Chemicals and media
All reagents and extender components were purchased from Sigma- Aldrich (St. Louis, MO, USA).
Spermatozoa collection and processing
The material used in this study was obtained and stored pursuant to the permit no. WPN.6401.170.2019.MH issued by the Regional Director for Environmental Protection in Wrocław.
In this study spermatozoa from 4 wisents (Bison bonasus, Linnaeus 1758) aged 4–8 years was collected from February to December 2019. Bulls were culled during planned eliminations due to injuries and reproductive disorders under the conditions regulated by Polish law (The Nature Conservation Act 2004). Immediately after animal death, testicles were removed from the scrotum and spermatozoa were obtained by performing multiple incisions in the epididymal tail and immersing the tissue in 4 ml of a sperm-optimized Tris-based extender (Tris (2.4 g), citric acid (1.4 g), glucose (0.8 g), penicillin (5000 IU) streptomycin (100 mg) and distilled water up to 100 ml) (33 °C) placed on glass Petri dishes. Petri dishes were placed on the warming platform for 10 minutes. After incubation, the samples were analysed.
Initial spermatozoa assessment
The male gametes obtained from each of eight epididymis were processed separately and treated as independent samples in the analyses.
Concentration per unit volume (106 cells/ ml) and motility was assessed using the phase contrast microscope (Nicon Eclipse E200) with warming stage.
To assess the percentage of motile spermatozoa ten microlitres of sample were placed on the slide and covered with a cover slip (× 400).
Concentration in sperm per million (× 106) was counted using Thoma chamber (× 400).
For further morphological assessment, smears from 10 μL of sperm-rich fluid were made and stained with Bydgoska method [37]. To assess the percentage of live and dead spermatozoa, smears from 10 μL of sample and 10 μL of eosin-nigrosin dye were prepared. The following morphological defects where evaluated: proximal droplet, head abnormalities, acrosome abnormalities, midpiece defects, dag-like defect, distal droplet, bent tail, detached head, coiled tail. As a morphological normal sperm were described spermatozoa which did not show these particular defects.
Preparation of semen
After the initial assessment (concentration and subjective motility) each sample was divided into two parts. One part was diluted with freezing extender I (Tris (2.4 g), citric acid (1.4 g), glucose (0.8 g), egg yolk (20% v/v), penicillin (5000 IU) streptomycin (100 mg) and distilled water up to 100 ml) [38] to obtain the concentration 200 × 106/ml.
The second aliquot was subjected to Percoll® density gradient centrifugation.
Percoll® density gradient centrifugation
Percoll® solutions (45 and 90%) were prepared as described by Lee et al. (2009) [6] with some modification. Percoll® solution (90%) was prepared by mixing Stock Isotonic Percoll® Solution (SIP) with Human Tubal Fluid (HTF) in a 9:1 proportion. Percoll® solution (45%) was prepared by mixing 90% solution with HTF in equal volume. Subsequently, a 2 ml sample was gently laid on 2 ml 45% Percoll® layer and 2 ml 90% Percoll® layer as presented in Fig. 6. Samples were centrifuged at 800×g for 35 min in a horizontal centrifuge. After removing the supernatant, the pellets were resuspended in 1 ml of HTF and then centrifuged for 5 min at 800×g. After centrifugation, the samples concentrations were diluted with freezing extender I to obtain the concentration 200 × 106 cells/ml.
Cryopreservation and thawing
The same cryopreservation procedure was used for both group of samples.
After dilution in freezing extender I at 22 °C the samples were kept in a water bath and then placed into a refrigerator and cooled down to 5 °C. Chilled freezing extender II (freezing extender I plus 6% glycerol) was added to obtain final concentration 160 × 106 cells/ml. Diluted spermatozoa were left for further equilibration for 90 min. Subsequently, samples were loaded into 0.25 ml straws (4.0 × 106 spermatozoa per straw). Each free end of the straw was closed with polyvinyl alcohol. Filled straws were frozen in liquid nitrogen vapours (15 min) and then immersed in liquid nitrogen and stored in tanks [38]. From a few weeks to 1 year from the collection day, two straws (one from Percoll® and one from control group) from each epididymis were thawed. The straws were placed in a water bath (37 °C) for 30 sec [38] and subjected to further analysis. To assess the percentage of spermatozoa with morphological defects and the percentage of live and dead gametes, smears were prepared using the same method as described above.
Assessment of thawed sperm movement parameters with CASA
Motility (MOT, %) and progressive motility (PMOT, %), parameters characterizing sperm movement: VCL (μm/s), VAP (μm/s), VSL (μm/s), LIN (%), STR (%), amplitude of lateral head displacement (ALH) μm), BCF (Hz), Rapid, Medium, Slow and static sperm subpopulations were assessed by using HTM IVOS version 12.2 (Hamilton-Thorne Biosciences Beverly, MA, USA).
The used CASA setups were as for bull spermatozoa: frame rate (60 Hz), frames acquired (30), minimum contrast (80), minimum cell size (5 pixels), low VAP cut-off (30 μm/sec) and low VSL cut-off (15 μm/sec) [38].
Assessment of the function and structure of spermatozoa by flow cytometry
Spermatozoa were assessed by flow cytometry before and after freezing-thawing process. Due to the long travel distance from the place where the material was obtained to the laboratory where the analyses were performed, the initial pre-freezing assessment was conducted 24 hr. after collection.
Wisent spermatozoa functionality was evaluated using Guava EasyCyte 5 (Merck KGaA, Darmstadt, Germany) cytometer. The fluorescent probes used in the experiment were excited by an argon ion 488 nm laser. Gametes acquisitions were analysed with the GuavaSoft™ 3.1.1 software (Merck KGaA, Darmstadt, Germany). The non-sperm events were gated out based on scatter properties and not analysed. A total of 10,000 events were analysed for each sample. Membrane integrity, acrosome integrity, mitochondrial activity, lipid peroxidation, apoptosis and membrane lipid disorders and chromatin status were assessed [39, 40].
Membrane integrity of wisent spermatozoa was assessed using SYBR-14 stain combined with propidium iodide (PI) (Life Technologies Ltd., Grand Island, NY, USA). 300 μL of sperm- rich fluid was incubated in the dark for 10 min with 5 μL of SYBR-14 working solution (0.1 μL SYBR14 + 4.9 μL TRIS III extender). The analysis was performed after 3 min of incubation with 3 μL of PI. Spermatozoa with intact membranes emit green fluorescence. Cells showing red fluorescence were classified as dead [41].
Acrosome integrity was assessed by lectin PNA stain from Arachis hypogaea Alexa Fluor® 488 conjugate (Life Technologies Ltd., Grand Island, NY, USA). Diluted samples were mixed with 10 μL of PNA working solution (1 μg/mL) and incubated for 5 min at room temperature in the dark. Before analysis, the samples were washed and 3 μL of PI was added [42].
Mitochondrial activity was determined using the JC-1 dye (Life Technologies Ltd., Grand Island, NY, USA). 500 μL aliquot of spermatozoa-rich fluid was stained with 0.67 μL JC-1 stock solution (3 mM stock solution of JC-1 in DMSO). The samples were incubated for 20 min at 37 °C in the dark. Spermatozoa emitting orange fluorescence were classified as having high mitochondrial membrane potential (HMMP). Spermatozoa emitting green fluorescence were defined as those with low mitochondrial activity [43].
Lipid peroxidation was evaluated with fluorescent lipid probe C11-BODIPY581/591 (Life Technologies Ltd., Grand Island, NY, USA). One μL of 2 mM C11-BODIPY581/591 in ethanol was added to the diluted sperm- rich fluid and incubated for 30 min at 37 °C in the dark. Subsequently, centrifugation at 500×g for 3 min was performed and the sperm pellets were resuspended in 500 μL of HTF extender. To determine viability, the spermatozoa were stained with PI and incubated for 5 min at room temperature. Spermatozoa which remain unstained were categorised as living population without LPO (L/LPO-) [44].
Apoptosis and membrane lipid disorder were evaluated with YO-PRO-1 dye (25 μM solution in DMSO) (Life Technologies Ltd., Grand Island, NY, USA) (4). 1 μL of YO-PRO-1 stain (final concentration: 25 nM) was added to 1 mL of diluted spermatozoa- rich fluid (500 μL HTF and 500 μL of spermatozoa solution). After incubation for 10 minutes, 3 μL of PI was added before cytometric analysis. Cells showing green fluorescence were classified as YO-PRO-1 positive. Spermatozoa which remain unstained were categorised as living population [44].
Chromatin status was established using the acridine orange dye (AO, Life Technologies Ltd., Grand Island, NY, USA. A spermatozoa-rich solution (100 μL) was subjected to brief acid denaturation by adding 200 μL of the lysis solution (Triton X-100 0.1% (v/v), NaCl 0.15 M, HCl 0.08 M, pH 1.4). After 30 seconds, 600 μL of AO solution (6 μg AO/mL in a buffer: citric acid 0.1 M, Na2HPO4 0.2 M, EDTA 1 mM, NaCl 0.15 M, pH 6) was added. The analysis was performed after 3 minutes of incubation. Spermatozoa with normal DNA configuration were characterised by green fluorescence. Gametes emitting red fluorescence were considered as a population of cells with denatured DNA (DFI) [45].
Statistical analysis
Statistical analyses were performed by using STATISTICA 13.3 StaSoft (USA). The results of quantitative data are presented as the mean and standard error. Shapiro-Wilk’s test was used to assess data normality. Where appropriate parametric tests (Student’s t-test and ANOVA) or nonparametric tests (Mann-Whitney U and Kruskal–Wallis) were used to evaluate differences between the groups. Differences were considered significant at p ≤ 0.05.
