Abstract

   The conservation of small and genetically vulnerable brown bear
   populations, such as the Cantabrian subpopulation in Spain, depends on
   developing species-specific assisted reproductive technologies and
   genetic resource banks. However, the lack of standardized sperm
   collection and cryopreservation protocols hinders their application.
   This study provides the first comparative analysis of sperm quality and
   proteomic profiles from three different origins: epididymal,
   pre-ejaculated, and ejaculated. Sperm quality parameters —motility and
   kinetic, viability, apoptosis, and oxidative stress— and protein
   expression were assessed. Although yields were similar, marked
   differences were observed in sperm quality and protein profiles.
   Sixty-three proteins involved in metabolism, stress response, and
   oxidative balance were differentially expressed depending on sperm
   origin. Epididymal sperm showed the highest viability and motility,
   lowest apoptosis, and a proteomic profile indicative of active
   spermatogenesis and enhanced oxidative stress defense. In contrast,
   ejaculated sperm had increased oxidative stress and reduced expression
   of metabolic proteins, while pre-ejaculated sperm exhibited lower
   motility, likely due to urine contamination and mitochondrial protein
   alterations, despite comparable viability and apoptosis. These findings
   offer novel insights into brown bear sperm biology and highlight the
   importance of sperm origin in developing optimized assisted
   reproduction strategies, ultimately supporting ex situ conservation
   efforts for this species.

1. Introduction

   The brown bear (Ursus arctos) is classified worldwide as a
   least-concern species by the International Union for Conservation of
   Nature (IUCN) Red List of Threatened Species, as its global population
   remains stable and is even increasing in some areas. The largest
   populations are in Russia, the United States (mainly Alaska), and
   Canada [[46]1]. However, in Central and Southern Europe, brown bear
   populations are small, fragmented, and highly vulnerable [[47]2].
   Consequently, the European Union lists brown bears as endangered
   (Council Directive 92/43/EEC, annexes II and IV), and Spanish law
   considers them at risk of extinction (Real Decreto 139/2011).
   Specifically, the Cantabrian brown bear is the only autochthonous bear
   population in the Iberian Peninsula, and might represent the last
   genetically pure lineage of the brown bear in the world [[48]3].
   According to the Fundación Oso Asturias, this population is currently
   estimated to be around 350 individuals, divided into two nuclei
   (eastern and western) in the northern Spanish mountains [[49]4].
   Although conservation measures have contributed to a recent population
   increase, the situation of the species remains critical due to the
   small size of both subpopulations and the high risk of inbreeding and
   genetic variability loss [[50]5].

   Genetic resource banks play a crucial role in preserving genetic
   diversity by enabling the long-term storage of gametes and embryos for
   potential use in recovery programs using assisted reproductive
   techniques [[51]3]. Nevertheless, the development of these banks is
   hindered by the limited knowledge of optimal protocols for sperm and
   oocyte collection, assessment, preservation, and application [[52]6].
   Traditionally, these procedures have been performed based on previous
   experience in domestic species, but the wide variety of methods and the
   different results obtained when applied to other species highlight the
   need for species-specific protocols [[53]7].

   Various methods have been documented for sperm collection in wild
   animals, grouped into three main types: (1) repeatable techniques in
   trained or conditioned animals, which include the use of artificial
   vaginas and phantoms [[54]8,[55]9,[56]10,[57]11,[58]12], induced
   spontaneous ejaculation [[59]13], and the manual stimulation of the
   penis or accessory sex glands [[60]14,[61]15,[62]16,[63]17]; (2)
   repeatable methods in anesthetized or sedated animals, involving
   electroejaculation [[64]18,[65]19,[66]20] or chemically induced
   ejaculation [[67]21,[68]22]; and (3) non-repeatable methods, such as
   sperm collection from the epididymis or vas deferens after castration,
   sterilization, or death [[69]23,[70]24,[71]25]. In bears,
   electroejaculation under anesthesia has been the sperm extraction
   method of choice over the years in several species, like the giant
   panda (Ailuropoda melanoleuca) [[72]26,[73]27], the Asiatic black bear
   (Ursus thibetanus) [[74]28,[75]29], the American black bear (Ursus
   americanus) [[76]30], the Malayan sun bear (Helarctos malayanus)
   [[77]31], the brown bear (Ursus arctos) [[78]6,[79]32,[80]33], and the
   polar bear (Ursus maritimus) [[81]34]. However, this technique presents
   some issues that should be considered, including expensive equipment,
   sudden arousal during anesthesia (with safety hazards), and the
   potential for urine contamination in the collected samples
   [[82]35,[83]36,[84]37,[85]38]. Moreover, the electroejaculation
   response can vary between species or males from the same species, and
   even between collections from the same male [[86]39]. To overcome these
   shortcomings, urethral catheterization has been explored as an
   alternative method for sperm collection in polar bears (Ursus
   maritimus) [[87]40] and Asiatic black bears (Ursus thibetanus)
   [[88]38]. This technique utilizes medetomidine, a potent α2-adrenergic
   agonist that induces the smooth muscle contraction of the vas deferens,
   facilitating semen release into the urethra [[89]41], but the quality
   of the sperm obtained via this method in bears remains poorly
   characterized. Additionally, as wild animals, valuable bears can die
   unexpectedly, making the collection of epididymal sperm the only way to
   preserve their gametes [[90]42]. Despite its importance, little
   information exists on bear epididymal sperm cryopreservation [[91]43].

   Thus, this study aimed to describe and compare the quality and
   proteomic profiles of brown bear (Ursus arctos) sperm from three
   different origins (epididymal, pre-ejaculated, and ejaculated) as a
   first step in developing adapted cryopreservation protocols that would
   help to create biological resource banks for this species.

2. Materials and Methods

2.1. Reagents and Media

   All chemicals used in this study were acquired from Sigma-Aldrich
   (Saint Louis, MI, USA), except the fluorescence probes Zombie Violet™
   Fixable Viability Kit and CellEvent™ Caspase-3/7 Green Detection
   Reagent, which were purchased from BioLegend (San Diego, CA, USA) and
   ThermoFisher (Waltham, MA, USA), respectively.

2.2. Animals

   A total of 14 sexually mature male brown bears were the subjects of the
   experiments during the breeding season (end of April to early July)
   from 2022 to 2024. Animals were housed in a half-freedom regime in
   Cabárceno Park (Cantabria, Spain; 43° 21′ N, 3° 50′ W; altitude 143 m)
   and fed a diet of chicken meat, bread, and fruits. Pre-ejaculated and
   ejaculated samples were obtained in one session, while epididymal sperm
   was collected from the same individuals in another session as part of a
   population control program in the park.

2.3. Sperm Collection

   Males were immobilized via teleanesthesia with 750 mg of zolazepam HCl
   + tiletamine HCl (Zoletil 100^®; Virbac, Carros, France) and 6 mg of
   medetomidine (Zalopin^®; Orion Pharma Animal Health, Espoo, Finland).
   Animals were weighed and monitored during anesthesia (pulse, peripheral
   oxygen saturation, and breathing).

2.3.1. Epidydimal Sperm

   The testicular region of 12 brown bears was shaved, washed, and
   disinfected, and the males were castrated or epididectomized.
   Epididymides were dissected and cleaned of connective tissue and
   superficial blood vessels to avoid contamination. Sperm were collected
   through several incisions on the cauda epididymis with a surgical
   blade, and the emerging fluid was deposited in 0.5 mL graduated
   microtubes (n = 12). Sperm yield and functionality evaluations were
   performed at this moment.

2.3.2. Pre-Ejaculated and Ejaculated Sperm

   The pubic region of 14 brown bears was cleaned, the penis was washed
   with sterile physiological saline, and the rectum was emptied of feces.
   The bladder was catheterized to prevent the urine contamination of
   ejaculated samples, and it was possible to obtain a pre-ejaculate sperm
   sample from the urinary catheter in some bears (n = 12). After this,
   electroejaculation was performed with a PT Electronics
   electroejaculator (PT Electronics, Boring, OR, USA) and a 320 mm-long
   and 26 mm-wide transrectal probe. Electric stimuli (10 V and 250 mA on
   average) were given until ejaculation (n = 14). Ejaculated sperm
   samples were collected in 15 mL graduated glass tubes as isolated
   fractions. Osmolarity was measured using a cryoscopic osmometer OSMOMAT
   3000 (Gonotec, Berlin, Germany), and subjective motility was assessed
   with a phase-contrast microscope (100× magnification) (Eclipse E400;
   Nikon, Tokyo, Japan). Fractions with a similar quality of the same
   electroejaculation were mixed and constituted one ejaculate.
   Pre-ejaculated and ejaculated sperm samples with a reduced
   concentration (<150 × 10^6 sperm/mL) were centrifuged at 600× g for 6
   min at room temperature, and the pellets were processed for sperm yield
   and functionality evaluations.

2.4. Sperm Yield Evaluation

   Immediately after sperm collection from the three origins, the volume
   was recorded via the graduation marks of the collection tubes, the
   sperm concentration was analyzed using a cell counter (NucleoCounter
   SP-100; ChemoMetec, Allerod, Denmark), and the total sperm output was
   calculated by multiplying the measured volume by the corresponding
   sperm concentration.

2.5. Sperm Functionality Evaluation

2.5.1. Sperm Motility and Kinetic Parameters

   Sperm motility and kinetic parameters were assessed through the
   Computer-Assisted Sperm Analysis (CASA) Sperm Class Analyzer^® (SCA^®),
   software version 6.4.0.89 (Microptic S.L., Barcelona, Spain). The
   system was set to capture a total of 50 frames at 100 frames/s and
   particles with an area of 12–62 µm^2. Samples were diluted to 25 × 10^6
   sperm/mL in a TES-Tris-Fructose extender with 1% clarified egg yolk
   (320 mOsm/kg, pH 7.2) and pre-warmed for 5 min on a 37 °C plate. A 5 µL
   drop from each diluted sample was then placed into a Makler chamber (10
   µm depth; Sefi Medical Instruments, Haifa, Israel) and analyzed on a
   warmed plate (37 °C) at a 100× magnification with a phase-contrast
   microscope and a BASLER acA1300-200uc digital camera (Basler Vision
   Technologies, Ahrensburg, Germany). At least 400 sperm from four
   randomly selected fields were captured and analyzed, removing non-sperm
   events. The kinetic parameters reported included linearity (LIN, %) and
   the amplitude of lateral head displacement (ALH, μm). Sperm exhibiting
   curvilinear velocities (VCLs) above 15 μm/s and 45 μm/s were classified
   as motile (total motility, TM) and progressively motile (progressive
   motility, PM), respectively.

2.5.2. Sperm Viability and Apoptosis

   Aliquots containing 2 × 10^6 sperm were washed with phosphate-buffered
   saline (PBS) (300 mOsm/kg, pH 7.2) via brief centrifugation (15 s;
   MiniSpin plus, Eppendorf, Hamburg, Germany), and the supernatant was
   discarded. The resulting pellets were then stained at room temperature
   for 30 min in the dark with Zombie Violet™ Fixable Viability Kit
   (plasma membrane integrity probe) (1:1000 final dilution in PBS) and
   CellEvent™ Caspase-3/7 Green Detection Reagent (apoptosis marker) (4 µM
   final concentration in PBS). After that, another washing step was
   performed to stop cell staining, and the stain was fixed in 500 µL of
   4% formaldehyde (ThermoFisher, Waltham, MA, USA) at room temperature
   and in darkness for 15 min. A new washing step was carried out, and the
   sperm pellets were resuspended in 1 mL of PBS. The samples were stored
   at 5 °C until delayed flow cytometry analysis, which occurred
   approximately four hours after staining and fixation upon arrival at
   the laboratory.

   Flow cytometry analyses were conducted in a CytoFLEX S (Beckman
   Coulter, Brea, CA, USA) equipped with four lasers emitting at 405
   (violet), 488 (blue), 561 (yellow), and 638 nm (red). A total of 40,000
   events per sample and at least 20,000 sperm were recorded at 200–300
   cells/s flow rate. Violet and green fluorescence were detected in FL5
   (excitation 405 nm, emission 450/45 band pass (BP) filter) and FL1
   (excitation 488 nm, emission 525/40 BP), respectively. Data were
   processed using FlowJo™ software version 10.8.1 (Ashland, Wilmington,
   DE, USA). Sperm viability was determined by the proportion of cells
   exhibiting low fluorescence with Zombie Violet™, indicating intact
   plasma membranes. Apoptotic cells were identified by their positive
   staining with CellEvent™ Caspase-3/7 Green, which marks caspase 3 and 7
   activity. The gating strategy used has been previously described in
   detail by Palacin-Martinez et al. [[92]44].

2.5.3. Sperm Oxidation–Reduction Potential

   The oxidation–reduction potential of the sperm samples was measured
   using the RedoxSYS™ diagnostic system (Luoxis Diagnostics, Englewood,
   CO, USA), a potentiometric device designed to evaluate the redox status
   of biological fluids or cell suspensions. To this end, 1 × 10^6 sperm
   from each sperm sample were washed with 100 µL of PBS to remove debris,
   and the resulting sperm pellet was resuspended in 20 µL of PBS. After
   that, this volume (20 µL) was applied to the system’s disposable
   sensor, which measures electron transfer in response to the redox
   environment of the sample. The RedoxSYS™ system provided two key
   parameters within 4 min: the static ORP index (sORP), the integrated
   balance of oxidants and reductants reported in millivolts (mV); and the
   capacitance ORP index (cORP), the amount of antioxidant reserves
   expressed in microcoulombs (µC).

2.5.4. Sperm Proteome

   Sperm samples were centrifuged at 10,000× g for 15 min at 4 °C. The
   supernatant was discarded, and the resulting pellets (200 × 10^6 sperm)
   were stored at −80 °C until further analysis. Phase-contrast microscopy
   was used to verify sample purity. The protein extraction,
   quantification, and digestion procedures followed the methodology
   previously established by Martín-Cano et al. [[93]45]. Briefly, sperm
   were lysed in 400 µL of Protein Extraction Reagent Type 4 (7.0 M urea,
   2.0 M thiourea, 40 mM Trizma^® base, and 1.0% C7BzO; pH 10.4) and
   incubated for one hour at 4 °C under constant agitation. Samples were
   then centrifuged at 17,000× g for 30 min at room temperature to remove
   cell debris, and the supernatant was collected. The protein
   concentration was determined using the 2-D Quant Kit (GE Healthcare,
   Sevilla, Spain), and 100 µg of protein from each sample was subjected
   to in-solution digestion with trypsin. Samples were analyzed in
   duplicate with an Agilent 1290 Infinity II UHPLC system coupled to an
   Agilent 6550 Q-TOF Mass Spectrometer via an Agilent Jet Stream Dual
   electrospray interface (Agilent Technologies, Santa Clara, CA, USA).
   Instrument control was managed using the MassHunter Workstation Data
   Acquisition software (Rev. B.06.01; Agilent Technologies, Santa Clara,
   CA, USA). Peptides (75 µg per sample) were injected into an Agilent
   AdvanceBio Peptide Mapping UHPLC column (2.7 µm, 150 × 2.1 mm; Agilent
   Technologies, Santa Clara, CA, USA) maintained at 55 °C with a constant
   flow rate of 0.4 mL/min. Chromatographic separation was achieved using
   a multi-step gradient: first, 2% of buffer B (water/acetonitrile/formic
   acid, 10:89.9:0.1) for 5 min, increasing to 45% over 40 min, followed
   by a ramp up to 95% B in 15 min, held constant for an additional 5 min.
   This 65 min run was followed by 5 min of using the initial condition
   for re-conditioning the column for the next run. The mass spectrometer
   operated in positive mode, with a nebulizer gas pressure of 35 psi, a
   drying gas flow of 10 L/min at 250 °C, and a sheath gas flow of 12
   L/min at 300 °C. The capillary spray voltage was set at 3500 V, the
   fragmentor at 340 V, and the octopole RF Vpp at 750 V. The profile data
   were acquired for both MS and MS/MS scans in the extended dynamic range
   mode with a mass range of 50–1700 m/z, and scan rates of 8 spectra/s
   for MS and 3 spectra/s for MS/MS. The Auto MS/MS mode was used, with
   precursor selection by abundance and a maximum of 20 precursors
   selected per cycle. A ramped collision energy was used with a slope of
   3.6, and an offset of –4.8 was applied. The same ion was rejected after
   two consecutive scans.

   Data processing and analysis were performed using Spectrum Mill MS
   Proteomics Workbench (Rev B.04.01; Agilent Technologies, Santa Clara,
   CA, USA). Briefly, the raw data were extracted under default
   conditions, with non-fixed or variable modifications, [MH] + 50–10,000
   m/z, maximum precursor charge +5, retention time and m/z tolerance ±60
   s, minimum signal-to-noise ratio (S/N) 25, and the identification of
   ^12C signals. The MS/MS search was conducted against the appropriate
   and updated protein database (in this case, UniProt/Bear) with the
   following criteria: non-fixed modifications, carbamidomethylated
   cysteines, and tryptic digestion with five maximum missed cleavages
   were selected as variable modifications. The ESI-Q-TOF instrument was
   set to a minimum matched peak intensity of 50%, maximum ambiguous
   precursor charge of +5, monoisotopic masses, peptide precursor mass
   tolerance of 20 ppm, product ion mass tolerance of 50 ppm, and to
   calculate the reversed database scores. The auto validation employed an
   auto-threshold method, in which the peptide score is automatically
   optimized for a target False Discovery Rate (FDR) of 1.2%.
   Subsequently, protein polishing validation was performed to increase
   the sequence coverage of the validated results, with a new maximum
   target FDR set at 0%.

2.6. Bioinformatic Analysis of Proteomic Data

   The software R version 4.1.2 (Aukland, New Zealand) was used to perform
   all bioinformatic analyses. The data were quantile normalized.
   Differentially enriched proteins among sperm origins (epididymal,
   pre-ejaculated, and ejaculated) were identified using linear models
   (limma version 3.50.3), and p values were adjusted for multiple
   comparisons by applying Benjamini–Hochberg correction. Heatmaps were
   generated using the heatmap3 version 1.1.9 package. Proteins identified
   in the different sperm origin samples were queried against the Ursidae
   database in String ([94]https://string-db.org/, accessed on 3 February
   2025) to identify the biological pathways likely to be active in brown
   bear sperm.

2.7. Statistical Analysis

   Statistical analyses of sperm yield and quality were performed and
   graphs were made using Prism 10 (GraphPad Software, San Diego, CA,
   USA). The homogeneity and normality of variables were examined via
   Levene’s and Kolmogorov–Smirnov tests. Normally distributed data were
   analyzed using one-way ANOVA, whereas non-normally distributed data
   were analyzed using the Kruskal–Wallis test. The results are displayed
   as the mean ± SEM (Standard Error of the Mean). Differences were
   statistically significant at p < 0.05.

3. Results

3.1. Sperm Collection Yield

   The sperm collection yield data are shown in [95]Table 1. The
   successful sperm collection rate (number of obtained sperm
   samples/number of attempts at sperm collection) was similar (p ≥ 0.05)
   for all sperm origins. However, the sperm sample volume obtained was
   significantly different (p < 0.05) among all sperm origins, with the
   lowest value for epididymal samples and the highest for ejaculated
   samples. The sperm concentration showed an opposite pattern to that of
   the volume, being significantly higher (p < 0.05) in epididymal samples
   and significantly lower (p < 0.05) in ejaculated samples compared to
   pre-ejaculated samples, which showed an intermediate result. Finally,
   the total sperm production was non-significantly different (p ≥ 0.05)
   among sperm origins.

Table 1.

   Sperm yield in brown bears using epididymal (EP), pre-ejaculated (PR),
   or ejaculated (EJ) collection methods.
   SPERM ORIGIN EP PR EJ
   Successful collection rate 100% (12/12) 85.71% (12/14) 100% (14/14)
   Volume (mL) 0.14 ± 0.03 ^a 1.93 ± 0.62 ^b 3.49 ± 0.34 ^c
   Concentration (×10^6 sperm/mL) 3509.00 ± 324.60 ^a 1399.00 ± 336.60 ^b
   191.30 ± 36.46 ^c
   Total output (×10^6 sperm) 509.60 ± 116.20 1089.00 ± 283.90 602.60 ±
   107.00
   [96]Open in a new tab

   Different lowercase letters (a, b, and c) indicate significant
   differences (p < 0.05) among the different sperm origins.

3.2. Sperm Functionality Evaluation

3.2.1. Sperm Motility and Kinetic Parameters

   Pre-ejaculated sperm samples showed significantly lower (p < 0.05) TM
   and PM than the other two sperm origins (epididymal and ejaculated)
   ([97]Figure 1A,B). Nevertheless, the LIN and ALH were similar among the
   different sperm origins ([98]Figure 1C,D).

Figure 1.

   [99]Figure 1
   [100]Open in a new tab

   Motility and kinetic evaluation of brown bear sperm. (A) Total motility
   (TM, %); (B) Progressive motility (PM, %); (C) Linearity (LIN, %); and
   (D) Amplitude of lateral head displacement (ALH, µm). Graph dots
   represent the individual values of each male. Different lowercase
   letters (a, b) indicate significant differences (p < 0.05) among the
   different sperm origins (EP, epididymal; PR, pre-ejaculated; and EJ,
   ejaculated).

3.2.2. Sperm Viability and Apoptosis

   Both sperm quality parameters determined via flow cytometry were
   significantly different (p < 0.05) in the ejaculated sperm samples
   compared to the epididymal ones. Specifically, sperm viability was
   significantly lower (p < 0.05) and sperm apoptosis was significantly
   higher (p < 0.05) in ejaculated sperm than in those obtained from
   epididymis ([101]Figure 2).

Figure 2.

   [102]Figure 2
   [103]Open in a new tab

   Flow cytometry evaluation of brown bear sperm. (A) Viable sperm (%)
   (Zombie Violet™); (B) Apoptotic sperm (%) (CellEvent™ Caspase-3/7
   Green). Graph dots represent the individual values of each male.
   Different lowercase letters (a, b) indicate significant differences (p
   < 0.05) among the different sperm origins (EP, epididymal; PR,
   pre-ejaculated; and EJ, ejaculated).

3.2.3. Sperm Oxidation–Reduction Potential

   Although the cORP index appeared similar (p ≥ 0.05) among the different
   sperm origins (epididymal, pre-ejaculated, and ejaculated) ([104]Figure
   3B), the sORP index was significantly higher (p < 0.05) in the
   ejaculated samples than in the pre-ejaculated samples, with
   non-significant differences (p ≥ 0.05) between the epididymal sperm
   samples and the other two origins ([105]Figure 3A).

Figure 3.

   [106]Figure 3
   [107]Open in a new tab

   RedoxSYS evaluation of brown bear sperm. (A) Static ORP index (sORP,
   mV); (B) Capacitance ORP index (cORP, µC). Graph dots represent the
   individual values of each male. Different lowercase letters (a, b)
   indicate significant differences (p < 0.05) among the different sperm
   origins (EP, epididymal; PR, pre-ejaculated; and EJ, ejaculated).

3.2.4. Sperm Proteome

   Changes in the relative amounts of proteins because of the sperm origin
   are presented as a heatmap ([108]Figure 4). A total of 63 proteins with
   significantly different expressions were identified among the different
   sperm origins (epididymal, pre-ejaculated, and ejaculated) with a
   q-value < 0.05 and a fold change > 1. Interestingly, proteins related
   to African trypanosomiasis, carbon metabolism,
   glycolysis/gluconeogenesis, the biosynthesis of amino acids, and the
   HIF-1 signaling pathway were decreased in the ejaculated sperm
   ([109]Figure 5).

Figure 4.

   [110]Figure 4
   [111]Open in a new tab

   Heatmap showing the impact of sperm origin on the brown bear sperm.
   Proteins are classified following hierarchical clustering. Rows
   correspond to individual proteins (labeled with their UniProt IDs), and
   columns represent samples from each sperm origin (EP, epididymal; PR,
   pre-ejaculated; and EJ, ejaculated). The color scale indicates
   normalized expression levels, with red areas representing larger
   amounts of protein and blue regions representing smaller amounts of
   protein. Proteins were normalized and filtered by a fold change > 1
   with q < 0.05.

Figure 5.

   [112]Figure 5
   [113]Open in a new tab

   KEGG pathway enrichment analysis of under-expressed proteins in
   ejaculated brown bear sperm. Pathways are sorted by signal strength to
   highlight the most biologically relevant terms. Circle size represents
   the number of proteins associated with each path, while the color
   gradient indicates the false discovery rate (FDR), with green colors
   representing lower FDR values and blue colors having higher FDR values.

   Moreover, a pairwise analysis of sperm origins (epididymal vs.
   ejaculated and pre-ejaculated vs. ejaculated) identified the proteins
   shown in [114]Figure 6 and [115]Figure 7 as having the lowest abundance
   in the ejaculated sperm samples, as well as the proteins whose
   expression is primarily associated with just one sperm origin
   ([116]Table 2).

Figure 6.

   [117]Figure 6
   [118]Open in a new tab

   Heatmap representing the differential protein expression between
   epididymal (EP) and ejaculated (EJ) brown bear sperm. Proteins are
   classified following hierarchical clustering. Rows correspond to
   individual proteins (labeled with their UniProt IDs) and columns
   represent samples from each sperm origin. The color scale indicates
   normalized expression levels, with red areas representing larger
   amounts of protein and blue regions representing smaller amounts of
   protein. Proteins were normalized and filtered by a fold change > 1
   with q < 0.05.

Figure 7.

   [119]Figure 7
   [120]Open in a new tab

   Heatmap representing the differential protein expression between
   pre-ejaculated (PR) and ejaculated (EJ) brown bear sperm. Proteins are
   classified following hierarchical clustering. Rows correspond to
   individual proteins (labeled with their UniProt IDs) and columns
   represent samples from each sperm origin. The color scale indicates
   normalized expression levels, with red areas representing larger
   amounts of protein and blue regions representing smaller amounts of
   protein. Proteins were normalized and filtered by a fold change > 1
   with q < 0.05.

Table 2.

   Under-expressed proteins in ejaculated brown bear sperm compared to
   epididymal (EP) and/or pre-ejaculated (PR) sperm.
   SPERM ORIGIN UniProt ID Protein Organism
   EP A0A384CWF6 Heat shock-related 70 kDa protein 2 Polar bear
   (Ursus maritimus)
   A0A384C157 Heat shock cognate 71 kDa protein Polar bear
   (Ursus maritimus)
   A0A384CR94 78 kDa glucose-regulated protein Polar bear
   (Ursus maritimus)
   A0A452S6N6 Phosphoglycerate kinase (PGK1) American black bear
   (Ursus americanus)
   A0A452RQD5 Actin, cytoplasmic 2 American black bear
   (Ursus americanus)
   A0A384BZ26 Actin, aortic smooth muscle Polar bear
   (Ursus maritimus)
   PR A0A384D8F3 Clusterin Polar bear
   (Ursus maritimus)
   A0A452SM85 Pyruvate kinase American black bear
   (Ursus americanus)
   A0A452TU63 Polar bear
   (Ursus maritimus)
   A0A452SA90 Glyceraldehyde-3-phosphate dehydrogenase American black bear
   (Ursus americanus)
   A0A384D5K5 Glutathione S-transferase Polar bear
   (Ursus maritimus)
   A0A452SSP4 Isoaspartyl peptidase/L-asparaginase American black bear
   (Ursus americanus)
   A0A384DG01 Polar bear
   (Ursus maritimus)
   [121]Open in a new tab

4. Discussion

   Sperm collection methods directly impact sperm quality and usability in
   assisted reproductive technologies, making them an essential
   consideration in wildlife conservation programs [[122]7]. Although
   electroejaculation has historically been the preferred method in bears,
   its limitations make it necessary to explore alternative approaches
   [[123]38,[124]40]. To our knowledge, this is the first study evaluating
   the quality and proteomic profiles of brown bear (Ursus arctos) sperm
   of three different origins (epididymal, pre-ejaculated, and
   ejaculated), contributing to the development of species-specific sperm
   manipulation and cryopreservation protocols.

   From a sperm yield perspective, our results indicated that the three
   techniques were similar for the brown bear, as no differences were
   observed in the collection success or the total sperm output. These
   findings contrast with those reported by other researchers and suggest
   that electroejaculation appears to be more effective in brown bears
   (90.5–100% success rate and 545.0 ± 51.9–1387.2 ± 2160.7 × 10^6 sperm,
   based on the data of [[125]46] and that of Ishikawa et al. [[126]33])
   than in other bear species, such as the Asiatic black bear (Ursus
   thibetanus) (53.8% success rate and 100.9 ± 70.0 × 10^6 sperm)
   [[127]38], the American black bear (Ursus americanus) (53.8% success
   rate, no sperm production data) [[128]30], or the polar bear (Ursus
   maritimus) (16.7% success rate and estimated 10.1 × 10^6 sperm)
   [[129]34]. This difference is probably due to the presence of adipose
   tissue surrounding the internal organs in species adapted to colder
   environments, which is non-conductive and increases electrical
   impedance, reducing electroejaculation’s efficiency [[130]47]. In
   contrast, the pre-ejaculated sperm collection method in our study
   yielded outcomes (85.7% success rate and 1089.0 ± 283.9 × 10^6 sperm)
   comparable to those reported for other bear species where this
   technique has been employed (92.3% success rate and 1196.6 ± 955.5 ×
   10^6 sperm in the Asiatic black bear, and 72.0% success rate with total
   sperm count data not provided in the polar bear) [[131]34,[132]38].
   Sperm collection via urethral catheterization is highly reliant on the
   adrenergic effects of the anesthetic drugs used, which are effective in
   a wide range of domestic [[133]22,[134]48,[135]49,[136]50] and wild
   felines [[137]21,[138]51,[139]52], dogs [[140]53], foxes [[141]54],
   ferrets [[142]55,[143]56], macaques [[144]57], rhinoceros [[145]58],
   and so on. Finally, in epididymal sperm collection, we also achieved
   better results than those reported by previous authors, with sperm
   successfully recovered from 100% of the males and a sperm production of
   509.6 ± 116.2 × 10^6 sperm. In contrast, Wojtusik et al. [[146]34]
   reported successful sperm rescue in only 57.1% of collections in polar
   bears without sperm concentration data. These differences may be
   attributed to our use of apparently healthy individuals as part of a
   reproductive control program, whereas the other researchers
   opportunistically sampled animals that had died naturally or were
   euthanized due to illness. Similarly, Archibald et al. [[147]59]
   successfully collected epididymal sperm via mincing in 78.6% of
   American black bears without reporting sperm production data, but these
   animals were legally killed by hunters outside the breeding season, so
   the lower success rate could be related to the timing of sperm
   collection.

   Concerning sperm quality, both the total and progressive motility of
   brown bear sperm displayed their lowest values in the pre-ejaculated
   samples, in contrast to other authors’ observations in similar studies
   on different species. In Asiatic black bears [[148]38] and rhinoceros
   [[149]58], no differences were found in the sperm motility status
   between samples obtained via urethral catheterization and those
   collected via electroejaculation. On the other hand, Prochowska et al.
   [[150]49] noticed a higher total motility in fresh urethral sperm from
   domestic cats compared to fresh epididymal sperm, with no differences
   in other motility or kinetic parameters assessed in their study.
   Despite the above, the motility values of brown bear epididymal and
   ejaculated sperm are consistent with the existing literature on this
   species [[151]43,[152]46,[153]60,[154]61,[155]62], and we associate the
   reduced motility of the pre-ejaculated sperm samples with the presence
   of urine, as reflected by the lower osmolarity in these samples
   compared to the ejaculated ones ([156]Figure A1). Urine contamination
   in urethral catheterization sperm collections has been previously
   reported by Wojtusik et al. [[157]34] in polar bears, linked to a rapid
   loss of motility and viability after collection. Similar findings have
   been described in wolves [[158]63] and dogs [[159]53], affecting up to
   44% of samples and reducing motility and progressive movement. However,
   the pre-ejaculated samples in our study did not differ from the other
   two sperm origins in terms of viability and apoptosis, although the
   sperm quality was lower for both parameters in the ejaculated samples
   than in the epididymal samples, as has been documented in other species
   due to differences in the sperm proteins involved in metabolism, stress
   response, and redox balance [[160]64,[161]65]. In this context, the
   RedoxSYS system is a novel technology that measures the static (sORP)
   and capacity (cORP) oxidation–reduction potential, with sORP referring
   to the passive or current state of activity between oxidants and
   antioxidants [[162]66,[163]67]. Brown bear sperm samples of different
   origins showed similar cORP values, but ejaculates exhibited the
   highest sORP index, indicating greater oxidative stress in these
   samples.

   Using proteomic strategies, 63 proteins with differential expression
   were identified among the different sperm origins (epididymal,
   pre-ejaculated, and ejaculated). Enrichment analysis revealed that
   pathways such as African trypanosomiasis, carbon metabolism,
   glycolysis/gluconeogenesis, the biosynthesis of amino acids, and HIF-1
   signaling were under-expressed in ejaculated brown bear sperm compared
   to epididymal and pre-ejaculated sperm. Sperm are highly specialized
   cells whose primary function is to deliver the male genetic material to
   the oocyte. Given their specialization and function, sperm exhibit high
   metabolic activity and require more energy than other cells [[164]68],
   so they can use both exogenous and endogenous carbohydrates, as well as
   other carbon-containing compounds such as lactate and pyruvate, to meet
   their energy demand via two main pathways: glycolysis (anaerobic
   pathway) and oxidative phosphorylation (aerobic pathway)
   [[165]69,[166]70]. Although the machinery for both processes is present
   in sperm from all species, the predominant ATP generation route appears
   to be species-specific [[167]71]. Little is known about sperm
   metabolism in wildlife and endangered species, where the impact of
   inbreeding on sperm function or the need to preserve genetic material
   requires knowledge of their metabolism, since it is relevant to sperm
   performance and use in assisted reproductive techniques [[168]72]. In
   this context, the identification of key glycolytic enzymes such as
   Glyceraldehyde-3-phosphate dehydrogenase and Pyruvate kinase suggests
   that glycolysis may play a major role in energy production and the
   maintenance of essential metabolic functions in brown bear sperm.
   Notably, these proteins were less active in ejaculated sperm than in
   epididymal and pre-ejaculated sperm. This finding is surprising and
   contrasts with results from bovine sperm reported by Zoca et al.
   [[169]65], where energy production, particularly through glycolysis, is
   more regulated in the epididymis than after ejaculation. Indeed, it is
   well established that sperm in the caudal epididymis remain in a
   quiescent state due to the presence of both quiescence and
   anti-premature sperm activation factors in the epididymal fluid
   [[170]73,[171]74,[172]75], which dilute at ejaculation by mixing with
   seminal plasma, leading to an increase in sperm metabolism and motility
   [[173]76]. Regarding amino acid biosynthesis, our results are in
   agreement with those of Setchell et al. [[174]77], who reported that
   the concentration of the amino acid glutamate in epididymal fluid was
   up to ten times higher than in testicular fluid and seminal plasma in
   rams. Lastly, the hypoxia-inducible factor-1 (HIF-1) signaling pathway
   is recognized as a key mediator of cellular adaptation to hypoxia
   [[175]78,[176]79]. Therefore, it appears normal that its activity was
   higher in epididymal and pre-ejaculated brown bear sperm. As sperm
   transit from the testis to the distal epididymal regions, they are
   thought to encounter a gradually increasing oxygen concentration within
   the luminal microenvironment [[177]80]. In this situation, sperm
   produce reactive oxygen species (ROS) and are highly susceptible to ROS
   oxidative damage, which may activate HIF-1 [[178]81]. Alternatively,
   certain areas of the epididymis may exhibit localized hypoxia. In
   particular, the distal epididymis exhibits a less dense capillary
   network compared to the proximal segments [[179]82], which may limit
   oxygen delivery [[180]83]. Moreover, the increased luminal diameter of
   the cauda epididymis contributes to greater diffusion distances for
   oxygen, potentially resulting in small local fluctuations in oxygen
   availability [[181]84]. These conditions, combined with the large
   number of sperm stored in this region, may demand enhanced protective
   mechanisms to cope with variable oxygen tensions. It has also been
   suggested that localized hypoxia may exist within densely packed sperm
   populations in the epididymal tail [[182]81], which could be another
   reason for the activation of HIF-1 in the brown bear epidydimal and
   pre-ejaculated sperm. This information could guide the design of
   cryopreservation media or pre-freezing treatments aimed at enhancing
   sperm resilience to freezing-induced damage, for example, through
   antioxidant supplementation or hypoxia-mimicking conditions. Further
   studies should explore whether the metabolic and molecular profiles
   observed in these sperm types confer better post-thaw quality, which
   would be particularly relevant for germplasm banking and assisted
   reproduction in endangered wildlife.

   A more detailed pairwise proteomic comparison between sperm origins
   (epididymal vs. ejaculated) identified the following proteins as
   predominantly expressed in epididymal sperm from brown bears: Heat
   shock-related 70 kDa protein 2; Heat shock cognate 71 kDa protein; 78
   kDa glucose-regulated protein; Phosphoglycerate kinase (PGK1); Actin,
   cytoplasmic 2; and Actin, aortic smooth muscle. Heat Shock Proteins
   (HSPs), particularly those belonging to the 70 kDa family (HSP70), are
   commonly present on the sperm’s surface across various species
   [[183]85,[184]86]. Specifically, Heat shock-related 70 kDa protein 2
   (HSP70-2) has been strongly associated with mammalian spermatogenesis
   [[185]87]. This complex process involves the mitotic proliferation of
   spermatogonia, the meiotic progression of spermatocytes, and the
   post-meiotic transformation of spermatids into mature sperm [[186]88].
   In human germ cells, HSP70-2 expression occurs notably at two specific
   phases: (1) during meiosis, where it is a component of the synaptonemal
   complex, and (2) in the final stages of spermiogenesis, where it is
   involved in spermatid elongation [[187]89]. In mice, HSP70-2 is
   essential for the progression of meiosis [[188]90,[189]91,[190]92].
   Moreover, HSP70-2 is crucial for sperm binding to the zona pellucida in
   humans [[191]89], boars [[192]93], and bulls [[193]94], highlighting
   its important role in male fertility. The 78 kDa glucose-regulated
   protein (GRP78) is another key member of the HSP70 family closely
   related to HSP70-2, which shares over 61% of the amino acid sequence
   similarity with it. In mouse testis sections, GRP78 shows strong
   immunostaining from pachytene spermatocytes to post-meiotic germ cells,
   but is absent in spermatogonia and other cell types [[194]95]. Wang et
   al. [[195]96] also demonstrated that GRP78 expression in human and rat
   testes increased with age, and that its localization in different
   epididymal regions indicated a dynamic secretory model, suggesting its
   involvement in mammalian spermatogenesis. Numerous studies have shown
   that GRP78 expression is linked to resistance to apoptotic cell death
   in somatic cells, particularly in progressively growing tumors
   [[196]97,[197]98,[198]99]. Like tumorigenesis, spermatogenesis is a
   dynamic and complex process involving extensive cell proliferation and
   differentiation, where apoptosis control is critical [[199]100]. It
   plays a crucial role in limiting the testicular germ cell population
   during male development and eliminating germ cells with altered DNA at
   various phases of spermatogenesis [[200]101,[201]102]. In our study,
   the predominant expression of these two heat shock proteins in
   epididymal sperm supports their role in brown bear spermatogenesis. In
   turn, Heat shock cognate 71 kDa protein (HSC71), a product of the HSP70
   family, appears to play a role in the terminal differentiation of
   mammalian male germ cells and/or in the function of mature sperm,
   particularly through their ATPase activity [[202]103,[203]104]. Related
   to this protein, Phosphoglycerate kinase (PGK) is an enzyme that
   catalyzes ATP production within the glycolytic pathway [[204]105]. PGK
   exists in two isoforms, PGK1 and PGK2, which support sperm function at
   different germ cell stages. PGK1, the isoform identified in this study,
   is primarily expressed in spermatogonial germ cells, while PGK2 is
   found in round spermatids, although low levels of PGK1 may persist
   after meiosis at levels sufficient for spermatid metabolism
   [[205]106,[206]107,[207]108]. It is unusual to find spermatids or
   immature sperm in the epididymal tail, so we hypothesized that the
   presence of PGK1 in brown bear sperm cells collected from the
   epididymis could be linked to increased sexual activity during the
   breeding season, similar to findings reported in rams [[208]109].
   Conversely, PGK2 detection, as expressed at late stages of
   spermatogenesis, has been used to predict the presence of more mature
   sperm [[209]110]. Finally, regarding the detection of actin in brown
   bear epididymal sperm samples, there are two different causes.
   Cytoplasmic Actin 2 is involved in germ cell movement, cargo
   transportation, and nuclear modification during spermatogenesis
   [[210]111], while Smooth Muscle Actin probably derives from the smooth
   muscle cells of the epididymal blood vessels due to tissue slicing for
   sperm retrieval [[211]112].

   Similarly, the pairwise comparison between pre-ejaculated and
   ejaculated sperm samples revealed only two proteins predominantly
   present in pre-ejaculated brown bear sperm: Glutathione S-transferase
   and Isoaspartyl peptidase/L-asparaginase. It has been well established
   that ROS play a dual role in sperm, having beneficial or harmful
   effects depending on their concentration, localization, or duration of
   exposure [[212]113]. In the testis, the continuous division of germ
   cells is accompanied by high mitochondrial activity, leading to
   elevated ROS production that can negatively impact spermatogenesis
   [[213]114]. Glutathione S-transferases (GSTs) are a large group of
   enzymes commonly implicated in controlling physiological ROS levels and
   protecting sperm and germ cells against oxidative stress by scavenging
   electrophilic substances [[214]115]. Different GST isoforms have been
   previously identified in sperm [[215]116], seminal plasma [[216]117],
   and male reproductive tissues [[217]118]. Although these enzymes
   generally operate as antioxidants in most mammalian cell types, sperm
   GSTs play a triple role: (1) in cell detoxification (preventing lipid
   membrane peroxidation and subsequent oxidative stress) [[218]119], (2)
   in cell signaling regulation (involved in spermatogenesis and sperm
   capacitation) [[219]120,[220]121], and (3) in fertilization (since
   specific GST members are involved in sperm–oocyte binding and the
   acceleration of sperm nuclear decondensation)
   [[221]122,[222]123,[223]124]. Thus, our findings support the relevance
   of GSTs in oxidative stress regulation during brown bears’
   pre-ejaculatory stages. Conversely, the function of the Isoaspartyl
   peptidase/L-asparaginase (ASRGL1) remains unclear, although it is known
   to localize in the middle part of the sperm tail, probably associated
   with mitochondria [[224]125]. Higher expression levels have been
   related to asthenozoospermia in humans [[225]125,[226]126], which,
   together with the lower osmolarity, could explain the reduced motility
   observed in the pre-ejaculated brown bear sperm samples.

5. Conclusions

   To sum up, although the three sperm origins (epididymal,
   pre-ejaculated, and ejaculated) showed similar sperm yield results,
   this study highlights key differences in sperm quality and protein
   expression in the brown bear. Epididymal sperm exhibited superior
   quality and a proteomic profile consistent with active spermatogenesis
   and oxidative stress protection mechanisms, whereas ejaculated sperm
   showed higher oxidative stress and reduced expression of essential
   metabolic proteins. Pre-ejaculated sperm, while comparable in yield,
   demonstrated compromised motility, likely associated with urine
   contamination and mitochondrial protein alterations. These findings
   offer novel insights into brown bear sperm biology and highlight the
   importance of sperm origin in developing optimized cryopreservation
   protocols and assisted reproduction strategies, ultimately supporting
   ex situ conservation efforts for this species.

Acknowledgments