CANINE SEMEN CRYOPRESERVATION AFTER SEMINAL PLASMA REMOVAL IN DOGS WITH BENIGN PROSTATIC HYPERPLASIA

TRADITION AND MODERNITY IN VETERINARY MEDICINE, 2025, vol. 10, No 2(19): 86–92 CANINE SEMEN CRYOPRESERVATION AFTER SEMINAL PLASMA REMOVAL IN DOGS WITH BENIGN PROSTATIC HYPERPLASIA Miroslav Genov1, Desislava Gradinarska-Yanakieva2 1Multidisciplinary Veterinary Clinic “BULGARIA”, 1614, Sofia, Bulgaria 2Institute of Biology and Immunology of Reproduction „Acad. K. Bratanov” – Bulgarian Academy of Sciences, 1113, Sofia, Bulgaria E-mail: [email protected] ORCID: 0000-0003-1096-0972 D. Y.-G. (Submitted: 29 January 2025; Accepted: 29 October 2025; Published: 27 November 2025) ABSTRACT Benign prostatic hyperplasia (BPH) can alter the composition of canine seminal plasma (SP) and specific prostatic proteins may impair sperm cryotolerance. The present study evaluated a large-volume cryopreservation protocol with SP removal in dogs with BPH. Whole ejaculates were collected from 10 healthy dogs and 10 dogs with BPH. SP was removed by brief low-speed centrifugation. Samples were dispensed into cryovials in 500 µL aliquots and subjected to equilibration. They were then frozen using an accelerated ultralow temperature profile and subsequently thawed for computer-assisted sperm analysis. Post-thaw progressive motility declined in both groups when compared to fresh samples (p < 0.001), while linearity, straightness, and wobble remained stable within groups and were comparable between groups. Curvilinear, straight-line, and average path velocities decreased after freezing compared to fresh controls (p < 0.05) in both groups. The amplitude of lateral head displacement and beat-cross frequency exhibited significantly greater post-thaw reductions in BPH dogs than in healthy controls (p < 0.05). These findings indicate that the developed cryopreservation protocol enables effective long-term storage of canine semen from patients with BPH, while maintaining key trajectory parameters despite cryoinjury. Key words: Canis familiaris, Spermatozoa, Cryotolerance, Computer-assisted sperm analysis, Sperm kinematics. Introduction Technologies for the long-term storage of genetic material at ultralow temperatures began to develop in the mid-20th century. They have proven their advantages in in vitro procedures used to enrich and improve gene pools in animals (Estudillo et al. 2021; Hiemstra et al. 2006; Mara et al. 2013). Nonetheless, cryopreservation can compromise the structure and function of spermatozoa, thereby reducing their viability and fertility (Lemma 2011; Muiño-Blanco et al. 2008). Furthermore, age-related susceptibility to cryodamage has been reported in dogs (de la Fuente-Lara et al. 2019; Hesser et al. 2017) by comparing the quality parameters of spermatozoa from fresh and frozen-thawed semen from dogs of different ages. Numerous studies on the role of different freezing rates and protective media in canine semen cryopreservation have been conducted, and it has been shown that the spermatozoa of this species are more sensitive to low temperatures, similar to other species such as boars and horses (Knox 2015; Oldenhof et al. 2012; Schäfer-Somi et al. 2022; Yeste 2016). With the rise in the population of domestic dogs, there has been a corresponding increase in reports of patients developing benign prostatic hyperplasia (BPH). Proteins specific to the seminal plasma (SP) of dogs with BPH that are not present in healthy individuals have been previously documented (Genov et al. 2020; Genov et Ivanova 2021; Gradinarska et al. 2019). Prostatic fluid Canine semen cryopreservation after seminal plasma removal in … 87 (PF), which forms the liquid phase of the ejaculate, has shown context-dependent effects on canine spermatozoa during cryopreservation. Some prostate components may have different positive or negative effects on sperm cell functionality following low-temperature storage or cryopreservation (Sirivaidyapong et al. 2001; Strzeżek et Fraser 2009). A positive effect on sperm motility and viability has been reported when PF is added to canine epididymal spermatozoa (Hori et al. 2005). However, such positive effects were not observed after semen freezing and thawing. Korochkina et al. (2014) suggested that some of the specific proteins present in PF are likely “detrimental” to sperm cell survival during low-temperature storage or cryopreservation. Thus, the role of SP in canine sperm cryotolerance remains unclear, especially in dogs with BPH. Despite extensive research on cryopreservation protocol optimisation, it remains unclear whether removing SP before freezing improves post-thaw function, specifically in semen from dogs with BPH, where SP composition is altered. To address this gap, we evaluated whether a largevolume cryopreservation protocol with SP removal preserves post-thaw motility and kinematic parameters in dogs with BPH compared with healthy controls. Materials and Methods Ejaculate collection and primary analysis Whole ejaculates were manually collected through digital stimulation from 20 dogs of various breeds aged between 4 and 7 years (10 healthy dogs and 10 dogs with BPH). BPH diagnosis was based on clinical evaluation, digital rectal palpation, laboratory tests (bloodwork and urinalysis), and diagnostic imaging (abdominal ultrasonography, radiography, and, when necessary, computed tomography) to confirm prostatomegaly and parenchymal changes. In cases where clinical signs and imaging findings were inconclusive, a prostatic biopsy was conducted to rule out prostatitis, abscessation, or neoplasia. Dogs that met these criteria and showed no signs of infection or neoplasia were categorised into the BPH group, whereas healthy dogs served as controls. All regulations and requirements regarding humane treatment and animal well-being were strictly followed. Primary semen analysis was performed using computer-assisted sperm analysis (CASA). The ejaculates from healthy (n=10) and BPH dogs (n=10) to undergo SP removal and freezing were selected to ensure comparable pre-freeze motility, specifically similar total and progressive motility. Semen extenders and dilution After the initial semen assessment, samples from both groups were centrifuged at 300 × g for 20 s to remove SP. After centrifugation of the ejaculates, the SP was discarded and sperm cell dilution was performed with cryoprotective medium (TRIS 3 g, citric acid 1.7 g, fructose 1.3 g, distilled water up to 100 ml, glycerol 8 ml, egg yolk 20% v/v). This dilution resulted in a final sperm concentration of 200-300×106/ml to ensure adequate sperm numbers post-thaw within dosing ranges reported for artificial insemination with frozen–thawed canine semen (Farstad 2009; Pesch et Hoffmann 2007; Silva et al. 2005). Freezing of canine semen samples The subsequent equilibration phase in sample processing was initiated by aliquoting 500 µL of each sample into a cryovial. This phase serves as the adaptation period for spermatozoa to acclimate to low temperatures in a protective medium containing cryoprotective agents. Each cryovial containing diluted semen was stored at 4°C for 180 min. 88 Miroslav Genov, Desislava Gradinarska-Yanakieva After the equilibration period, cryovials were subjected to accelerated freezing at ultralow temperatures in a biofreezer at -150°C. A temperature probe was used to monitor freezing rate consistency. Accelerated freezing started from +4°C (the temperature of the samples after equilibration) and reached -130°C for 29 min at an average rate of temperature decrease over the entire period of - 4.6°C/min (Gradinarska 2019). The samples were then stored at -150°C. Thawing of cryovials with canine semen samples Each semen sample was thawed in a water bath at 65°C for 60–80 sec, with the cryovials being gently agitated until the solid phase was completely dissolved. The samples were then incubated at 37°C in a temperature-controlled incubator and assessed using CASA. Computer-assisted sperm analysis Thawed samples were assessed by CASA for motility, progression, velocity, and kinematic parameters of frozen-thawed spermatozoa. The Sperm Class Analyser® CASA System (Microptic, Spain) and "Motility and Concentration" software module were used in the present study. The CASA software was configured for canine semen analysis using a Nikon BM 10× objective, with coverslip dimensions of 18 × 18 mm and a droplet volume of 8 µL. For each sample, a minimum of 1000 spermatozoa were analysed across at least five captured fields. The basic parameters measured were concentration, motility, progression, velocity, and head area. Kinematic parameters were evaluated for the entire sample and within specific sperm subpopulations classified as static, slow, medium, or rapid. These included the curvilinear velocity (VCL), straight-line velocity (VSL), and average path velocity (VAP). Additional kinematic parameters included linearity (LIN), straightness (STR), wobble (WOB), amplitude of lateral head displacement (ALH), and beat-cross frequency (BCF). Statistical analysis Data are reported as mean ± standard error of the mean (SEM). A two-tailed analysis was performed. Differences between the healthy and BPH groups (separately for fresh and frozenthawed samples) were assessed using the non-parametric Mann–Whitney U test. Within-group changes (fresh vs. post-thaw) were assessed using the Wilcoxon signed-rank test. Differences were considered statistically significant at p < 0.05. All analyses were performed using Statistica v.6.0 (StatSoft Inc.). Results and Discussion In the present study, the evaluated cryopreservation protocol used pre-freeze SP removal via gentle 300 × g for 20 s separation. Low g-force and brief duration were chosen to minimise mechanical stress and potential structural damage to spermatozoa, particularly in BPH cases (Koderle et al., 2009; Lazov et al. 2019; Tsvetkov et Daskalova 2023). Accelerated freezing results in a rapid transition of cells from partial to complete anabiosis, thus shortening the time for the main damaging factors to cause effects during cryopreservation (Eilts 2005; Kim et al. 2012). Subsequent rapid thawing at higher temperatures also minimises the duration of ice crystal growth and recrystallisation during the warming phase, which can cause severe mechanical and osmotic damage to spermatozoa (Mazur 1984; Watson 2000). In this protocol, the combination of high-temperature thawing and precise timing ensures that gametes pass quickly through the critical temperature zone between −50°C and −5°C, thereby improving the preservation of structural integrity and post-thaw functionality of the sperm. Canine semen cryopreservation after seminal plasma removal in … 89 Comparative CASA results for motility, progression, and velocity of spermatozoa in healthy dogs and dogs with BPH, before and after freezing, are presented in Table 1. Ejaculates from healthy and BPH dogs to undergo this study were selected to ensure comparable baseline motility. Prior to freezing, the two groups showed no significant differences in the main CASA parameters used for post-thaw interpretation (e.g. total and progressive motility). Exception to this was a larger rapid subpopulation in healthy dogs before freezing (15.73±3.16 % vs 8.91±2.11 % in BPH group), indicating greater energetic potential of the spermatozoa (Table 1). Table 1: CASA of motility, progression, and velocity of fresh and frozen spermatozoa in dogs with BPH and healthy dogs. Group State Static (%) Non-progressive (%) Progressive (%) Rapid (%) Medium (%) Slow (%) BPH Fresh 35.03±7.79§ 52.87±4.69 12.10±3.47§ 8.91±2.11§ 10.09±3.29 45.96±3.85 Frozen 72.87±8.42# 25.77±8.01 1.36±0.54# 1.58±1.13 2.20±1.48 23.35±6.13 Healthy Fresh 31.38±8.19§ 57.84±7.34 10.77±3.32§ 15.73±3.16# 11.61±4.46 41.27±6.65 Frozen 60.45±7.21# 37.68±6.76 1.87±0.62# 0.70±0.25 2.18±0.65 36.67±6.49 Note: Data are presented as mean ± SEM (n=10 per group). Symbols (§, #) were used to highlight specific differences. Within columns, values with different superscript symbols differ significantly (p < 0.05). After thawing, both groups showed the expected decline in total and progressive motility. The cryopreservation process results in the deterioration of the general biological parameters of spermatozoa and an increase in static spermatozoa (Korochkina et al. 2014), which is the reason for the significant decrease in motility after thawing in both groups compared to fresh spermatozoa (p < 0.001). Post-thaw progressive motility did not differ between BPH and healthy dogs (Table 1). The head area of spermatozoa in fresh ejaculates was larger in healthy dogs than in dogs with BPH. Post-thaw, between-group differences in head area were not observed (healthy: 22.19 ± 2.16 µm²; BPH: 20.04 ± 0.96 µm²). These results indicate that the freezing protocol preserved head morphometry to comparable levels in both groups. Prior observations of BPH-related changes in SP proteins (Gradinarska et al. 2019) and of prostatic or SP components which can modulate postthaw sperm quality (Korochkina et al., 2014; Neagu et al., 2011) suggest a plausible role of SP composition in the preservation of structural and functional integrity of spermatozoa during cryopreservation that should be tested in future targeted studies. The results from the CASA of sperm kinematics from healthy dogs and dogs with BPH before and after freezing are presented in Table 2. Both groups demonstrated similar velocity kinematics before cryopreservation. After thawing, a significant decrease in the values of VCL, VSL, and VAP of sperm cells from healthy dogs and dogs with BPH was observed compared to the pre-freeze values (p < 0.05) (Table 2). These results are natural consequences of the detrimental effects of low temperatures during freezing. No significant differences were observed between BPH and healthy spermatozoa in the post-thaw velocity parameters (Table 2). 90 Miroslav Genov, Desislava Gradinarska-Yanakieva Table 2: CASA of kinematic parameters of fresh and frozen spermatozoa in dogs with BPH and healthy dogs. Group State VCL (µm/s) VSL (µm/s) VAP (µm/s) LIN (%) STR (%) WOB (%) ALH (µm) BCF (Hz) BPH Fresh 42.07±6.32§ 21.39±3.31§ 28.69±4.65§ 50.41±2.25 74.90±1.56 67.22±2.4 3.21±0.13 10.84±0.25 Frozen 22.30±1.85# 9.79±1.57# 13.85±1.67# 43.42±4.93 68.73±4.19 61.42±4.09 1.53±0.42§ 5.97±1.86§ Healthy Fresh 48.92±7.52§ 25.05±4.31§ 33.00±4.74§ 53.19±6.49 74.69±4.65 68.84±4.47 2.49±0.46 9.62±1.25 Frozen 25.80±1.19# 11.30±0.80# 16.72±1.01# 43.49±1.67 67.33±1.54 64.46±1.35 2.82±0.17# 9.51±0.73# Note: Data are presented as mean ± SEM (n=10 per group). Symbols (§, #) were used to highlight specific differences. Within columns, values with different superscript symbols differ significantly (p < 0.05). Post-thaw trajectory parameters demonstrated that movement quality was largely maintained across the groups (Table 2). Both STR and progressive motility in dogs with BPH remained comparable to those of healthy controls after freezing, despite the expected overall decline in both groups. LIN, STR, and WOB remained stable in the healthy and BPH groups at both the pre-freeze and post-thaw assessments, showing no within-group change and no between-group differences. A notable post-thaw divergence was that ALH and BCF exhibited a greater decrease in BPH than in healthy dogs (p < 0.05), indicating greater susceptibility of the flagellar beat pattern to cryoinjury in this group. These findings indicate that cryopreservation after gentle SP removal preserved the biological potential of spermatozoa from dogs with BPH, with post-thaw motility and kinematic profiles comparable to those of healthy controls. 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