Cryopreserved sperm does not affect larval ontogeny and quality in Rhamdia quelen

Abstract

Fish sperm cryopreservation is an important technique for optimizing juvenile production in aquaculture stations and laboratories and contributing to the conservation of endangered species. Despite its benefits, the cryopreservation process can cause cellular damage, affecting spermatozoa quality and offspring viability. This study aimed to evaluate the larval development of jundiá Rhamdia quelen originating from cryopreserved sperm. Larvae were obtained from artificial reproduction using oocyte samples from four females combined with fresh (Control) or cryopreserved/thawed sperm. The semen was diluted in the cryoprotective solution (1:3 ratio) consisting of skimmed milk powder (5%), methanol (10%), and fructose (5%), and was packaged into 0.25 mL straws. The straws were then stored and cooled in liquid nitrogen vapor for 18 h. The straws were individually warmed in a water bath at 25 °C for 10 s to thaw the samples. The experiments were performed in triplicates. Sperm quality, fertilization, hatching, and larval development were evaluated. After larval hatching, six larval collections were performed (5, 10, 15, 20, and 25 days after hatching), and 15 larvae were sampled per collection per treatment. Cryopreservation reduced sperm motility (70.48 ± 7.70 fresh to 41.36 ± 4.80 cryopreserved semen), progressivity (3874 fresh to 2505 cryopreserved semen), and beat cross frequency (55.83 ± 155 fresh to 50.22 ± 190 cryopreserved semen). Increased the percentage of sperm with abnormal morphology and increased most sperm pathologies. Furthermore, the fertilization rate was lower in the cryopreserved group (63.1 ± 18, and 83.72 ± 7.59 for fresh semen), while hatching was not different between groups (65.3 ± 18.05 fresh, 48.89 ± 21.77 cryopreserved semen) Otherwise, the initial larval development morphology showed no difference in the appearance of structures such as the presence of the vitelline structure, pigmentation pattern, development of the anal pore, embryonic membrane, eye, barbells, notochord flexion, and fin rays, for both treatments. There was no significant difference in the frequency of structures between larvae from fresh and cryopreserved/thawed sperm, revealing a similar developmental pattern in both treatments. In conclusion, the cryopreservation protocol affects sperm quality; however, those sperm able to fertilize the oocytes originate normal larvae with regular larval development of R. quelen up to 25 days old.

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Fish Physiol Biochem (2025) 51:42
https://doi.org/10.1007/s10695-025-01455-5
RESEARCH
Cryopreserved sperm does notaffect larval ontogeny
andquality inRhamdia quelen
VanessaConceiçãoCoimbra· JeaneRodrigues· RaquelSantosdosSantos· RômuloBatistaRodrigues·
DaniloStreit‑Jr· AnaLuizadeSouzaCaldas· EduardoSilvadoNascimentoAlbuquerque·
EvagnoJuniordaSilvaFerreira· CaioMaximino· DiógenesHenriquedeSiqueira‑Silva
Received: 20 September 2024 / Accepted: 23 January 2025 / Published online: 5 February 2025
© The Author(s), under exclusive licence to Springer Nature B.V. 2025
Abstract Fish sperm cryopreservation is an impor-
tant technique for optimizing juvenile production
in aquaculture stations and laboratories and con-
tributing to the conservation of endangered species.
Despite its benefits, the cryopreservation process
can cause cellular damage, affecting spermatozoa
quality and offspring viability. This study aimed to
evaluate the larval development of jundiá Rham-
dia quelen originating from cryopreserved sperm.
Larvae were obtained from artificial reproduction
using oocyte samples from four females combined
with fresh (Control) or cryopreserved/thawed sperm.
The semen was diluted in the cryoprotective solu-
tion (1:3 ratio) consisting of skimmed milk powder
(5%), methanol (10%), and fructose (5%), and was
packaged into 0.25mL straws. The straws were then
stored and cooled in liquid nitrogen vapor for 18h.
The straws were individually warmed in a water bath
at 25 °C for 10 s to thaw the samples. The experi-
ments were performed in triplicates. Sperm quality,
fertilization, hatching, and larval development were
evaluated. After larval hatching, six larval collec-
tions were performed (5, 10, 15, 20, and 25days after
hatching), and 15 larvae were sampled per collection
per treatment. Cryopreservation reduced sperm motil-
ity (70.48 ± 7.70 fresh to 41.36 ± 4.80 cryopreserved
semen), progressivity (3874 fresh to 2505 cryopre-
served semen), and beat cross frequency (55.83 ± 155
fresh to 50.22 ± 190 cryopreserved semen). Increased
the percentage of sperm with abnormal morphology
V.C.Coimbra· J.Rodrigues· R.SantosdosSantos·
A.L.deSouzaCaldas· E.S.doNascimentoAlbuquerque·
E.J.daSilvaFerreira· D.H.deSiqueira-Silva(*)
Group ofStudies On theReproduction ofAmazon Fish
(GERPA/LaNeC), Biology Faculty (FACBIO), University
Federal ofSouth andSouthern ofPará (Unifesspa),
Marabá, Pará, Brazil
e-mail: diogenessilva@unifesspa.edu.br
V.C.Coimbra· J.Rodrigues·
R.SantosdosSantos· A.L.deSouzaCaldas·
E.S.doNascimentoAlbuquerque· E.J.daSilvaFerreira·
C.Maximino· D.H.deSiqueira-Silva
Neuroscience andBehavior Laboratory Frederico
Guilherme Graeff (LANEC), Institute ofHealthy
andBiologics Studies, Psychology University, Federal
University ofSouth andSouthern ofPará, Av. Dos Ipês,
Marabá, S/NPará68507-590, Brazil
V.C.Coimbra· J.Rodrigues· E.J.daSilvaFerreira·
D.H.deSiqueira-Silva
Graduate Program inAnimal Reproduction intheAmazon
(ReproAmazon), Federal Rural University oftheAmazon
(Ufra) andFederal University ofPará (UFPA), Av.
Presidente Tancredo Neves, N° 2501, Terra Firme, Belém,
Pará66.077-830, Brazil
R.SantosdosSantos· D.Streit-Jr
AQUAM (Aquatic Species Production andConservation)
attheAquaculture Laboratory oftheDepartment
ofAnimal Science, Federal University ofRio Grande Do
Sul (UFRGS), PortoAlegre, RioGrandeDoSul, Brazil
R.B.Rodrigues· D.Streit-Jr
Veterinary Science Research Program, Federal University
ofRio Grande Do Sul, PortoAlegre, RioGrandeDoSul,
Brazil

Fish Physiol Biochem (2025) 51:42
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and increased most sperm pathologies. Furthermore,
the fertilization rate was lower in the cryopreserved
group (63.1 ± 18, and 83.72 ± 7.59 for fresh semen),
while hatching was not different between groups
(65.3 ± 18.05 fresh, 48.89 ± 21.77 cryopreserved
semen) Otherwise, the initial larval development
morphology showed no difference in the appear-
ance of structures such as the presence of the vitel-
line structure, pigmentation pattern, development of
the anal pore, embryonic membrane, eye, barbells,
notochord flexion, and fin rays, for both treatments.
There was no significant difference in the frequency
of structures between larvae from fresh and cryopre-
served/thawed sperm, revealing a similar develop-
mental pattern in both treatments. In conclusion, the
cryopreservation protocol affects sperm quality; how-
ever, those sperm able to fertilize the oocytes origi-
nate normal larvae with regular larval development of
R. quelen up to 25days old.
Keywords Appearance· Cryopreservation· Fish
conservation· Larval structures· Morphological
description
Introduction
Cryopreservation involves storing and preserv-
ing cellular and tissue viability at low temperatures,
inducing almost a suspension of cellular metabolism
(Mazur etal. 1984). When applied to preserving male
fish gametes, this biotechnique plays a crucial role
in juvenile production in aquaculture facilities and
laboratories, optimizing the use of breeders (Galo
et al. 2018). A fundamental concern in aquaculture
is ensuring the quality of gametes to achieve high
fertilization rates (Lahnsteiner et al. 1998; Ottesen
et al. 2012), consequently, optimal larval develop-
ment (Lahnsteiner et al. 1998; Ottesen et al. 2012;
Ottesen and Babiak 2007) to meet the demands of
breeding systems (Cabrita et al. 2010; Goes et al.
2017). Cryopreservation also facilitates the safe trans-
fer of genetic material between different facilities,
preventing contamination, enabling the synchronous
acquisition of male and female gametes, and optimiz-
ing sperm (Cabrita etal. 2010; Koch etal. 2024). In
this sense, the formation of germplasm banks allows
the conservation of gametes from animals with
advantageous characteristics, such as fast growth and
disease resistance, or those at risk of extinction (Tsai
and Lin 2012; Taylor etal. 2019; Zaniboni-Filho and
Ribolli 2024).
Despite the benefits associated with the cryopreser-
vation of male gametes, cryoinjuries can occur to
sperm cells (De Jesus Paula etal. 2019). The poten-
tial risks involve excessive cell dehydration, osmotic
stress, and the formation of small ice crystals that can
rupture the membrane (Silva and Guerra 2011). This
is attributed to exposure to low temperatures and sub-
sequent warming, as well as contact with cryoprotect-
ants (Gosden 2011), whose function is to protect the
cell or tissue against dehydration, cooling, and dam-
age caused by the extreme reduction in temperature
(Santos etal. 2008). Despite the advantages offered by
cryoprotectant agents (Streit etal. 2024), it is crucial
to consider their toxicity, a limiting factor for the suc-
cess of a cryopreservation protocol. The selection of
the type and concentration of a cryoprotectant, aim-
ing for reduced toxicity, is conditioned by the type of
cell and tissue to be cryopreserved (Fuller and Paynter
2004). These factors can consequently lead to issues
for the offspring. For instance, cells that damage the
DNA molecule may alter gene expression in larvae
(Cabrita etal. 2011; Pérez-Cerezales etal. 2011).
Most cryopreservation studies focus on techniques
used to assess the quality of gametes in fish, primarily
relying on morphological analyses of gametes (Costa
2013; Lopes etal. 2014; Lazarotto 2021) and fertili-
zation rates (Graham and Mocé 2005; Partyka etal.
2012; Asturiano etal. 2016). However, few of these
studies have monitored the subsequent larval develop-
ment. The evaluation of offspring development is of
utmost importance to qualify cryopreserved sperm.
Observing changes in development at an indi-
vidual level is a crucial assessment approach. For an
accurate interpretation of these changes, the use of
methods and measures that are sufficiently sensitive
becomes necessary, capable of detecting alterations at
the level of an organism, species, population, or com-
munity (Sanseverino and Messimian 2008). Among
the tools used for this type of study, we can include
macroscopic analyses such as larval morphology and
the description of structures that stand out through
the analysis and comparison of biological form, ena-
bling the understanding of structure changes (Roth
and Mercer 2000).

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The jundiá (Rhamdia quelen), belonging to the
Heptapteridae family and the Siluriformes order
(Silva 2007), is a native fish species in the Southern
region of Brazil, with a Neotropical distribution rang-
ing from the southwest of Mexico to the central part
of Argentina (Perdices etal. 2022). Widely exploited
in aquaculture due to its adaptability to cultivation
systems in temperate or subtropical climates, the R.
quelen is included in the list of fish species raised
with commercial potential in Brazil (Montanha etal.
2011), consequently becoming a target of research in
several areas such as nutrition (Camargo etal. 2005),
physiology (Montanha et al. 2011), reproduction
(Gomiero etal. 2007), behavior assessments (Abreu
etal. 2016), among others. It is also widely used in
studies related to cryopreservation, such as in the
performance of cryopreservation methods for testicu-
lar tissue and spermatogonial stem cells (Rosa etal.
2023). In another study, the impacts of cryopreserva-
tion on morphological changes in spermatozoa were
reported (Da Costa etal. 2019). Additionally, another
research has addressed the effect of post-thaw dilu-
tion of R. quelen spermatozoa in a 1.1% NaCl solu-
tion on quality and reproductive capacity (Gomiero
etal. 2007). Aiming to complement and contribute to
the study of this species and investigate the potential
effects of cryopreservation on sperm and its conse-
quence for the derived offspring, this study aimed to
evaluate the initial larval development and ontogeny
of R. quelen derived from cryopreserved sperm. For-
mer studies have already described the early ontogeny
of R. quelen larvae (Pereira etal. 2006; De Amorim
etal. 2009) including in different incubation tempera-
tures (21, 24, 27, and 30 °C) (Rodrigues-Godinho
etal. 2010). However, none have reported it from lar-
vae prevenient from cryopreserved sperm.
Materials andmethods
Ethical considerations
All animal handling during experimentation was
conducted by the AQUAM group (Production and
Conservation of Aquatic Species) at the Aquacul-
ture Laboratory of the Department of Animal Sci-
ence at the Federal University of Rio Grande do Sul
(UFRGS), Porto Alegre, Brazil, with prior approval
from the Ethics Committee (CEUA-UFRGS,
Protocol No. 35329). The experiments followed
procedures consistent with the ARRIVE guidelines
and the National Institutes of Health Guide for the
Care and Use of Laboratory Animals (NIH Publica-
tions No. 8023, revised in 1978).
Experimental design
Rhamdia quelen larvae were produced from cryo-
preserved sperm and had their initial ontogeny com-
pared with larvae produced from fresh sperm. For
that, four females were spawned. The four spawn-
ing were divided into two experimental groups: (a)
treatment 1 (control) larvae originating from fresh
sperm; and (b) treatment 2—larvae prevenient from
cryopreserved sperm, and fertilization was per-
formed in triplicate for each spawning. Six larval
collections were performed from the larva hatching,
with a five-day interval between them.
Animal maintenance and management
The silver catfish specimens were kept in water
recirculation systems containing constant tempera-
ture, aeration, and biological filtration. The fish
used in reproduction were fed twice a day (8 am
and 4 pm) with commercial feed (32% crude pro-
tein, Acqua Fish, Supra®, Alisul, Brazil) until
apparent satiety. They were previously selected
according to the appearance of secondary sexual
characteristics, sperm release for males, and bulg-
ing belly for females, weighing 66.3 ± 184.7 g and
655.8 ± 212.3 g, respectively. The water parameters
were maintained at water temperature (24 ± 2 °C),
pH (7.0 ± 0.5), dissolved oxygen (> 5.5 ± 1 mg/L),
and natural photoperiod (12h light/12h dark). The
fry from the reproduction was maintained in four
1000-L incubators per treatment (one for each rep-
licate/female), under the same conditions, for one
month being fed every three minutes on the first day,
every hour on the second day, and three times a day
from day 3 to 30th until apparent satiation with brine
shrimp nauplii, cooked egg yolk, and crushed feed.
Cleaning and dirt (feces and feed residue) removal
were performed whenever necessary, with water
renewal of approximately 10% of the total volume. In
addition, the filter was cleaned periodically.

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Gametes acquisition
Induction was performed according to Woynarovich
and Horváth (1983) and Bombardelli et al. (2006).
Females (4 animals) were induced with 5.5 mg/kg
hormone/fish weight of carp pituitary extract divided
into two doses (0.5 and 5.0 mg/kg hormone/fish
weight, in the first and second doses, respectively)
with a 12-h interval between them. For males (10
animals), only one dose of 3.0mg/kg hormone/fish
weight of carp pituitary extract was applied at the
moment of the second female dose. Sperm and eggs
were collected after a period of 240 accumulated
thermal unit hours through gentle abdominal massage
in the cephalocaudal direction.
Sperm collection was performed using a 15 mL
Falcon conical tube and then kept at 4°C until the
collection of all samples before freezing. The first
portion of sperm was discarded to avoid contamina-
tion with urine, feces, or blood. The sperm pool was
formed from the 10 different males. The pooling was
done to reduce the male effect and to enable simul-
taneous fertilization of the 4 females and subsequent
larval evaluation. This approach allowed fertilization
of the 4 females (using two incubators per female,
one for each treatment) within the incubator system,
which consisted of eight incubators in total. The
absence of sperm motility and the ability to initiate
motility were verified in the collected samples, using
a microscope. The subjective motility and sperm
concentration were assessed using an optical micro-
scope (Nikon Eclipse E200, Tokyo, Japan) with a
40 × objective.
Sperms were collected in a 1-L beaker. One part
was fertilized with fresh sperm and another with cry-
opreserved/thawed sperm.
Sperm cryopreservation
The collected sperm was cryopreserved following the
Adames etal. (2015) protocols. In summary, the cry-
oprotective solution used consisted of skimmed milk
powder (5%), methanol (10%), and fructose (5%).
After diluting the semen in the cryoprotective solu-
tion at a ratio of 1:3 (sperm:cryoprotective solution),
it was packaged into 0.25mL straws. The straws were
then stored and cooled in liquid nitrogen vapor (dry
shipper) for 18h. After this period, the straws were
transferred to a cryogenic storage dewar immersed in
liquid nitrogen (− 196°C). The straws were individu-
ally warmed in a water bath at 25°C for 10s to thaw
the samples. After fertilizing the oocytes with cryo-
preserved and fresh sperm, the embryos were incu-
bated in continuously circulating water maintained
at 24.0 ± 0.5°C in four replicates for each treatment
(fresh and cryopreserved sperm).
Sperm quality assessment
Motility andduration ofmotility
The kinetic parameters of spermatozoa were analyzed
by CASA, as reported by Wilson-Leedy and Inger-
mann (2007). The sperm parameters analyzed were
motility rate (MOT), curvilinear velocity (VCL),
mean path velocity (VAP), straight-line velocity
(VSL), sperm path straightness (STR), oscillation
(WOB), and cross-beat frequency (BCF). An aliquot
of 1 μL of fresh sperm was pipetted into a plastic
tube (2mL), then 600μL of distilled water (0mOsm/
kg) at 25 °C were added, reaching a ratio of 1:600
(sperm:activator). For post-thawed sperm, an aliquot
of 20μL from the straws was activated with 100μL
of distilled water (25°C), reaching a final ratio of 1:5
(thawed sperm:activator). After activation, 5 μL of
the mix was pipetted into a Neubauer chamber under
an optical microscope (Bel Solaris, Milan, Italy) at
100 × magnification with a camera (Basler AC640-
120uc, 658 × 492 pixels, 120 frames per second (fps),
Ahrensburg, Germany) attached for video record-
ing. Videos of spermatic movements were captured
at 100 fps. This video was recorded by a computer
connected to the camera using Pylon Viewer 4 soft-
ware (Version 4.1.0.3660 64-Bit; Basler, Ahrensburg,
Germany). Subsequently, the videos from 10s after
activation were edited into 50 images, representing
0.5 s of video, with the VirtualDub software (Ver-
sion 1.10.04; Microsoft Virtual Studio, Redmond,
USA). The images were analyzed using the Com-
puter-Assisted Sperm Analyzer (CASA) free plug-in
software from ImageJ (Version 1.53e 64-Bit, National
Institutes of Health, USA). The following input vari-
able was used in the CASA plug-in: a = 1, b = 40,
c = 100, d = 12, e = 3, f = 10, g = 15, h = 5, i = 1, j = 15,
k = 15, l = 25, m = 80, n = 80, o = 50, p = 60, q = 100,
r = 556.24, s = 0, t = 0, u = 0. Fresh (n = 10), cryopre-
served (n = 8).

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Sperm morphology
For sperm morphology analysis, the sperm sample
was previously fixed in buffered saline formalin solu-
tion (4.6%) in a ratio of 1:500 (sperm:formaldehyde).
Subsequently, the fixed sample was mixed with Rose
Bengal dye (4%) in a microtube (1.5mL) at a dilution
of 1:50 (Rose Bengal: sperm fixed in buffered forma-
lin 10%) and slides were then prepared using 20μL
of the stained sample. The morphology of 200 sperm/
slide was evaluated under an optical microscope
(Eclipse E200, Nikon) at 1000 × magnification. The
percentage of normal and abnormal sperm was quan-
tified, following the methodology of Da Costa etal.
(2019), and the types of pathologies identified (mac-
rocephaly, microcephaly, degenerated head, loose
head, distal gout, proximal gout broken tail, curled
tail, short tail, and folded tail). Fresh (n = 12), cryo-
preserved (n = 12).
Larvae production
For fertilization assessment, samples of 0.1 mL of
spawning (on average 180 oocytes) were placed in
50 mL plastic cups (three replicates per spawning of
each female for each treatment), and the sperm dosage
was added to these oocytes. A volume of post-thaw
sperm and fresh sperm was pipetted into each cup in
the fixed ratio of 70,000 motile spermatozoa per oocyte
(Neumann etal. 2019). The activation of the sperm and
hydration of the oocytes was carried out with 10mL
of distilled water (24 ± 1°C) and gentle mixing for one
minute, followed by the transfer of eggs to an incuba-
tion system. The incubation was performed in small
circular sieves (5.5 × 3 cm) with nylon nets in plastic
tanks (500L), with constant water quality control, tem-
perature (24 ± 0.5°C), 7.2 ± 0.2 pH and 4.9 ± 0.5mg/L
of dissolved oxygen. The measurement of the rates
of fertilization was performed after the closing of the
embryonic blastopore (Pereira etal. 2006), by counting
all embryos from each experimental unit (sieve). The
result is given by the formula: Fertilization (%) = (num-
ber of fertilized/total oocytes) × 100.
The hatching and normal larvae rates were evalu-
ated 30 h after fertilization. Larvae without head,
spine, or yolk sac abnormalities (Jezierska etal. 2009)
were considered as normal. The ratio of hatched lar-
vae was evaluated by counting the larvae that rup-
tured the chorion, and the percentage was obtained
by the following equation: Hatching (%) = (number of
larvae/total oocytes) × 100.
The larval morphological evaluation was accessed
simultaneously with the evaluation of the larvae hatch-
ing rate. The following equation was used to obtain the
percentage of hatched larvae with normal morphology:
Normal Larvae (%) = (number of normal larvae/ total
larvae) × 100. These analyses were performed using a
binocular stereomicroscope (Q7740SZ-T, Quimis, Dia-
dema, Brazil) at 10 × with the aid of an adapted glass
plate and manual counter. Exogenous feeding was initi-
ated 55h after fertilization.
Sample collection and morphological measurement
For each treatment, 15 larvae were sampled by collec-
tion (six with 5-day intervals between them counting
from the hatching day until 25days after hatching) and
fixed in Karnovsky’s solution (25mL 8% paraformalde-
hyde, 5mL 25% glutaraldehyde, 20mL 0.2M phosphate
buffer, pH 7.0). They were prepared for assessment of
morphology and initial larval development (Fig.1).
The morphological analysis involved comparing 90
individuals from each treatment, starting from their ini-
tial stage (day 0), to identify possible changes in their
body morphology. This analysis and larval classifica-
tion into different stages were based on Ahlstrom and
Ball (1954), modified by Nakatani etal. (2001), with
some adaptations for the species. It included vitelline
larval stage (VL)—developmental stage from hatching
until the onset of exogenous feeding and the absence
of yolk; pre-flexion stage (PF)—developmental stage
extending from the start of exogenous feeding until
the onset of notochord flexion with the appearance
of supporting elements that give rise to the caudal fin
(caudal rays); flexion stage (FL)—developmental stage
characterized by the initiation of notochord flexion, the
appearance of supporting elements of the caudal fin,
until complete flexion, and the appearance of the dorsal
and anal fin buds for some species; post-flexion stage
(PF)—developmental stage characterized by complete
notochord flexion and the appearance of dorsal and
anal fin buds until the total formation of fin rays.
The developmental sequence was determined pri-
marily by the degree of flexion of the final notochord
structure, the formation of fin rays, and their support-
ing elements. The description of each stage was based
on the degree of development and the occurrence of
major morphological events.

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Statistical analysis
A frequency analysis of body characteristics (fin buds
and fin rays, and pre-anal structure (PreAL)) was
conducted and expressed as percentages (%) by the
formula: Sum: (F2:F16)*100/15. The data statistical
analysis was performed using JAMOVI 2.4.14 soft-
ware, conducted through the Chi-Square Test. Fifteen
larvae for sample by treatment were used.
Statistical analyses of sperm evaluation and ferti-
lization data were performed as follows. Levene and
Shapiro–Wilk tests were used to test the homogeneity
of variances and normality of data, respectively. When
the statistical assumptions were met, Student’s t test
was performed to compare the fresh and cryopreserved
groups. For non-parametric data, Mann–Whitney anal-
ysis was applied to compare the groups. A significance
level of 5% was considered in all tests. The analyses
and creation of graphs were performed using the statis-
tical software GraphPad Prism 9.0.
Results
Sperm quality assessment
The results of the analyses of sperm motility param-
eters and kinetics can be seen in Fig.2. The percent-
age of sperm motility (Fig. 2A), beat cross frequency
(Fig. 2B), and progressive motility (Fig. 2H) were
significantly higher in fresh sperm, while STR (Fig.2F)
was significantly higher in the cryopreserved group.
The other variables did not differ between the experi-
mental groups. The results of the sperm morphology
analysis can be seen in Fig.3. The percentage of nor-
mal sperm was higher (p < 0.0001) in fresh sperm with
80.75 ± 8.92% when compared to cryopreserved sperm
(25.42 ± 7.89%), as can be seen in Fig. 3A. The cryo-
preservation process caused a reduction of more than
50% of sperm with normal morphology, demonstrat-
ing the cryobiological damage caused to sperm cells.
There was a difference between the experimental groups
(p < 0.0001) for all head pathologies, with cryopreserved
sperm presenting the highest percentage of these pathol-
ogies, when compared with fresh sperm (Fig. 3B–E).
There was no significant difference between the experi-
mental groups for the rate of sperm with proximal gout
(Fig.3F), while the percentage of distal gout was higher
in the cryopreserved group (Fig.3G). There was a sig-
nificant difference between the experimental groups for
all tail pathologies, with cryopreserved sperm presenting
the highest percentage of the distal curled tail (Fig.3H),
strongly curled tail (Fig.3I), broken tail (Fig. 3J), and
short tail (Fig.3L). While fresh sperm showed a higher
percentage of folded tail (Fig.3K).
Fertilization assessment
A higher fertilization rate was observed in the group
with fresh sperm, compared with the cryopreserved
Fig. 1 Illustration depict-
ing the experimental design,
highlighting the collection
time of Rhamdia quelen
larvae from fresh and
cryopreserved sperm. (T)
treatment; (D.A.H) day(s)
after hatching; (N) number
of selected larvae

Fish Physiol Biochem (2025) 51:42 Page 7 of 16 42
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group (Fig.4A). The hatching rate did not differ sig-
nificantly between the experimental groups (Fig.4B).
Morphological analysis
The assessment of larval stages revealed notable
characteristics at each stage of development. Mor-
phological events such as the presence of the vitelline
structure, pigmentation pattern, development of anal
pore, embryonic membrane, eye, barbels, notochord
flexion, and fin ray were equivalent for both treat-
ments (Table1 and Fig.5).
Frequency analysis
There was no significant difference in the frequency
of structures between larvae from fresh and cryo-
preserved semen, revealing a similar developmental
pattern in both treatments. The pre-anal structure is
present in the larvae at zero days post-hatching, with
Fig. 2 Sperm motil-
ity and kinetic param-
eters. A Sperm motility
(p < 0.0001); B BCF
(p < 0.0001); C VCL
(p = 0.2767); D) VAP
(p = 0.6701); E VSL
(p = 0.9885); F STR
(p = 0.0206); G WOB
(p = 0.0531); H PROG
(p = 0.0002). Significant
difference by Student’s
t test (*p < 0.05; ***
p < 0.001; **** p < 0.0001).
Mean ± SD. Fresh (n = 10),
Cryopreserved (n = 8)

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Fish Physiol Biochem (2025) 51:42 Page 9 of 16 42
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full expression (100%) on the fifth day. The presence
of rays in the caudal fin was observed 5days post-
hatching, reaching full expression (100%) at 15days.
The development of the caudal fin, including the
bifurcation process, was evident at 20 days post-
hatching, with a predominant bifurcation expression
(90%) at 25days. The appearance of dorsal fin buds
and rays occurred at five days post-hatching, with
full expression (100%) for buds and for rays (90%) at
25days post-hatching. In the anal fin, buds emerged
at 15 days post-hatching, reaching full expression
(100%) at 25days, while rays manifested at 20days,
with higher expression (80%) in larvae from cryo-
preserved semen compared to fresh semen (73%) at
25days post-hatching (Fig.6).
Discussion
The cryopreservation of gametes offers several ben-
efits; however, this process can cause damage to cel-
lular structure, and the objective of different cryo-
preservation protocols is to minimize cellular injury
during the freezing and thawing stages (Ribeiro
etal. 2012). In this study, we investigated how the
potential effects of sperm cryopreservation may
impact the larval development of R. quelen. The
unprecedented results for this species address one
of the key questions regarding the use of cryopre-
served reproductive cells, revealing that, at least for
this species, no significant morphological altera-
tions were observed in larvae originating from cryo-
preserved sperm.
Here in our study, we observed that cryopreserva-
tion impaired sperm motility (a reduction of almost
30% compared to fresh sperm). Furthermore, pro-
gressive motility and beat cross frequency were also
worse in cryopreserved samples than in fresh sperm
samples. The difference between motility observed
in our study between cryopreserved sperm and fresh
sperm is smaller than that observed by Da Costa etal.
(2021) and França etal. (2023), with approximately
a 40% reduction in motility after cryopreservation in
both studies, with the same species, or that observed
for other South American species Piaractus orino-
quencis (34.67%, Medina-Robles et al. 2023). The
sperm motility analysis is extremely important for
evaluating the effects of fish sperm manipulation,
such as in cryopreservation, and is generally cor-
related with fertility (Kime et al. 2001). This influ-
ence of motility on the sperm’s ability to fertilize the
oocyte was also observed in our study, because in
addition to the drop in motility after cryopreservation,
the fertilization rate was also impacted, with a drop of
approximately 20% in the cryopreserved group, when
compared to the fresh group. Despite the difference
in fertilization, the hatching rate in this study was the
Fig. 3 Evaluation of sperm morphology and pathologies.
A Normal spermatozoa (p < 0.0001); B macrocephaly (%)
(p < 0.0001); C microcephaly (%) (p < 0.0001); D loose head
(%) (p < 0.0001); E degenerated head (%) (p < 0.0001); F
proximal gout (%) (p = 0.2745); G distal gout (%) (p < 0.0001);
H distal curled tail (%) (p = 0.0028); I strongly curled tail
(%) (p < 0.0001); J broken tail (%) (p < 0.0001); K folded tail
(%) (p = 0.0021); L short tail (%) (p < 0.0001). Significant
difference by Mann–Whitney test (*p < 0.05; ** p < 0.01;
*** p < 0.001; **** p < 0.0001). Graphs: Box and Whisk-
ers = median, max, and min. n = 12
◂
Fig. 4 Results of the fertilization experiment with fresh and cryopreserved sperm. A Fertilization rate (p = 0.0163). B Hatching rate
(p = 0.1748). Significant difference by Student’s T test (*p < 0.05). Mean ± SD. N = 12

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same between the cryopreserved and fresh groups.
This means that at the end of the cryopreserva-
tion process used in this protocol, the cryopreserved
sperm can produce the same number of larvae as the
fresh sperm.
In addition to negatively affecting sperm motil-
ity and fertilizing capacity, cryopreservation caused
harm to sperm morphology. Similarly, to the obser-
vation of Galo etal. (2018) and Atencio-García etal.
(2023) for other South American species, Piaractus
mesopotamicus and Sorubim cuspicaudus, respec-
tively, here we observed a significant reduction in
the percentage of sperm with normal morphology,
and consequently an increase in practically all sperm
pathologies, in the cryopreserved samples, when
compared to fresh samples. Damages caused by cryo-
preservation on sperm morphology of R. quelen were
observed by Da costa etal. (2019), which reported
an increase in morphological alterations in the spe-
cies’ sperm, indicating a substantial sperm fragility
concerning the cryopreservation process. In another
study by Da Costa etal. (2020), the authors observed
the sperm cryopreservation of zebrafish (Danio rerio)
resulted in a reduction in sperm motility, the num-
ber of morphological normal cells, and membrane
integrity, along with an increase in the percentage of
morphological sperm abnormalities. Those studies
indicate that cryopreservation can influence sperm
morphology, despite being a well-established tech-
nique with relative success in many temperate climate
fish species (Cabrita et al. 2011). However, studies
about the interference of sperm cryopreservation on
progeny morphology are still scarce. Thus, describing
the morphological characteristics found throughout
the development of larval stages, prevenient from cry-
opreserved sperm, is fundamental to providing infor-
mation about the potential effects of cryopreservation.
Larvae from fresh and cryopreserved sperm
exhibited similar characteristics during the develop-
mental stages, with no statistical differences in their
Table 1 Characterization of larval stages of silver catfish (Rhamdia quelen) from fresh and cryopreserved semen. (DAH) days after
hatching
Stages DAH Description Image
Vitelline Larval 0 Initiation, shortly after hatching, in which the larvae do not have a fully apparent oral cav-
ity, possess an optic vesicle with little pigmentation, the presence of the vitelline structure
with a yellowish color, transparent body with low pigmentation, and a straight notochord.
Structures such as the anal pore, embryonic membrane, and barbel pores are observable
A
Pre-flexion 05 Presents an open mouth, pigmented eyes with a rounded formation, the digestive tract
appeared more defined, absence of yolk, underdeveloped caudal rays, pigmentation is
more focused in the head region than in the notochord, and a subtle flexion of the noto-
chord. Additionally, it exhibits an anal pore, barbels, and an embryonic membrane
B
Initiation of flexion 10 Presents an open mouth, pigmented eyes, absence of the yolk sac, and caudal rays, with
concentrated pigmentation more noticeable in the head region compared to the notochord.
The anal pore, barbels, embryonic membrane, and partial flexion of the notochord are
notable
C
Flexion 15 Has an open mouth, pigmented eyes, no presence of yolk, increased development of caudal
rays with a size increase, subtle development of the dorsal and anal fin buds, concentrated
pigmentation more noticeable in the head region compared to the notochord. Addition-
ally, it exhibits an anal pore, barbels, and an embryonic membrane, and the notochord is
more flexed
D
Initiation of post-flexion 20 Presents an open mouth, pigmented eyes, absence of yolk, caudal rays develop initiating
the bifurcation process. Pigmentation is more concentrated in the head region compared
to the notochord. Additionally, the anal pore, barbels, and an embryonic membrane are
identifiable. The notochord shows a greater flexion, and the presence of rays in both the
dorsal and anal fins is noticeable
E
Post-flexion 25 Has an oral cavity, pigmented eyes, absence of yolk, while the caudal rays are developed,
continuing the bifurcation process. The rays of the anal and dorsal fins continue to
develop, and the head and notochord show evident pigmentation with a higher concentra-
tion in the head region. It has the presence of an anal pore, barbels, embryonic membrane,
and total flexion of the notochord
F

Fish Physiol Biochem (2025) 51:42 Page 11 of 16 42
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emergence or the frequency of manifestation, and is in
line with the observation of Pereira etal. (2006) and
De Amorim etal. (2009) for the initial larval devel-
opment in the same species. Studies on fish species
such as turbot (Scophthalmus maximus) (Chereguini
et al. 2001) and the Rainbow trout (Oncorhynchus
mykiss) (Labbe etal. 2001) presented similar results.
In those studies, no differences were observed in mor-
phological characteristics such as length and weight
(Chereguini et al. 2001) or survival, and the occur-
rence of abnormalities in progeny, even with DNA
damage of nearly 16% in the nucleus of cryopreserved
sperm (Labbe etal. 2001). When fertilization occurs,
there may be efficient repair mechanisms, as oocytes
can repair DNA damage in sperm for some species of
animals, and in human beings (Newman etal. 2021).
Rana and McAndrew (1989) demonstrated that the
survival and growth of tilapia (Oreochromis spp.) fry
at 30days post-hatching were not negatively affected
using thawed sperm. Another study using R. quelen
(Araújo 2023) proved that there were no alterations in
the behavior of offspring from cryopreserved semen
Fig. 5 Morphological characteristics of Silver catfish (Rham-
dia quelen) larvae in six described stages of early develop-
ment: A yolk larva, B pre-flexion, C early flexion, D flexion, E
early post-flexion, F post-flexion. ov optic vesicle, n notochord,
double arrow barbel pores, dashed arrow anal pore, y yolk, ef
embryonic fin, dashed arrow anal pore, M mouth, b barbels,
arrowhead anal rays, dt digestive tract, thick arrow dorsal com-
plex, hollow arrow anal complex, asterisk dorsal rays. (n = 15
per treatment)

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up to 20days post-hatching, suggesting good-quality
progeny.
Larval survival post-thaw and developmental com-
petence can be influenced by several factors such as
the type of cryoprotectant agent used, gamete qual-
ity, and embryonic developmental stage (Ferreira
etal. 2015). Consequently, the formation of structures
throughout development may reflect larval adaptation
and their survival in larval stages (Taguti etal. 2009).
In the vitellogenic larval stage, larvae from both treat-
ments exhibited a transparent aspect, an important
characteristic for the animal at this phase, represent-
ing a strategy to avoid predation (Almeida 2016). As
Nakatani etal. (2001) described in the Pimelodidae
family, a scarcity of pigmentation in the eyes, and a
non-functional mouth and intestine, is common in
this phase. The yolk is abundant, serving as the main
source of nutrition until the onset of exogenous feed-
ing (Mello 2015).
In the pre-flexion stage, R. quelen larvae from
both treatments, already exhibit a complete mouth
opening, engage in exogenous feeding with a defined
digestive tract, and demonstrate pronounced pigmen-
tation in the eyes. There is also an increase in size
and development of the caudal fin and barbels. These
characteristics indicate the development of many
structures necessary for swimming, sight, prey cap-
ture, and movement perception. Despite being frag-
ile, these active structures enhance their chances of
survival. Junior etal. (2021), in a study on the visual
development of Brycon orbignyanus larvae, reported
that the presence of cones and rods enhances the
larvae visual capacity, which is crucial for evading
predators and capturing food. This, in turn, increases
the species’ chances of survival during its early life
stages.
In the flexion stage, larvae continue developing
their structures, but maintain the same pigmenta-
tion pattern, with greater concentration around the
head region. This pigmentation pattern displayed by
R. quelen larvae can be a crucial characteristic for
taxonomic studies, aiding in species identification
(Godinho and Santos 2003). In the post-flexion stage,
pigmentation intensifies throughout the R. quelen lar-
vae’s body. The fins continue to develop fully, except
for the embryonic fin, which gradually regresses. This
embryonic fin is present since the vitellogenic larval
stage and holds significant importance for locomotion
Fig. 6 Frequency of pre-anal structures (PreAl, (a)) and
fins(Caudal fin, (b), Dorsal fin (c), Anal fin (d)) observed in
larvae of Jundia (Rhamdia quelen) from fresh (F) and cryopre-
served (C) semen. (D.A.H) Days after hatching; (%) percent-
age. (n = 15 per treatment)

Fish Physiol Biochem (2025) 51:42 Page 13 of 16 42
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and cutaneous respiration in the early stages (Almeida
2016; Lima 2011).
In summary, we observed that the cryoinjuries
caused by the cryopreservation process affected some
sperm quality variables, such as motility and morphol-
ogy, and the fertilization rate. However, the evaluation
of larval development demonstrated that despite the
negative effects on sperm, this does not cause damage
to the larvae. These pieces of evidence indicate that lar-
vae derived from cryopreserved sperm did not experi-
ence a compromise in their ontogeny during the initial
stages due to the sperm freezing and thawing process.
Considering that cryopreservation protocols are spe-
cies-specific, this study demonstrated that the protocol
applied to R. quelen did not affect the initial morphol-
ogy and ontogeny of the individuals. These findings
are promising and significant for research and practical
aquaculture and genetic conservation applications.
Acknowledgements The authors would like to thank: the
“Fundação Amazonia de Amparo a Estudos e Pesquisas
(FAPESPA)”, and “Conselho Nacional de Desenvolvimento
Científico e Tecnológico (CNPq)” for granting the students’
scholarship through the intermediary of “Pró-Reitoria de Pós-
Graduação, Pesquisa e Inovação Tecnológica da Unifesspa”.
Research fellows Danilo P. Streit Jr. (CNPq grant 305387/2022-
7), Romulo Batista Rodrigues (CNPq grants 141717/2019-0
and 200285/2021-1), Diógenes H. Siqueira-Silva (CNPq grants
313053/2022-7 and 442763/2023-9), and CNPq for funding
project 300189/2022-2.
Author contributions VC: Conceptualization, data curation,
investigation, methodology, visualization, roles/ writing—origi-
nal draft. JR: Data curation, investigation. RS: Data curation,
investigation. RR: Conceptualization, data curation, investiga-
tion, methodology, visualization, roles/ writing—original draft.
DSJ: Conceptualization, data curation, investigation, method-
ology, visualization, roles/ writing—original draft. AC: Data
curation, investigation. ESNA: Data curation; investigation. EF:
Data curation, investigation. CM: Formal analysis; resources;
DSS: Conceptualization, methodology, formal analysis, Project
administration, resources, supervision, validation, visualization,
roles/writing—original draft, writing—review and editing.
Funding The work was supported by Fundação Amazo-
nia de Amparo a Estudos e Pesquisas (FAPESPA), and Con-
selho Nacional de Desenvolvimento Científico e Tecnológico
(CNPq) Grant term n° 050/2021.
Data availability No datasets were generated or analysed
during the current study.
Declarations
Competing interests The authors declare no competing interests.
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