Reagents and media
All the products used in this paper were of at least reagent grade and were acquired from Sigma (Madrid, Spain), unless otherwise stated. Fluorescence probes YO-PRO-1 and Hoechst33342 were purchased from Invitrogen (Barcelona, Spain). Stock solutions of the fluorescence probes were: PI: 7.5 mM; PNA-FITC: 0.2 mg/mL; YO-PRO-1: 50 μM. All fluorescent stocks were prepared in DMSO —except for PI, Hoechst 33,342 and PNA-FITC, which were prepared in water— and kept at −20 °C in the dark until needed, with the exception of the Hoechst, which was stored at 5 °C. Flow cytometry equipment, software and consumables were purchased from Beckman Coulter (Fullerton, CA, USA) or Becton Dickinson (San Jose, CA, USA). The medium for cytometry assessment was bovine gamete medium (BGM-3) composed of 87 mM NaCl, 3.1 mM KCl, 2 mM CaCl2, 0.4 mM MgCl2, 0.3 mM NaH2PO4, 40 mM HEPES, 21.6 mM sodium lactate, 1 mM sodium pyruvate, 50 μg/mL kanamycin, 10 μg/mL phenol red and 6 mg/mL BSA (pH 7.5).
For fixing samples, a 2% Glutaraldehyde solution was used, composed of BL-1 medium (glucose 14.6 mM, sodium citrate anhydrous 3.4 mM, sodium bicarbonate 2.4 mM.
Animals and sperm collection
Animal handling and electroejaculation were performed in accordance with Spanish Animal Protection Regulation RD53/2013, which conforms to European Union Regulation 2010/63/UE. All experiments were approved by the Ethical Committee for Experimentation with Animals of León University, Spain (03–02/2010).
Ejaculates from 6 sexually mature (≥ 6 years old) brown bear (Ursus arctos) males were obtained by electroejaculation, during the breeding season (end of April to early July). The animals were housed in a half-freedom regimen in Cabarceno Park (Cantabria, Spain; 43° 21′ N, 3° 50′ W; altitude: 143 m), and fed with a diet based on chicken meat, bread, fruits and vegetables.
The animals were immobilized by teleanaesthesia and the electroejaculation was carried out as described by [2]. Immediately after collection, the volume and concentration of each ejaculate were recorded. Sperm concentration, sperm motility kinematics, and urospermia content were assessed as previously described by [2]. Low motility (< 50%) or urine-contaminated (>80 mg urea/dL) samples were discarded. The selected ejaculates were centrifuged at 600×g for 6 min at room temperature. The supernatants were discarded and each pellet was resuspended with the same volume of TES-Tris-Fructose (TTF) extender at room temperature (dilution 1:1), and cooled at −0.25 °C/min to a final temperature of 5 °C. The extender was prepared using TTF (300 mOsm/kg, pH 7.1); 20% egg yolk, supplemented with 2% EDTA, 1% Equex (Equex STM Paste; Minitüb, Tiefenbach, Germany), 0.302 mg penicillin G sodium salt/mL and 0.625 mg dihydrostreptomycin sesquisulfate/mL. The glycerol concentration was adjusted to a final concentration of 6%.
Sperm cryopreservation
Sperm samples were diluted to a final concentration of 100 × 106 sperm/mL. After 2 h of equilibration at 5 °C, 0.25 ml straws were frozen in a programmable biofreezer (Kryo 560–16 Planer™, Planer plc, Sunbury-On-Thames, UK) at −20 °C/min up to −100 °C, transferred to liquid nitrogen containers and stored for a minimum of one year. Thawing was performed by dropping straws in water at 65 °C for 6 s.
Experimental design
Seven straws from each male were thawed and pooled to avoid differences in the starting concentration. An aliquot of 150 μL was used as an unselected control. Three proportions of Androcoll-Bear were assessed, at 80, 65 and 50%.
A further 6 aliquots of 150 μL were each layered on top of a column (1 mL of Androcol-Bear in a 1.5 mL Eppendorf® tube; 2 per each concentration (80, 65 and 50%)). The centrifugation was carried out with two different protocols; CM1: 300 x g/20 min and CM2: 600 x g/10 min. The resulting sperm pellets were harvested and resuspended in 50 μL of TES-Tris-Fructose. Selected samples and unselected samples were assessed, then incubated for 2 h at 37 °C and re-assessed. The number of spermatozoa was counted before and after single layer centrifugation (SLC) using a Nucleocounter®SP-100 (Chemometec, Allerod, Denmark) to determine the yield of the separated sperm population.
Computer-assisted sperm analysis
Total motility (%; TM), progressive motility (%; PM), curvilinear path velocity (μm/s; VCL) and linearity (%; LIN) were assessed using a CASA system (Computer Assisted Sperm Analysis), as described by [21].
Image sequences were saved and analysed afterwards using the editing facilities provided by ISAS (v. 12, Integrated Semen Analyser System; Poriser, Valencia, Spain). Sperm were considered motile when VCL > 10 μm/s and progressive if VCL >25 and straightness (STR) >80%. The progressive sperm subpopulations were classified according to velocities as follows: Slow (VCL <25), Medium (VCL >25 and <65), Rapid (VCL >65). Other events different from spermatozoa were removed, and settings were adjusted in each case to assure a correct track analysis.
Evaluation of sperm viability, apoptotic markers and acrosomal status
Several physiological traits (viability, apoptosis and acrosomal status) were assessed using fluorescent probes and flow cytometry, as described in a previous study Anel-Lopez et al. [21]. Spermatozoa stained in these two solutions were incubated for 10 min in the dark before analysis with a CyAn ADP flow cytometer (Beckman Coulter, Brea, CA, USA), equipped with semiconductor lasers emitting at 405 nm (violet; Hoechst 33,342) and 488 nm (blue; YO-PRO-1, PNA-FITC and PI). Filters used for each fluorochrome were 450/50 (blue) for Hoechst 33,342, 530/40 (green) for YO-PRO-1 and PNA-FITC and 613/20 (red) for PI.
The sperm subpopulations obtained go as follow; PI (+) (dead sperm), PI (−) (Viable sperm), PNA-FITC (+) (Acrosoma damaged), PNA-FITC (−) (Acrosoma non damaged), Yo-Pro1 (+) (early permeability changes in the membrane).
Morphometric assessment
The sperm samples (before and after Androcoll-Bear) were fixed in 2% glutaraldehyde and smeared on microscope slides, air dried and then stained using Diff-Quick (QCA, Tarragona, Spain). For staining, slides were immersed for 10 min in solution A, for 15 min in solution B and then rinsed using distilled water, air dried and mounted with Entellan (Sigma-Aldrich). Samples were examined using a Nikon Eclipse E600 microscope (Nikon, Tokyo, Japan) equipped with a ×60 bright field objective (×500). An average of 200 cells in each sample were photographed using a Nikon DF1200 digital camera (Nikon, Tokyo, Japan) and the pictures were processed using the NIS Elements v.3 image analysis system. The morphometric analysis was carried out using a semi-automatic macro that allowed the operator to discard the sperm heads in the image that did not meet the technical requirements for the study (e.g. overlapping cells and the presence of staining artefacts). For each cell, the following parameters were calculated: area (A; μm2), length (L; μm), perimeter (P; μm), width (W; μm) and elongation (L - W)/(L + W).
Statistical analysis
Data were analysed using the SAS™ V.9.1. Package (SAS Institute Inc., Cary, NC, USA). Results are shown as means and standard errors of the mean. Data were tested for normality (Shapiro-Wilk test) and arcsin square-root transformation was used to normalise the data before analysis when necessary. Analyses of the data were carried out using linear mixed-effects models (MIXED procedure, ML method), including proportion of Androcoll-B (80, 65 and 50%), centrifugation protocols (V1 and V2) and incubation time after thawing (0 vs. 2 h) as fixed factors, and males as random effect. Significant fixed effects were further analysed using multiple comparisons of means with Tukey contrasts. A significance level of P < 0.05 was used.
