miliorini et al., 2011). Primary abnormalities are related to
spermatogenesis and secondary abnormalities are related to environ-
mental factors and reproductive management (da Costa et al., 2019).
Macrocephaly, degenerated head and broken tail are classied as pri-
mary defects (Miliorini et al., 2011). Nevertheless, in our study, those
abnormalities were only present in post-thawed sperm, indicating that
they were caused by cryogenic processes. Similarly, other authors have
indicated that sh sperm abnormalities, such as curly tail and macro-
cephaly (da Costa et al., 2019), as well as broken tail, short tail and
microcephaly, which are classied as primary in mammals, may be
related to factors such as: osmotic shock from exposure to cryoprotective
solutions; toxicity of cryoprotectants and time of exposure; and the
freezing and thawing rates applied during the cryopreservation process
(Streit et al., 2009). Consequently, special care must be taken with the
classication of sh sperm abnormalities.
This is the rst study showing the ultrastructural morphology of
P. orinoquensis spermatozoa through electron microscopy, as well as the
sperm changes observed after cryostorage. The morphology and
morphometry of the spermatozoon of P. orinoquensis is quite similar to
the pattern described for Piaractus mesopotamicus (Cruz-Landim et al.,
2003; Gusm˜
ao-Pompiani et al., 2009). The sperm cells of these species
showed simple structures with ovoid heads, a centriolar complex
composed of a proximal and a distal centriole that lie entirely inside of
the nuclear fossa, and a cylindrical mid-piece which is surrounded by the
cytoplasmic channel. In the present study, two mitochondria were
observed in fresh spermatozoa, while in P. mesopotamicus there is
probably only a single long mitochondrion forming a ring around the
cytoplasmic channel (Gusm˜
ao-Pompiani et al., 2009). The number of
mitochondria is one of the most characteristic variables recorded in sh
spermatozoa. In bown (Amia calva), 12–16 mitochondria were found
arranged in two rings around the two centrioles (Jamieson, 2009); while
salmonid spermatozoa are characterized by a single mitochondrion,
typically forming a complete ring round the mid-piece (Lahnsteiner and
Patzner, 2008; Sandoval-Vargas et al., 2022). The spermatozoa of
P. orinoquensis have a agellum that contains the typical 9 +2 axoneme
and a pair of lateral ns, as reported in P. mesopotamicus (Cruz-Landim
et al., 2003; Gusm˜
ao-Pompiani et al., 2009). The sperm ultrastructure of
P. orinoquensis is also in agreement with other studies of freshwater
Characiformes with a uniagellated, anacrosomal Type I aquasperm
(Faustino et al., 2015; Solis et al., 2017; Quagio-Grassiotto et al., 2020).
The most severe ultrastructural damage determined by TEM in
frozen-thawed sperm was observed in samples cryostored for 12 months.
Thus, we found loss of continuity and increased thickness of the plasma
membrane, decreased chromatin density, loss of agellum, uncoupling
of the mid-piece, decrease in the number of mitochondria and damage to
mitochondrial cristae. The number of mitochondria in cryopreserved
spermatozoa varied between 1 and 2. This difference could be due to the
swelling and rupture of the mid-piece, expelling the mitochondria into
the extracellular medium. This, together with the structural damage to
the mitochondria observed in sperm cryostored for 6 and 12 months,
may have led to a decrease in mitochondrial activity and therefore in the
production of ATP and agellar movement, while motility was unaf-
fected by shorter periods of cryopreservation.
Previous studies have also reported that cryopreservation procedures
cause substantial morphological alterations in sh spermatozoa (He and
Woods, 2004; Liu et al., 2007; Balamurugan et al., 2019; Figueroa et al.,
2019). In fact, this damage may occur after the dilution of the milt in the
cryopreservation medium, and increase with deep-freezing (Billard,
1983). It seems that the severity of the damage may depend on the
species – and of course, on the cryopreservation protocols. The most
severe damage was reported by Billard (1983) in brown trout (Salmo
trutta fario) and Lahnsteiner et al. (1996) in rainbow trout (Oncorhynchus
mykiss); in the latter case, the authors found that 57 ±14% of the
spermatozoa were completely damaged during cryopreservation.
In conclusion, our results demonstrate that the cryopreservation
procedure used in this study affected signicantly the motility and
viability of P. orinoquensis spermatozoa from the rst assessment after
24 h cryostorage; nevertheless, those variables, as well as the motility
duration, pH, and total antioxidant capacity, did not differ signicantly
across the year of cryostorage included in the study. On the contrary,
both DNA integrity and ATP content were stable in samples cryostored
for 24 h and 1 month, but decreased drastically in samples cry-
opreserved for 6 and 12 months, consistent with the substantial ultra-
structural damage observed during the same period. This could imply
that cryostorage in liquid nitrogen did not completely stop the metabolic
processes of the spermatozoa. Finally, further studies should be con-
ducted to determine the effects of long-term cryopreservation on prog-
eny development.
Declaration of Competing Interest
The authors declare that they have no known competing nancial
interests or personal relationships that could have appeared to inuence
the work reported in this paper.
V.M. Medina-Robles et al.
Aquaculture Reports 29 (2023) 101477
9
Data availability
Data will be made available on request.
Acknowledgements
This work was supported by the Social Fund for Higher Education of
the government of Meta, through the doctoral scholarship awarded to
the author Víctor Mauricio Medina Robles, in the call "Training of high-
level human capital 2015 - Governor’s Ofce of Meta". The authors
would like to thank the Institute of Aquaculture of Universidad de los
Llanos, as well as the other members of the GRITOX Research Group, for
the logistic support provided for the execution of the research project.
We also thank the common equipment laboratory of the Faculty of
Medicine of Universidad Nacional de Colombia, the Microbiology lab-
oratory of Universidad de Ciencias Aplicadas y Ambientales - UDCA, the
electron microscopy unit of Fundaci´
on Santaf´
e de Bogot´
a and the INTEK
Group company, the Animal Reproduction laboratory of the Corpo-
raci´
on Colombiana de Investigaci´
on Agropecuaria, AGROSAVIA, at La
Libertad campus, and the embryology laboratory of Universidad Militar
Nueva Granada for their valuable help with laboratory analysis.
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