This study was performed in accordance with valid legislative regulations in Czech Republic (law No. 166/1996 and no. 246/1992); the permit was issued to No. 2293/2015-MZE-17214 and No. 55187/2016-MZE-17214 in project NAZV QK1710310. All the samplings were carried out with the relevant permission from the Departmental Expert Committee for Authorization of Experimental Projects of the Ministry of Education, Youth and Sports of the Czech Republic (permit No. MSMT 4394/2017-2).

Semen was collected during the natural spawning season (February–April) from six apparently healthy and mature sterlet males (6–7 years, 2–3 kg). The fish were kept in 4 m3 plastic tanks with pond water at a constant flow of 20 l/min (8–10°C) in the hatchery of the Research Institute of Fish Culture and Hydrobiology, Vodnany, Czech Republic. Prior to the experiment, the water temperature was gradually increased to 14–15°C over the course of 24 h. The fish were kept at the above temperature for another 4 days until the beginning of the experiment. Spermiation was induced by intramuscular injection of carp pituitary powder dissolved in 0.9% NaCl solution at a dose of 4 mg/kg body weight. After 24 h, semen was collected at the uro-genital sinus by aspiration with a 4 mm plastic catheter attached to a plastic syringe. The samples were stored on ice before and during the experiments.

Six freshly collected sperm samples with motility greater than 90% (sperm concentration: 0.55–0.75 × 10⁹ spermatozoa mL^–1) were diluted separately (1:1) in an extender composed of 23.4 mM sucrose, 0.25 mM KCl, 30 mM Tris-HCl, pH 8.0, 10% methanol (Glogowski et al., 2002; modified by Xin et al., 2018a,b) with/without 10 μg/mL AFPI (optimized protocol according to Xin et al., 2018a). The diluted sperm from each male were immediately loaded into 0.5 mL plastic straws (CRYO-VET, France) and placed on a 3 cm thick polystyrene raft on ice for 10 min of equilibration. The polystyrene raft containing the straws was then transferred and frozen in liquid nitrogen vapor in a styrofoam box. After 10 min, the straws were directly immersed in liquid nitrogen and stored in plastic goblets. Finally, the samples were thawed in a water bath at 40°C for 6 s and then immediately used for further assessments and sample processing.

Fresh, cryopreserved, and cryopreserved sperm with an addition of 10 μg/mL AFPI (A/F Protein Inc., Waltham, MA, United States) (cryopreserved + AFP) were activated in an activation medium (AM) consisting of 10 mmol/L Tris-HCl buffer, pH 8.0, 2 mmol/L CaCl2, and 0.125% pluronic acid. For this purpose, the sperm to AM ratio was maintained at approximately 1:50. The sperm was mixed with AM on a 24 × 32 mm microscopic glass slide (ISO 8037/I, Thermo Fisher Scientific, Waltham, MA, United States) at room temperature (21°C). Spermatozoa motility parameters were recorded on the top and bottom of the AM using a CCD video camera (Sony, SSCDC50AP, Tokyo, Japan) operating in 25 frame per second (fps) mode, mounted on an inverted microscope (Olympus IX83, United Kingdom) with a 20 × NIC contrast objective lens (Dadras et al., 2019a). Analysis of recorded videos for curvilinear velocity (VCL; time-averaged velocity of a sperm head along its actual curvilinear path, μm/s), linearity [LIN; defined as straight-line velocity (VSL)/curvilinear velocity (VCL);%] and total motility rate (%) were performed for approximately 100–150 spermatozoa per microscopic field in triplicate (three fields/sample) using ISAS software (PROISER, Paterna, Spain) 15 s after activation. Spermatozoa concentration was determined according to Xin et al. (2018b).

Flow cytometry was used to investigate the integrity of the plasma membrane of fresh and cryopreserved spermatozoa with/without AFPI. 10,000 cells were analyzed for each sample by flow cytometry. Two fluorescent dyes were used to distinguish spermatozoa with intact and impaired membrane integrity. SYBR Green binds to spermatozoa (live and dead), but only dead spermatozoa were labeled with propidium iodide (PI). A total of 2 μL of fresh or cryopreserved sperm samples with/without AFPI were prediluted in 2 mL of phosphate-buffered saline (PBS) (PH 7.4). Then 5 μL of 1:500 diluted SYBR Green (Sigma-Aldrich, St. Louis, MO, United States) was added to the diluted sperm samples. After 5 min, 5 μL of propidium iodide (PI) (Sigma-Aldrich, St. Louis, MO, United States) was added to the sperm diluted with PBS and SYBR Green. After incubating the sperm samples with both fluorescent dyes for 30 min at room temperature, flow cytometry was performed using a fluorescence-activated cell sorter (FACS) Canto II flow cytometer (BD Biosciences). FACS Diva software, v. 5.0 (BD Biosciences) was then used for data analysis. The number of SYBR Green positive events in the FITC channel and the number PI positive events in the PE -Texas Red channel were used to determine dead sperm. Intact plasma membranes (live spermatozoa) were determined as SYBR Green positive and PI negative events. Each analysis was performed in triplicate.

Sperm samples (fresh; sperm concentration: 0.55–0.75 × 10⁹ spermatozoa mL^–1 and cryopreserved; sperm concentration: 0.27–0.37 × 10⁹ spermatozoa mL^–1) sperm with/without AFPI] were centrifuged at 500 × g at 4°C for 20 min to obtain seminal fluid (SF) and a spermatozoa pellet. The pellet was suspended in 50 mM potassium phosphate (KPi) buffer, pH 7.0, containing 0.5 mM EDTA and homogenized in an ice bath using a Sonopuls HD 2070 ultrasonicator (Bandelin Electronic, Berlin, Germany). The homogenate pellet was centrifuged at 10,000 × g at 4°C for 10 min to obtain supernatant. The obtained supernatant (containing spermatozoa) and SF were used to measure the thiobarbituric acid reactive substances (TBARS) and the activities of superoxide dismutase (SOD) and catalase (CAT). The protein concentration of the samples was measured using a BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA, United States).

The CAT activity was determined spectrophotometrically at 240 nm according to the method described by Marklund et al. (1981). The reaction medium consisted of 10 mM K + phosphate buffer containing 0.1 mM EDTA, pH 7.4, and 15 mM H₂O₂ was used. The activity of CAT was calculated from the H₂O₂ decomposition rate using the molar extinction coefficient 43.6 M^–1 cm^–1, and the specific activity was expressed as micromole/min × mg protein.

The activity of SOD was determined spectrophotometrically at 420 nm as described by Marklund and Marklund (1974). The activity of SOD was measured by the inhibition of autoxidation of pyrogallol by the sample. The addition of the analyzed sample led to the inhibition of autoxidation of 0.2 mM pyrogallol in air-equilibrated 50 mM Tris-HCl buffer containing 1 mM EDTA, pH 8.2. The amount of enzyme inhibiting the reaction (pyrogallol autoxidation) by 50% is considered as one unit of enzyme. The specific activity of SOD was expressed as unit/mg protein.

The TBARS level was determined spectrophotometrically (Asakawa and Matsushita, 1980): briefly, 0.025 mL of a solution of butylated hydroxytoluene (22 mg in 10 mL ethanol), 0.025 mL of a ferric chloride solution (27 mg FeCl₃ 6H₂O in 10 mL water), 0.375 mL of a 0.2 M glycine-hydrochloric acid buffer, pH 3.6, and 0.375 mL of TBA reagent (0.5% TBA and 0.3% sodium dodecyl sulfate) were added to 0.4 mL of the supernatant and heated in a boiling water bath for 15 min. The samples were allowed to cool, then 0.25 mL of glacial acetic acid and 0.5 mL of chloroform were added. After vigorous shaking, the samples were centrifuged at 1,500 × g for 10 min. The absorbance was measured at 535 nm. TBARS content was determined using a molar extinction coefficient of 1.56 105 M^–1 cm^–1 and expressed as nmol/mg protein sample. All measurements were performed in triplicate for each sample.

DNA integrity of fresh (sperm concentration: 0.57–0.70 × 10⁹ spermatozoa mL^–1) and cryopreserved sperm with/without AFPI was assessed using the comet assay. Sperm samples from each male were diluted 1:300 with pre-chilled PBS and stored at 4°C. Agarose gel (0.6% NuSieve GTG Agarose OxiSelect™ with low melting point; Cell Biolabs, Inc., San Diego, United States) was melted at 90°C and cooled to 37–40°C. The agarose gel was mixed with diluted sperm at a ratio of 9:1. Then, 50 μL of the prepared sperm samples were pipetted into each well of the slides (OxiSelect™; Cell Biolabs, INC., United States) and spread over the well. The slides were allowed to solidify at 4°C and then immersed in lysis buffer prepared according to the protocol for the OxiSelect Comet assay with the addition of proteinase K (1 mg/mL of lysis solution). The slides were incubated in the lysis buffer for 5 h at 37°C. After incubation, the lysis buffer was carefully aspirated and replaced with electrophoresis solution (90 mM Tris Base, 90 mM boric acid, 2.5 mM EDTA, pH 8.0). After incubation in the electrophoresis solution for 20 min at room temperature, the slides were carefully transferred to the electrophoresis chamber filled with the electrophoresis solution. Electrophoresis was performed at 35 V and 170 mA for 15 min. The slides were then transferred to a small container of distilled water for 5 min. The slides were then drained well, dehydrated by immersion in 70% ethanol for 5 min, and air-dried. Once the agarose slides were completely dried, they were stained with DAPI (50 μL, 1 μg/mL). The slides were observed under an Olympus Fluoview microscope (Olympus Corp., Tokyo, Ja-pan) using filters with an excitation wavelength of 450–480 nm and recorded with a SONY DXC -9100D video camera (SONY, Tokyo, Japan). Spermatozoa DNA damage (Tail DNA, %) was assessed in one hundred cells for each sample using CASP software (version 1.2.3., 2001).

For scanning electron microscopic (SEM) observations, sperm samples (fresh and cryopreserved sperm with/without AFPI) were fixed in freshly prepared 2.5% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4) at 0–4°C for 3 h, rinsed in the same buffer, and immersed for 2 h in 1.0% osmium tetroxide in 0.1 M phosphate buffer (pH 7.4) at room temperature for 2 h. After rinsing in the buffer, samples were dehydrated in a graded acetone series (30, 50, 70, 80, and 100% for 10 min each). Then, the samples were dried using a critical point dryer (XD -1, Eiko) and coated with gold using a coater (IB -3, Eiko). Finally, the samples were examined and photographed using a scanning electron microscope (Hitachi-S570, Tokyo, Japan and JSM 6300, JEOL Ltd., Akishima, Tokyo, Japan).

For transmission electron microscopy (TEM), the sperm samples were centrifuged at 200 × g for 30 min, and the supernatant was discarded. The samples were fixed with the same solutions used for SEM. Then the samples were dehydrated in a graded acetone series and embedded in Epon812. The solidified blocks were sectioned at 50–70 nm and the sections mounted on copper grids. Post-staining with 2% uranyl acetate in 50% ethanol was performed, followed by staining with Reynolds’ lead citrate (Golpour et al., 2016a,b; Dadras et al., 2019b). The prepared grids were examined and photographed by transmission electron microscopy (JEM -1230, Tokyo, Japan and JEOL 1010, JEOL Ltd., Tokyo, Japan). For TEM, specimens were processed separately for each treatment and then approximately 400 electron micrographs were taken for each treatment. For SEM, about 100 electron micrographs were taken. The electron micrographs were examined for changes in the structure of the spermatozoa. Finally, the best images showing normal structure in the control treatment or altered spermatozoa structure in both treatments (with/without addition of AFP1) were selected.

For quantitative analysis, thirty TEM micrographs of each group studied were evaluated to determine the morphological abnormalities. Quantification of the abnormalities in the micrographs was performed using ImageJ v1 software (Wayne Rasband, United States). The analyses were performed in triplicate.

Experimental values for each studied parameter were obtained by averaging the results of six male fish with three replicates for each male. The mean values of the males were calculated and used for statistical analyses. The data distribution characteristics and homogeneity of dispersion were evaluated using Shapiro-Wilk and Levene’s tests, respectively. The normally distributed data with similar dispersion values were analyzed using one-way ANOVA followed by Tukey’s honest significant difference test. Non-homogeneous data (Tail DNA) were analyzed using a non-parametric Kruskal-Wallis ANOVA followed by multiple comparison of mean ranks for all groups. Statistical significance was accepted at p < 0.05. Data are presented as box plots and bar graphs (mean ± SD). All analyses and plots were performed using STATISTICA v. 12 (Statsoft Inc.)¹ and GraphPad Prism v. 8.

After dilution with AM, the fresh spermatozoa exhibited 93.3 ± 5%, 182.2 ± 17.8 μm/s and 62 ± 8% for motility rate, VCL and LIN, respectively (Figures 1A–C). A significant decrease in motility rate was observed in cryopreserved spermatozoa (with 10 μg/mL AFPI: 80 ± 6%; without 10 μg/mL AFPI: 78.3 ± 7%) compared with fresh spermatozoa (Figure 1A). The curvilinear velocity (VCL) showed a significant difference between fresh spermatozoa (182.2 ± 17.8 μm/s) and cryopreserved spermatozoa with/without 10 μg/mL AFPI (with AFPI: 141.5 ± 11.4 μm/s; without AFPI: 155.8 ± 8.3 μm/s) (Figure 1B). No significant differences were observed in LIN between fresh and cryopreserved spermatozoa with/without the addition of the antifreeze protein (Figure 1C).

The integrity of the plasma membrane of fresh spermatozoa was 96 ± 2.2%. The spermatozoa with intact plasma membranes were significantly decreased in cryopreserved sperm with (70.4 ± 2.4%) and without AFPI (70.3 ± 3.6%) (Figure 2). No significant difference in spermatozoa membrane integrity was observed between cryopreserved sperm with and without the addition of AFPI (Figure 2).

The protein concentrations of seminal fluid and spermatozoa were in the range of 0.17–0.8 and 0.58–1.71 mg/mL, respectively. The CAT and SOD activities in the spermatozoa and seminal fluid of each group are shown in Figures 3A,B, respectively. No significant difference was observed among the studied groups with regard to the enzymatic antioxidant activities (SOD and CAT). The TBARS value also showed no difference among the groups (Figure 3C).

An illustration of the assay used to assess DNA damage for all groups is shown in Figures 4A–C. The evaluation of tail DNA%, which is representative of DNA damage, showed that the level of DNA fragmentation was significantly higher in cryopreserved spermatozoa with (4.8%) and without (4.9%) AFPI than in fresh spermatozoa (2.2%) (Figure 5). The cryopreserved spermatozoa supplemented with 10 μg/mL AFPI showed no difference in tail DNA% compared to those without AFPI (Figure 5).

Sterlet spermatozoa consist of an elongated head with an acrosome, a cylindrical midpiece, and a flagellar region surrounded by the plasma membrane (Figures 6A, 7A, 8A, 9A). The SEM micrographs show no damage to the fresh spermatozoa, while morphological damages such as swelling of midpiece and detachment of the plasma membrane of flagellum were clearly observed in cryopreserved spermatozoa with/without AFP (Figures 6A–C).

Most of the visualized fresh spermatozoa showed normal morphology and ultrastructure (Figure 7A). The homogeneous nucleus was surrounded by the nuclear membrane (Figure 7B), and the mitochondria were irregularly dispersed in the cytoplasm with smooth matrix structures (Figures 7C,D). The flagellum was separated by the cytoplasmic channel as in other sturgeon spermatozoa (Figure 7E). The ultrastructure of the spermatozoa was partially affected by the standard cryopreservation procedure with/without AFPI. In both cases, visible changes were observed in the structure of the spermatozoa (Figures 8B–D, 9B–E). Partial damage to the structure of the midpiece and flagellum was visible (Figures 8C,D, 9D,E). Abnormalities of varying severity were observed, such as changes in the structure of the midpiece and detachment of the plasma membrane in the flagellum (Figures 8B,C, 9C,D). In addition, changes in the position of the mitochondrion and, in some cases, the absence of this organelle were observed (Figure 9D). Moreover, partial rupture of the plasma membrane in the flagellum of spermatozoa was observed after the standard cryopreservation procedure with/without AFPI (Figures 8D, 9E).

Partial morphological abnormalities of the mitochondria were observed in a small number of fresh spermatozoa (15 ± 15.07%) (Figure 10A). Significant mitochondrial damage (p < 0.05) was observed in cryopreserved spermatozoa with (49.16 ± 18%) and without AFPI (54.28 ± 15.11%) (Figure 10A). Moreover, the percentage of fresh spermatozoa with morphological changes in the flagellum was 9.16 ± 9.96% (Figure 10B). However, increased morphological abnormalities of the flagellum were observed in cryopreserved spermatozoa with (55 ± 13.22%) and without AFP (57.14 ± 12.53%) (Figure 10B).