Seminal characteristics and sensitivity of Astyanax lacustris (Characiformes: Characidae) sperm to cryoprotective solutions based on dimethylsufoxide and methylglicol

This study aimed to determine the semen characteristics of Astyanax lacustris after hormonal induction and to evaluate the sensitivity of the species sperm to cryoprotective solutions based on the cryoprotectants dimethyl sulfoxide and methyl glycol. Volume, color, sperm concentration, total motility and aspects of sperm movement were analyzed using “Integrated Semen Analysis System”. Three different extenders were tested: A) glucose 5%+egg yolk 10%, B) BTS® 5% and C) glucose 5% and two permeable cryoprotectants: dimethyl sulfoxide (Me2SO) and methyl glycol (MTG). Fresh A. lacustris semen presented total motility of 76.6±11.2%, motility duration of 33.0±2.2s, sperm concentration of 7.22±3.2×109sptz/mL and seminal osmolality of 219±0.03mOsm/ kg-1. The toxicity test showed the highest total motility values at the MTG15%+A, Me2SO15%+B and Me2SO10%+C dilutions, and the Me2SO10%+C and Me2SO15%+C dilutions presented the highest values for curvilinear velocity, linear velocity and average velocity. The tested protocol was not effective at maintaining the viability of A. lacustris semen after freezing because no motility was observed in any of the dilutions. However, the Comet Assay demonstrated that cryoprotectant solutions were effective in protecting the genetic material of cells, as DNA damage levels were low, with no difference between control and Me2SO10% + A, dilutions MTG10%+C, Me2SO10%+B and Me2SO15%+B.


INTRODUCTION
The yellow-tailed lambari Astyanax lacustris (Lütken, 1875) (= Astyanax altiparanae), belonging to the Characidae family, is a small species native to the upper Paraná basin, with omnivorous eating habit, easy environmental adaptation, rapid growth and spawn more than once a year, so it is used as a biological model (Sabbag et al., 2011;Gonçalves et al., 2012a,b;Lucena, Soares, 2016). With great economic potential, this species can be used as a sport bait, as well as being well accepted as a snack, which is why production is growing in aquaculture (Gonçalves et al., 2012a).
Considering their biological and economic importance, the development of technologies that catalyze the reproductive process of captive species is necessary, in order to support the projects of environmental preservation and commercial production. Technologies such as methods for the cryopreservation of fish sperm have the objective of facilitating and maximizing the reproduction of species of commercial interest, either threatened or endangered, and favoring the processes of genetic improvement in these situations (Cárton-Garcia et al., 2013). However, the freezing and thawing processes can cause decreased sperm motility, loss of functional and structural integrity of the plasma membrane, morphological changes in gametes and changes in sperm fertilization capacity (Streit Jr. et al., 2006;Viveiros et al., 2009;Ciereszko et al., 2014;Viveiros et al., 2015;Yang et al., 2016;Galo et al., 2019).
Due to the problems mentioned above, cryoprotective substances are added to the semen, whose main functions are to decrease the freezing point of the cell and protect the cell membrane during the cryopreservation process (Maria et al., 2006).
To develop a cryoprotectant solution that maintains the seminal characteristics and fertilization capacity of the sperm species, it is necessary to evaluate the tolerance of sperm cells to exposure to different cryoprotectant substances, so that there is effective sperm protection during the cryogenic process, since this tolerance varies according to the species of fish used (Judycka et al., 2018;Pereira et al., 2019).
Based on the assumptions that there is no concrete scientific information about seminal characteristics of Astyanax lacustris and no effective cryopreservation protocol has been established for semen of the species, this work aimed to determine the characteristics of A. lacustris semen after hormonal induction, and to evaluate the sensitivity of the species' sperm to cryoprotectant solutions based on dimethyl sulfoxide and methyl glycol cryoprotectants, in order to contribute to the development of a cryopreservation protocol for semen of this species.

MATERIAL AND METHODS
Animals and obtaining gametes. Thirty-eight sexually mature specimens were used of Astyanax lacustris, with a total mean length of 10.4 ± 0.5 cm, a standard length of 8.6 ± 0.5 cm and an average mass of 16 ± 3.1 grams, belonging to the Laboratório de Ictiologia Neotropical (L.I.NEO), Universidade Estadual Paulista "Júlio de Mesquita Filho", Ilha Solteira Campus. The fish were kept in 4,500L circular PVC tanks, at a ratio of 2:1 (male/ female), with an average temperature of 27.7ºC, pH 7.6 and ammonia of 0.00 ppm.
Determination of fresh sperm characteristics. Thirty-eight males were used to determine sperm characteristics. Staining was visually estimated considering the translucent and white patterns and the ejaculated volume measured by micropipettes. To determine seminal plasma osmolarity, semen was centrifuged at 3,000 rpm for 15 minutes, and the supernatant was collected and analyzed using an osmometer (OSMOMAT ® model 030, Berlin, Germany). Sperm concentration (sperm/mL) was measured using the Neubauer hematimetric chamber. For this, semen was diluted in formalin-saline solution at a ratio of 1:1000 (Bashiyo-Silva, 2014). Sperm motility characteristics were evaluated using the CASA (Computer Assisted Sperm Analysis) system (ISAS ® Integrated Semen Analysis System, Proiser, Valencia, Spain) coupled to a phase contrast microscope UB200i (UOP/Proiser) with a 10x negative phase contrast objective. The images were captured with ISAS 782C camera (Proiser, Spain) and processed with CASA software, recorded at 25 image per seconds (fps). Sperm activation was performed by adding 30 µl of distilled water to 0.5 µl of semen using a Makler ™ camera (Sefi Medical Instruments Ltd, Israel), and after the analyzes were performed every 10 seconds. Total motility (MOT, %), progressive motility (PRG, %), porcentagem fast sperm (VAP=50-100 μm/s), medium (VAP=25-50 μm/s) and slow (VAP=10-25 μm/s), curvilinear velocity (VCL, μm/s), linear velocity (VSL, μm/s), and mean velocity (VAP, μm), the linearity coefficient (LIN, %), the rectilinearity coefficient (STR, %), the average oscillation of the sperm spatial trajectory (WOB, %), the lateral displacement amplitude of the head (ALH, μm) and the beat cross frequency (BCF, Hz) were also evaluated. Straightness percentage (STR) >80% was used for sperm to be considered progressive. Sperm with VCL <10 μm/s were considered immovable.
Cryoprotectant toxicity test. Two internal cryoprotectants were tested, dimethyl sulfoxide (Me 2 SO) and methyl glycol (MTG) (Sigma-Aldrich), at concentrations of 10 and 15%, in combination with three diluent solutions: A) composed of 5% glucose + 10% egg yolk; B) consisting of 5% Bestlvile Thawing Solution -BTS ® (composition: 79.9% glucose, 12.71% sodium citrate, 2.65% EDTA, 2.65% sodium bicarbonate and potassium chloride 1.59%, gentamicin sulfate 0.5%) (Minitub, Tiefenbach, Germany), and C) composed of only 5% glucose, thus resulting in twelve treatments, in addition to the control group (fresh semen). Before the toxicity test, the osmolality of solutions A, B and C and the twelve treatments were measured (Tab. 1).  Semen was diluted 1:25 (semen/cryoprotectant solution) in all treatments and, after 15 minutes (equilibration time), 1µl of diluted semen was placed in a Makler chamber, activated with distilled water and analyzed using the sperm analysis program (ISAS ® , CASA). All motility parameters mentioned in item 2.4 were measured using four repetitions (fish) for each treatment.
Freezing. For freezing, semen was diluted 1:25 (semen: solution) in eight cryoprotectant solutions selected in the toxicity test (Zanandrea et al., 2016). This dilution was used due to the low amount of semen of the species, approximately 30 µl. It was then placed into 0.25mL plastic straws (Minitub) at room temperature (28°C); after a 15 minute equilibration period, the straws (8) were placed on a steel mesh tray about 1 cm away from the surface of liquid nitrogen (Ninhaus-Silveira et al., 2006a) contained in a 33 liter Styrofoam insulating box for seven minutes until reaching a temperature of -70ºC (Thermocouple -OMEGA ® RDXL4SD) (Viveiros et al., 2000). Subsequently, the samples were immersed in liquid nitrogen and transferred to a cryogenic liquid container (ABS -Pecplan, model ABS20 -MVE). After one month, the samples were thawed in a water bath at 36ºC for seven seconds (Ninhaus-Silveira et al., 2006b), and motility was checked using the integrated system (ISAS ® CASA). Four fish (repetitions) were also used per treatment.
DNA damage assessment. The Comet Assay methodology for determining the DNA integrity of cryopreserved sperm was based on the protocol proposed by Singh et al. (1988) and Klaude et al. (1996), and analyzed three repetitions (fish) by treatments. To perform the analyses, histological glass slides were prepared with a thin layer of normal melting agarose (NMP). Next, 10µl of diluted semen and 120µl of low melting temperature agarose (LMP) were added to a microtube. These samples were placed on slides previously prepared with normal agarose, covered with a coverslip and refrigerated for about 30 minutes to solidify. After this time, the slides were immersed in pH 10 lysis solution (2.5M NaCl, 100mM EDTA, 10mM Tris, 35mM Lauril) for one hour at 4°C.
Subsequently, the slides were placed in an electrophoresis vat that was filled with pH 13 solution (200mM EDTA, 10N NaOH), and kept for 30 minutes at 4ºC. Then, the electrophoresis process was performed for a period of 20 minutes at 25V/300mA. After this process, the slides were covered with pH 7.5 neutralization solution (0.4M Tris) for five minutes (3x), fixed by immersion in ice-cold absolute ethanol for five minutes, and dried at room temperature. To visualize the comet, the slides were stained by adding 100µl of ethidium bromide solution onto each slide, covered with coverslips and analyzed in a 40x objective with the aid of a fluorescence microscope (Olympus, BX-FLA, Japan). In total, 100 cells per sample were analyzed and grouped into damage class according to tail size and intensity as follows: class 0 -no apparent damage, and class 1 to 3 -increasing DNA damage, related to tail length and intensity (Kobayashi et al., 1995) (Fig. 1).
The damage index (total DI) was obtained by multiplying the number of comets in each class by the denominator digit of each class, according to the formula below, where "n" represents the number of cells in each damage class: DI total = 0 x (n Class 0) + 1 x (n Class 1) + 2 x (n Class 2) + 3 x (n Class 3) 6/20 ni.bio.br | scielo.br/ni Statistics. Statistical analysis was performed following the subdivided parcel model by analysis of variance (ANOVA). For results showing a significant difference (p<0.05), the Scott-Knott test was applied to compare control and treatments, and verify the difference between treatments. For nonparametric data, the Kruskal-Wallis test was applied (p<0.05). Data were analyzed using the R Studio statistical program (RStudio Team ,2015).. The fish was considered as the repetition with five fish per treatment.

Features of Fresh Semen.
Fresh semen was translucent, with little viscous consistency and a seminal volume of about 30 µL. The parameters for osmolality, sperm motility and concentration for Astyanax lacustris semen are presented in Tab. 2.
The total motility (MOT) differed statistically between the control and treatment groups, but no statistically significant differences were observed in the comparison between treatments. Treatments T4, T6 and T7 were the ones that provided higher    values of total motility ( Fig. 2A). Regarding progressive motility (PRG), the highest values were obtained in treatments T7, T8 and T6, respectively; however, there was no statistical difference between them ( Fig. 2A).
Regarding the percentage of fast, medium and slow sperm, there was a marked decrease in fast sperm and an increase in medium and slow sperm after dilution in cryoprotective solutions (Tab. 4). However, no statistical difference was observed 8/20 ni.bio.br | scielo.br/ni between the control and treatments T7, T8, T1, T2 and T5 for fast sperm, but treatment T7 had the highest number of fast spermatozoids, highlighting that treatments T3, T4 and T6 had less than 10% of fast spermatozoa differing statistically from the control. In the average sperm there was no difference between any of the treatments, but there was an increase in the percentage of average sperm compared to the control in all treatments with the exception of T7 and T8. The same pattern was observed for slow sperm that significantly increased in relation to the control, with the highest percentage found in the T3 treatment that differed statistically from the control (Tab. 4).  For the VCL, VSL and VAP parameters, a significant difference was found between the control and treatment groups (Figs. 2B-D). Regarding VCL, treatments T7, T8 and T1 presented the highest values, but did not differ statistically from the other treatments (Fig. 2B). For VSL and VAP, T7 and T8 represented the treatments with higher velocities, being statistically different from the others. The lowest velocities for both VSL and VAP were obtained at T3 and T4 (Figs. 2C-D).
For LIN and STR, there was only a statistical difference between treatments, with no difference between control and treatment groups, while for WOB there was a statistical difference in the control-treatment comparison and between treatments. For these three parameters, T5, T6, T7 and T8 presented the highest values, differing significantly (p<0.05) from the other treatments; it can also be shown that T4 was the treatment that provided the lowest values (Figs. 3A-C). An increase of LIN values was also observed for treatments T6, T7 and T8 in relation to the control. This increase was also found for STR in treatments T5, T6, T7 and T8, but only in treatment T7 for WOB. Regarding the amplitude of lateral displacement of the head (ALH), treatments T1, T2, T3 and T4 presented the highest values, not differing significantly from each other (p<0.05); in the other treatments (Fig. 3D), these values were also larger than those obtained in the control (1.14 μm). For the beat cross frequency (BCF), T1, T5, T6, T7 and T8 presented the highest values, without differing significantly from each other (p<0.05). It is important to highlight that these values were also higher than the value found in the control (7.0 Hz), where T4 was the group that provided the lowest value for BCF (Fig. 3E).
Semen post-thawing motility. After the thawing of A. lacustris semen, no mobile sperm were observed in any of the treatments tested.
DNA integrity. DNA integrity analysis showed that, in terms of possible DNA degradation, there was no difference between the control and T1, T3, T5 and T6 treatments, but the T5 treatment was responsible for presenting a lower DNA damage index (ID) (51 ± 34.5) (Fig. 4). The highest IDs were found in treatments T7 and T8, showing lower DNA protection compared to other treatments.
For the T4 treatment, compaction of the sperm nucleus was observed (Fig. 5). This compaction is possibly due to the chemical components of this treatment, which did not allow the exposure of nuclear DNA and, consequently, an evaluation of its damage.
Permeable cryoprotectants used in sperm freezing require low toxicity, small molecules, low molecular weight and high solubility (Chao, Liao, 2001) to reduce freezing points and cause cell dehydration, thus minimizing the cell formation of ice crystals. These substances are combined with non-permeable cryoprotectants which are large molecules that aid in cell dehydration, protect the plasma membrane and assist in osmotic homeostasis (Watson, 1995).
In the present study, the cryoprotectant toxicity test showed that the cryoprotectant solution composed of Me 2 SO10% + glucose 5% best met these characteristics, presenting  the highest values for total (MOT) and progressive (PRG) motility of spermatozoa of A. lacustris, as well as a greater amount of fast sperm. The higher PRG obtained with this extender is an important factor, since this parameter has a high correlation with the fertilization rate (Gallego, Asturiano, 2017).
The effectiveness of Me 2 SO is attributed to its low molecular weight and ability to easily bind to sperm membrane components, such as lipids and proteins, allowing molecular membrane reorganization after thawing (Pereira et al., 2019). Also, regarding total and progressive motility, the use of 5% BTS with methyl glycol, regardless of concentration, was toxic to A. lacustris sperm, causing mortality after dilution. Viveiros et al. (2009) found that the use of this cryoprotective solution caused a reduction in sperm motility in Salminus brasiliensis from 85% to 60% after dilution, and that a total loss of sperm motility was found after thawing.
However, despite this result, other studies with Characiformes such as Brycon nattereri Günther, 1864 (Oliveira et al., 2007), Brycon insignis Steindachner, 1877 (Viveiros et al., 2011), and Brycon orbignyanus (Viveiros et al., 2015) have shown that the use of BTS ® combined with methyl glycol was beneficial, with results superior or equal to the other solutions. In addition to these species, this freezing medium has also been tested for Steindachneridion scriptum (Miranda Ribeiro, 1918) siluriform (Pereira et al., 2019) and has also been positive, which further confirms the idea that cryoprotective solutions tend to be species-specific.
The toxicity caused by the cryoprotectant solution BTS ® + methyl glycol also shows that there is an interaction between permeable and impermeable cryoprotectants, since the use of BTS ® + Me 2 SO (T5 and T6) did not cause total loss of motility, and the use of methyl glycol + yolk + glucose (T4) resulted in the second highest total motility (61%) obtained in this study. For Brycon nattereri and Piaractus brachypomus (Cuvier, 1818), semen, an interaction between Me 2 SO and methyl glycol cryoprotectants with BTS ® and Saad, 154mM NaCl, 200mM NaCl and 277mM glucose dilutors was also observed (Oliveira et al., 2007;Nascimento et al., 2010).
The analysis of fast, medium and slow sperm revealed that there was a negative effect of cryoprotective solutions on these aspects, characterized by an increase in medium and slow sperm, possibly this increase occurred due to the sensitivity of sperm to cryoprotectants (Chao and Liao, 2001) or as an effect of energy loss by the cell, which has a significant effect on semen quality and motility after thawing (Bondarenko et al., 2013;Figueroa et al., 2013).
The velocity values (VCL, VSL and VAP) were higher for T7 (Me 2 SO10% + 5% glucose) and T8 (Me 2 SO15% + 5% glucose) sperm, which clearly indicates that these extenders provide higher sperm quality when compared with the other extenders used in this work. Sperm velocity is also an indicator of semen quality (Król et al., 2018), and these velocities are correlated with fertilization (Gallego et al., 2013). VCL is one of the parameters that is most closely related to fertilization capacity, as sperm with more 13/20 ni.bio.br | scielo.br/ni circular motions are theoretically easier to find the micropile (Leite et al., 2018).
In addition, a higher curvilinear velocity is also considered to be characteristic of spermatozoa with faster trajectories, while higher VSL means that the sperm trajectory is more linear and regular, with less lateral movements, which would indicate the sperm's ability to move in a straight line (Chiacchio et al., 2017). For some treatments, LIN, STR and WOB values were not affected by extender dilution, and an increase in these parameters after dilution has already been reported for other species after freezing, such as Acipenser baerii Brandt, 1869 and Acipenser ruthenus Linnaeus, 1758 (Sieczyński et al., 2015).
Unlike studies already performed with other fish species (Cejko et al., 2012(Cejko et al., , 2013) that associate higher ALH values with higher VCL values, our study showed an opposite behavior, in which higher ALH values were found in treatments with lower VCL. According to Cejko et al. (2012), high ALH and BCF values would indicate better sperm quality, since high ALH values indicate sperm maturity (Cejko et al., 2018;Król et al., 2018).
After semen thawing, no sperm motility was observed for any of the treatments. Among the factors considered for this result, we can point out the low percentage of average total sperm motility (76.6%) and the low amount of fast sperm (46.0%); the published studies for species of the same order have a 90-100% total motility for fresh semen (Lopes et al., 2014(Lopes et al., , 2018Chiacchio et al., 2017;Paula et al., 2019).
Animals were provided with a balanced feed and disposition in a controlled environment (pH, O 2 , TºC, Conductivity, ammonia) during the experiment, without sudden changes that could affect sperm quality. In addition, during collections, all recommended measures to maintain sperm quality were taken, such as prior sedation, avoiding contamination with urine, feces or blood, and avoiding the early activation of sperm. As there are no data in the scientific literature regarding seminal characteristics for the species or for specimens of the genus Astyanax, it did not allow a comparison to determine whether these motility characteristics are inherent of this genus or species.
In addition, the amount of fast sperm decreased significantly in the diluted semen, which probably contributed to the loss of motility after freezing and thawing, since this decrease can be attributed to the loss of cell energy due to damage to mitochondria, thus limiting movement sperm. Several studies have shown that sperm viability is directly affected by damage to mitochondria and consequent loss of cellular energy (Liu et al., 2007;Bondarenko et al., 2013;Figueroa et al., 2013;Zilli et al., 2014;Yusoff et al., 2018).
Other factors are mentioned in the literature as possible causes of loss of motility after freezing, such as the freezing rate used which is conditioned by the volume and height of the nitrogen straws and the amount of straws by freezing because the species-specific freezing rate and affects sperm survival after thawing (Chao, Liao, 2001;Ninhaus-Silveira et al., 2006b;Butts et al., 2010).
Although the thawed semen does not show motility, the DNA integrity test demonstrated that the use of these cryoprotective solutions were effective in maintaining the DNA integrity of the sperm cell. And the treatment composed of BTS and DMSO (T5) had the lowest ID (Damage Index) among all treatments, a fact also observed for treatments T1, T3 and T6 that also had low ID. For Steindachneridion scriptum, the use of this extender also resulted in low DNA fragmentation (0.051%). BTS ® + Me 2 SO 14/20 ni.bio.br | scielo.br/ni was also used in Prochilodus lineatus freezing but there was DNA fragmentation for all treatments analyzed in the study of Paula et al. (2019), unlike in our work, which demonstrates the influence of the species as well as the freezing method used, as the protocol applied in this study was different.
The use of a simple extender composed of glucose and ME 2 SO resulted in higher DNA fragmentation (T7 and T8), showing that this extender presented lower DNA protection efficiency. Nevertheless, the use of this extender for Salmo salar Linnaeus, 1758 was beneficial, generating low DNA fragmentation of only 4.8 ± 2.58% (Figueroa et al., 2016). DNA fragmentation due to the cryopreservation process using the same Me 2 SO cryoprotectant has been reported for several species such as Dicentrarchus labrax (Linnaeus, 1758) (Zilli et al., 2003), rainbow trout (Oncorhynchus mykiss (Walbaum, 1792)) (Cabrita et al., 2005) and Prochilodus magdalenae Steindachner, 1879(Martínez et al., 2012; however, the thinners used for these species differ from those used in our study. There is some controversy as to the effect of such DNA damage on fertilization success. It is known that the beginning of embryonic development is controlled only by inherited maternal information (Zilli et al., 2003). Thus, the effects of DNA fragmentation would not be manifested at this stage. In addition, DNA damage can be repaired by the genetic repair mechanism during early embryogenesis (Cabrita et al., 2005), without affecting offspring quality. This was found for rainbow trout where, although the cryopreservation process induces increased sperm DNA fragmentation, such damage did not affect the survival or quality of the offspring (Labbe et al., 2001).
However, some studies show that DNA damage affects both fertilization and offspring quality. Pérez-Cerezales et al. (2011) found that the use of cryopreserved trout sperm with damaged DNA for fertilization caused an increase in telomere length, as well as causing alterations in gene expression in surviving embryos and larvae, thereby negatively affecting offspring development. For goldfish semen, there was a relationship between fertilization and DNA damage, where treatment with higher DNA fragmentation was responsible for the lowest fertilization rate (Nathanailides et al., 2011).
DNA integrity analysis also showed that only the treatment consisting of 5% glucose + 10% egg yolk + 15% methyl glycol (T4) contained highly compacted nuclei, indicating that there was no cell lysis, which consequently prevented the analysis of this treatment for DNA damage caused by the cryopreservation process. This compaction is the result of a chemical reaction caused by the increased concentration of methyl glycol.
In conclusion, in the present study, the toxicity test showed that the use of 5% glucose and 10% Me 2 SO resulted in better maintenance of motility, progressivity and sperm speeds in A. lacustris semen. It is emphasized that there is a great sensitivity of the spermatozoa to methyl glycol combined with BTS and glucose. In addition, the cryoprotective solutions used did not lead to DNA fragmentation, thus maintaining the genetic integrity of A. lacustris sperm. However, A. lacustris sperm did not support the freezing protocol tested in this study, losing sperm viability and the ability to fertilize after thawing. Further studies are necessary to determine an effective protocol for cryopreservation the species semen.