Intraepididymal platelet-rich plasma improves semen cryoresistance via antioxidant, lipid and molecular modulation during the non-breeding season in rams
Serap Dayan Cinkara, Nida Badıllı, İbrahim Halil Güngör, Aslıhan Çakır Cihangiroğlu, Tutku Can Acısu, Görkem Kırmızıkaya Özmen, Gözde Arkalı, Mustafa Sezer Bulan, Ahmet Tektemur, Edip Toraman, Şeyma Özer Kaya, Mustafa Sönmez, Seyfettin Gür, Abdurrauf Yüce, Ökkeş Yılmaz
TL;DR
Injecting platelet-rich plasma into rams' epididymis improves frozen semen quality by reducing damage through antioxidants and molecular changes.
Contribution
This study demonstrates that intraepididymal PRP enhances ram semen cryoresistance via antioxidant, lipid, and molecular modulation.
Findings
Intraepididymal PRP improved post-thaw sperm motility and reduced acrosome damage in rams.
PRP increased catalase activity, cholesterol levels, and key protein expressions like StAR and HSD3β1.
PRP modulated microRNA and ion channel mRNA levels, enhancing cryopreservation outcomes.
Abstract
This study aimed to investigate the effectiveness of intraepididymal platelet-rich plasma (PRP) administration in preventing cryopreservation-induced sperm damage in rams. Twelve adult rams were randomly assigned into two groups (n = 6) in the non-breeding season. Rams in the PRP group received 0.2 ml/per epididymis (150–200 × 10⁶ platelets) of PRP every 15 days for a total of six injections, while control group received the same volume of saline. Semen samples were collected biweekly and pooled within each group before undergoing standard cryopreservation procedures. Post-thaw analyses included morphological, functional, biochemical, and molecular assessments. Compared to the control, intraepididimal PRP significantly increased hypo-osmotic swelling (HOS) response, total and progressive motility, rapid sperm percentage, and kinetic parameters (VCL- curvilinear velocity, VSL-…
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TopicsSperm and Testicular Function · Periodontal Regeneration and Treatments · Reproductive Physiology in Livestock
Introduction
The epididymis is an organ responsible for the transport, concentration, storage, and maturation of spermatozoa. Epididymal maturation includes the acquisition of motility and fertilization capacity by spermatozoa (Cooper and Yeung 2006). During this transit, spermatozoa undergo numerous biochemical and molecular modifications such as chromatin condensation, increases in overall surface negative charge and disulfide bonds, changes in the composition of plasma membrane proteins and lipids (including phospholipid content and cholesterol/phospholipid ratio), surface remodeling, elimination or modification of surface proteins and antigens (Sullivan 1999), structural modifications of the perinuclear cytoplasmic droplet (Müjica et al. 2003), and the ability to respond to hypoosmotic stress (Sahin et al. 2009).
One of the most significant changes occurring in the sperm plasma membrane during epididymal transit involves alterations in the lipid composition. In rams, at the end of epididymal maturation, spermatozoa typically retain low levels of cholesterol and total phospholipids, while the content of polyunsaturated fatty acids (PUFAs) remains high. This lipid profile renders ram spermatozoa highly susceptible to environmental stress, leading to membrane destabilization and ultimately contributing to infertility (Shan et al. 2021).
Cryopreservation, defined as the preservation of viable spermatozoa within semen by freezing, aims to maintain the structural and functional integrity of cells at ultra-low temperatures for long-term storage (Trounson 1990). However, it is well-known that cryopreservation induces irreversible structural, functional, and molecular damages in spermatozoa. Such damages include reduced motility, increased rates of dead and morphologically abnormal spermatozoa, compromised plasma and acrosomal membranes, disturbances in oxidative–antioxidant balance, increased global DNA methylation and apoptosis, and changes in the expression of miRNAs and ion channels (Güngör et al. 2021), altered concentrations of lipids, vitamins, and amino acids (Güngör et al. 2025). To mitigate these effects, the efficacy of various additives continues to be investigated.
Platelet-rich plasma (PRP) is a blood-derived product obtained by centrifugation of whole blood and contains a higher concentration of platelets than normal plasma. In addition to their role in coagulation, platelets play a key role in tissue repair and regeneration by releasing growth factors from their granules, which promote cell proliferation, re-epithelialization, vasculogenesis, and angiogenesis (Marx 2004). These granules are also rich in antioxidant molecules, bioactive lipids, and ions (McNicol et al. 1999; Hoeferlin et al. 2015).
While PRP has been widely applied in human medicine (Turgut et al. 2020), particularly in orthopedics and dermatology, its veterinary use has been primarily focused on the treatment of joint and muscle disorders in performance horses (Lam et al. 2007). In recent years, PRP has attracted increasing attention for its potential applications in male reproduction. Studies in humans (Somova et al. 2021; Masterson et al. 2023) and various animal species (Sayed and Elzainy 2021; Farhan and Al-Maathidy 2022; Kashkool and Al-Delemi 2023) have demonstrated that intratesticular, subscrotal, and intracavernosal applications of PRP can positively affect spermatogenesis, sperm quality, and fertilization outcomes. Moreover, in vitro supplementation of semen extenders with PRP has been shown to reduce cryopreservation-induced damage (Alcay et al. 2022; Türk et al. 2025).
In our recent study, we demonstrated that intraepididymal application of PRP—an approach differing from commonly studied local routes—resulted in morphological, functional, and biochemical improvements in fresh ram semen (Dayan Cinkara et al. 2025). Considering the membrane remodeling and functional maturation processes that occur during epididymal transit, PRP’s beneficial role in supporting these changes is noteworthy. Therefore, the present study aimed to investigate the efficacy of intraepididymal PRP administration in mitigating certain morphological, functional, biochemical, and molecular damages caused by cryopreservation in ram spermatozoa.
Materials and methods
Animal grouping
In this study, a total of 12 Akkaraman rams, approximately 1-year-old, were used. Prior to the experiment, the animals were housed in the paddocks of Fırat University Animal Hospital for a one-week adaptation period and remained there throughout the study. All rams were clinically healthy and weighed 45–50 kg. Scrotal palpation and semen examinations were performed, and normozoospermia was observed. The grouping process was carried out randomly as the live weights were close. All rams were provided with high-quality roughage and concentrate feed, and had free access to water. The animals were randomly divided into two groups: a control group and a PRP group, with six rams in each.
Preparation and intraepididymal administration of PRP
To avoid immune reactions, autologous PRP was used. Blood collection and PRP preparation were done carefully under sterile conditions to prevent contamination. Blood was drawn six times every 15 days during the study. PRP was prepared at room temperature using a two-step centrifugation method based on Ding et al. (2009). First, 20 ml of blood from each animal was centrifuged at low speed (312 × g) for 10 min, separating blood into three layers: red blood cells at the bottom, a thin middle layer rich in white blood cells, and plasma on top. The top and part of the middle layers were collected and centrifuged again at higher speed (1248 × g) for 10 min. After this, the upper part, poor in platelets, was removed, and the bottom part was taken as pure PRP.
Platelet counts in PRP samples were determined using an automated hematology analyzer (BC-5000Vet, Mindray, Guangdong, China) to quantify the platelet concentration in PRP intended for injection. To keep the PRP’s quality and growth factors stable, injections were made quickly, within 20 min. Because the epididymis is small, 0.2 ml of PRP (about 150–200 million platelets) was injected per epididymis to avoid swelling. The PRP group received six injections every 15 days, matching the epididymal sperm transport time in rams. This period was determined taking into account epididymal sperm transport in rams. Before injections, the scrotum was shaved and cleaned with 10% povidone-iodine, and testes were manually compressed from the spermatic cord to make the medially located corpus epididymis more prominent. Using a fine needle (30G), PRP was intraluminally injected into the middle of the corpus epididymis and spread toward both ends (Fig. 1). With the help of ultrasound, it was confirmed that the needle tip was in the lumen of the corpus epididymis. Control animals had the same treatment but received 0.2 ml of saline instead of PRP.Fig. 1. The experimental design summarising the preparation process and application of PRP (A) blood collection from the V. jugularis of rams using a sterile syringe, (B) centrifugation of the collected blood, (C) plasma from the centrifuged blood, (D) separation of PRP using a sterile syringe, (E) measurement of platelets in whole blood and PRP using a haematology device, (F) PRP ready for intraepididymal application, (G) application of PRP into the corpus epididymis lumen
Semen collection, dilution, freezing and thawing
A total of six ejaculates were collected from each ram at 15-day intervals (excluding the day of the first injection and including 15 days after the last injection) using an artificial vagina. On each collection day, a fresh Tris-egg yolk extender [297.58 mM Tris (hydroxymethyl aminomethane), 96.32 mM citric acid, 82.66 mM fructose, 1.000.000 IU penicillin (Penicillin G Potassium, 1.000.000 IU, İ.E. Ulagay, Istanbul, Türkiye), 100 mg streptomycin (Crystallized Streptomycin Sulfate, 1 g, İ.E. Ulagay, Istanbul, Türkiye), 15 ml egg yolk, and distilled water to make up 100 ml] was prepared. Eggs were obtained daily from the same farm throughout the study.
Ejaculates from rams in both the control and PRP groups were pooled at 38 °C. The pooled semen was initially diluted with half of the total extender volume to reach a concentration of 400 million sperm per ml. The pre-diluted semen was slowly cooled to 4 °C. Once cooled, the second half of the extender, which contained 10% glycerol (resulting in 5% final concentration), was added drop by drop over 15 min for final dilution and glycerolization.
The processed semen was kept at 4 °C for at least 3 h for equilibration. Then, it was filled into 0.25 ml straws, and the ends were sealed with polyvinyl alcohol powder. The straws were frozen in an automatic freezing machine (Microdigitcool, IMV, Neuilly-sur-Seine, France) using liquid nitrogen vapor at −140 °C. The freezing rate was set to 1 °C per minute from 4 °C to −20 °C, and 25 °C per minute from − 20 °C to −140 °C. Finally, the straws were stored in liquid nitrogen container at −196 °C until analysis. After freezing, and following a minimum storage period of 24 h, the straws from both the PRP and control groups were thawed for various analyses by placing them in a water bath at 38 °C for 25 s (Güngör et al. 2021; 2025).
Motility, kinetic, membrane integrity and morphologic analyses
Sperm motility and kinetic parameters were evaluated using a computer-assisted sperm analysis (CASA) system (ISASv1, Arquimea Agrotech, Paterna, Spain). Prior to analysis, semen samples were diluted in a pre-warmed (38 °C) Tris-buffer solution containing 3.63 g Tris (hydroxymethyl) aminomethane, 0.50 g glucose, 1.99 g citric acid, and distilled water to a final volume of 100 ml. Dilution was adjusted to yield approximately 100–150 spermatozoa per field of view. For CASA evaluation, 3.5 µl of the diluted semen was placed on a pre-warmed specialized counting chamber (Spermtrack, 20 μm depth, Arquimea Agrotech) and examined under a phase-contrast microscope equipped with a heated stage. Total motility (%), progressive motility (%), and proportions of rapid, medium, slow, and static sperm (%), standard kinetic parameters including VCL (curvilinear velocity, µm/s), VSL (straight-line velocity, µm/s), VAP (average path velocity, µm/s), LIN (linearity, %, VSL/VCL), STR (straightness, %, VSL/VAP), WOB (wobble, %, VAP/VCL), ALH (amplitude of lateral head displacement, µm) and BCF (beat cross frequency, Hz) were recorded. Sperm velocity thresholds specific to ram semen, as defined by the manufacturer, were used: static (< 10 μm/s), slow (10–44 μm/s), medium (45–74 μm/s), and fast (≥ 75 μm/s). The CASA system’s particle detection range was set to 15–70 μm (Özer Kaya et al. 2021).
Membrane integrity was evaluated using the hypo-osmotic swelling (HOS) test. For this, 10 µl of semen was diluted in Tris-buffer at 38 °C, and 25 µl of the diluted sample was then added to 250 µl of hypo-osmotic solution (composed of 0.49 g citric acid and 0.9 g fructose in 100 mL of distilled water). The mixture was incubated at 38 °C for 60 min. After incubation, 30 µl of the sample was placed on a slide and examined under a phase-contrast microscope at 400× magnification. As a result of the examination, 400 spermatozoa were counted and the percentage of spermatozoa with a spiral tail, indicating intact plasma membranes, was recorded (Özer Kaya et al. 2021).
For morphological assessment, 50 µl of the diluted semen was used to prepare air-dried smears. These were stained using a commercial Diff-Quick staining kit (Gündüz Kimya, İstanbul, Türkiye), following the manufacturer’s instructions. Slides were examined under a light microscope at 600× magnification, and 1000× was used when higher resolution was needed. A total of 200 spermatozoa per slide were evaluated. The percentages of spermatozoa with head, tail, and total morphological abnormalities were determined.
Flow-cytometric analyses
Viability, acrosomal damage, high- and low-mitochondrial membrane potential (HMMP and LMMP), and apoptosis in frozen-thawed semen samples were evaluated using a flow cytometer (CytoFLEX, Beckman-Coulter, California, USA). All reagents used in these assays were obtained from Thermo Fisher Scientific (Waltham, Massachusetts, USA). Each analysis was conducted on 1 ml of diluted semen adjusted to a concentration of 1 × 10⁶ spermatozoa/ml.
Sperm viability was assessed using the LIVE/DEAD™ Sperm Viability Kit (L7011) by incubating samples with 5 µl of SYBR-14 and 5 µl of propidium iodide (PI). Acrosomal damage was determined by staining with 5 µl of peanut agglutinin (PNA) conjugated with Alexa Fluor™ 488 (L21409) and 3 µl of PI. For evaluation of HMMP and LMMP, 2.5 µl of JC-1 dye (T3168) was added to the semen samples. All samples were vortexed and incubated at 38 °C for 30 min prior to flow-cytometric analysis. The device was set to count 10,000 spermatozoa. In the first graph, the total spermatozoa density was determined using a comparison of FSC (Forward Scatter) and SSC (Side Scatter). Subsequently, the cells filtered from this comparison were subjected to FSC-Area (Forward Scatter-Area) and FSC-Height (Forward Scatter-Height) comparisons to separate the double cells from the single cells. After this stage was completed, the separated single cells were subjected to PE (Phycoerythrin) and FITCH (Fluorescein Isothiocyanate) comparisons, and the flow cytometric results were expressed as a percentage according to cell differentiation (Güngör et al. 2024a, b a).
Apoptosis was detected using the Annexin V Apoptosis Detection Kit (eBioscience™, 88–8005-72). For this, 50 µl of semen was diluted with 950 µl of phosphate-buffered saline (PBS), centrifuged at 400 × g for 5 min, and the supernatant was discarded. The pellet was re-suspended in 500 µl of 1X Binding Buffer, followed by the addition of 5 µl Annexin V and 5 µl PI. After a 5-minute incubation at room temperature, samples were analyzed via flow cytometry. Spermatozoa in the Annexin V⁺/PI⁻ and Annexin V⁺/PI⁺ regions were identified as apoptotic, and their combined percentage was reported as the total apoptotic sperm population (Güngör et al. 2024a, b a).
Oxidative stress analyses
Lipid peroxidation (LPO) was evaluated by quantifying malondialdehyde (MDA), a secondary end-product of LPO, following the method described by Placer et al. (1966). Results were expressed as nmol/ml. For the analysis of glutathione (GSH) and glutathione peroxidase (GPx), samples were treated with 50% trichloroacetic acid to precipitate proteins, followed by centrifugation at 1000 × g for 5 min. The supernatant was used for further analysis. GSH concentration was measured according to the procedure outlined by Sedlak and Lindsay (1968), and expressed in nmol/ml. GPx activity was determined following the method of Lawrence and Burk (1976), and results were expressed in IU/g protein. Catalase (CAT) activity was measured by monitoring the decomposition of hydrogen peroxide, following the protocol described by Goth (1991), and results were reported as kU/g protein.
Lipid analyses
Lipid extraction was performed following the method of Hara and Radin (1978), wherein the samples were homogenized with a 3:2 (v/v) mixture of n-hexane and isopropanol for 30 s, followed by centrifugation at 4500 × g for 10 min. The resulting pellet was collected and used for fatty acid and cholesterol analyses.
For fatty acid profiling, total lipids were first trans-esterified into fatty acid methyl esters (FAMEs). This was achieved by treating the lipid extract with 2% methanolic sulfuric acid and 5% sodium chloride. The resulting FAMEs were transferred into auto-sampler vials and analyzed by gas chromatography (GC) in accordance with the method of Christie (1990). The separation of saturated (SFA), monounsaturated (MUFA), and polyunsaturated (PUFA) fatty acids was performed using a Shimadzu GC-2010 Plus system (Tokyo, Japan) equipped with an Rtx 2330 column (30 m × 0.25 mm × 0.20 μm). The GC oven was programmed from 120 °C to 220 °C, with injector and detector temperatures set at 240 °C and 280 °C, respectively. Nitrogen served as the carrier gas.
Cholesterol levels were quantified using high-performance liquid chromatography (HPLC) as described by Lopez-Cervantes et al. (2006), utilizing a Shimadzu HPLC-L214555 system (Tokyo, Japan). Chromatographic separation was achieved with a Wakosil II HG column (25 × 4.6 cm, 5 μm), using an acetonitrile/methanol (60:40, v/v) mobile phase at a flow rate of 1 ml/min. Detection was carried out at a wavelength of 202 nm using a photodiode array detector.
ELISA analyses
Prior to analysis, the samples were further diluted with PBS and centrifuged at 1500 × g for 10 min at room temperature to remove residual egg yolk content. The levels of steroidogenic acute regulatory protein (StAR, Cat. No: 201-07-3136 and 201-07-2219), 3β-hydroxysteroid dehydrogenase-1 (HSD3β1, Cat. No: 201-07-1037 and 201-07-2585), cation channel of sperm-1 (CatSper1, Cat. No: 201-07-2135), insulin-like growth factor-1 (IGF1, Cat. No: 201-07-0005), transforming growth factor-β (TGFβ, Cat. No: 201-07-3014), vascular endothelial growth factor (VEGF, Cat. No: 201-07-2673), platelet-derived growth factor (PDGF, Cat. No: 201-07-3618), platelet-derived growth factor receptor (PDGFR, Cat. No: 201-07-3826) and fibroblast growth factor (FGF, Cat. No: 201-07-0030) were analyzed using sheep specific kits following the protocols provided by the manufacturers (Sunred Biological Technology, Shanghai, China).
qRT-PCR analysis
Before commencing the analytical procedures, the samples underwent additional dilution with PBS and were subsequently centrifuged at 1500 ×g for 10 min at room temperature to separate and remove the yolk content. Total RNA extraction was carried out using a modified protocol based on the method described by Tektemur et al. (2021). To eliminate somatic cell contamination, the samples were initially washed with PBS and then treated with 1 ml of 0.5% Triton X-100. Following another PBS wash and centrifugation, the supernatant was discarded, and the resulting pellet was lysed using Trizol reagent (Thermo Fisher Scientific, Waltham, MA, USA) to complete the RNA isolation process. RNA concentration and purity were assessed using a Nano-Drop spectrophotometer, and the isolated RNA samples were stored at −80 °C until further analysis.
For complementary DNA (cDNA) synthesis targeting ion channel gene expression, 10 µl of purified RNA was combined with 2 µl of 10× RT random primers, 2 µl of 10× RT buffer, 0.8 µl of 25× dNTP mix, 4.2 µl of nuclease-free water, and 1 µl of MultiScribe™ reverse transcriptase (Thermo Fisher Scientific, Waltham, MA, USA). The reaction mix was incubated in a thermal cycler, with the final temperature maintained at 4 °C, and the synthesized cDNA was stored at −20 °C. For miRNA expression profiling, RT primers specific to target miRNAs (TaqMan MicroRNA RT Kit, Applied Biosystems, Thermo Fisher Scientific) were used instead of random primers.
qRT-PCR was performed using TaqMan-based assays with gene-specific primers. The expression levels of ion channel genes were analyzed in sperm-derived cDNA, with GAPDH serving as the internal control. For miRNA analysis, hsa-miR-191 was used as the reference gene. Relative gene expression levels were calculated using the 2^−ΔΔCT^ method. Amplifications were carried out using the Applied Biosystems 7500 Real-Time PCR System (Thermo Fisher Scientific, Waltham, MA, USA). Detailed information on the primers used in qRT-PCR analyses is presented in Table 1.Table 1. Primers used in qRT-PCR analyses1Gene SymbolAssay IDPart NumberMarkaCATSPER2Oa04827348_m14448892Applied Biosystems by Thermo Fisher Scientific2CATSPER3Oa04840552_m14448892Applied Biosystems by Thermo Fisher Scientific3CATSPER4Oa04739603_g14448892Applied Biosystems by Thermo Fisher Scientific4TRPM3Oa04910877_m14448892Applied Biosystems by Thermo Fisher Scientific5TRPV5Oa04791756_m14448892Applied Biosystems by Thermo Fisher Scientific6hsa-miR-26a4054427975Applied Biosystems by Thermo Fisher Scientific7hsa-let-7a3774427975Applied Biosystems by Thermo Fisher Scientific8oar-miR-21468542_mat4440886Applied Biosystems by Thermo Fisher Scientific9oar-miR-376a-5p463277_mat4440886Applied Biosystems by Thermo Fisher Scientific10oar-miR-125b469391_mat4440886Applied Biosystems by Thermo Fisher Scientific11bta-miR-1078229_mat4440886Applied Biosystems by Thermo Fisher Scientific12oar-miR-25468990_mat4440886Applied Biosystems by Thermo Fisher Scientific13bta-miR-22-3p242214_mat4440886Applied Biosystems by Thermo Fisher Scientific14oar-miR-10a468629_mat4440886Applied Biosystems by Thermo Fisher Scientific15oar-miR-10b470024_mat4440886Applied Biosystems by Thermo Fisher Scientific16mmu-miR369-5p13094440886Applied Biosystems by Thermo Fisher Scientific17oar-miR-200a468123_mat4440886Applied Biosystems by Thermo Fisher Scientific18oar-miR-23b468816_mat4440886Applied Biosystems by Thermo Fisher Scientific19oar-miR-409-3p463381_mat4440886Applied Biosystems by Thermo Fisher Scientific20oar-miR-1278411_mat4440886Applied Biosystems by Thermo Fisher Scientific21cfa-miR-30a1847_mat4440886Applied Biosystems by Thermo Fisher Scientific22hsa-miR-1524754427975Applied Biosystems by Thermo Fisher Scientific23hsa-miR-323-3p22274427975Applied Biosystems by Thermo Fisher Scientific24hsa-miR-148a4704427975Applied Biosystems by Thermo Fisher Scientific25hsa-miR-27a4084427975Applied Biosystems by Thermo Fisher Scientific26oar-miR-3958-3p464958_mat4440886Applied Biosystems by Thermo Fisher Scientific27rno-miR-494462468_mat4427975Applied Biosystems by Thermo Fisher Scientific28hsa-miR-41012744427975Applied Biosystems by Thermo Fisher Scientific29hsa-miR-1504734427975Applied Biosystems by Thermo Fisher Scientific30hsa-miR-181a4804427975Applied Biosystems by Thermo Fisher Scientific31hsa-miR-119728104427975Applied Biosystems by Thermo Fisher Scientific32cfa-miR-1994405_mat4440886Applied Biosystems by Thermo Fisher Scientific33bta-miR-4857093_mat4440886Applied Biosystems by Thermo Fisher Scientific
Western blot analysis
Before commencing the analytical procedures, the samples underwent additional dilution with PBS and were subsequently centrifuged at 1500 ×g for 10 min at room temperature to separate and remove the yolk content. Western blot analysis was performed to evaluate the expression levels of CatSper3, HSD3β2, and selected growth factor proteins. Initially, the total protein content of the samples was quantified spectrophotometrically using the bicinchoninic acid (BCA) assay (Pierce™ BCA Protein Assay Kit, 500 ml, Thermo Fisher Scientific, Cat. No: 23227, Waltham, MA, USA), in accordance with the method described by Smith et al. (1985). After protein quantification, samples containing equal protein amounts were separated via SDS-PAGE and subsequently transferred to PVDF membranes, following the protocols of Laemmli (1970) and Towbin et al. (1979).
Membranes were incubated overnight at 4 °C with primary antibodies specific to each target protein, diluted in 5% non-fat dry milk: CatSper3 (ab197924, 1:350), HSD3β2 (ab75710, 1 µg/ml), VEGFA (ab52917, 1:7000), PDGFB (ab178409, 1:5000), TGFβ1 (ab9758, 1:500), FGF10 (ab227102, 1:500), and GAPDH (ab22555, 1:5000) (all from Abcam, Cambridge, UK). After primary antibody incubation, the membranes were washed and then incubated with the appropriate HRP-conjugated secondary antibodies: Goat Anti-Rabbit IgG (ab97051, 1:12000) or Goat Anti-Mouse IgG1 (ab97240, 1:12000), also diluted in 5% milk solution. Protein bands were visualized using the ChemiDoc™ XRS+ imaging system (Bio-Rad, Hercules, CA, USA), and band densities were quantified using Image Lab™ Software (v5.2.1, Bio-Rad). Protein expression levels were normalized against GAPDH, which served as a loading control, and results were expressed relative to the control group as a percentage (Kielkopf et al. 2021).
Statistical analysis
SPSS software package (IBM SPSS Statistics for Windows, Version 22.0, IBM Corp., Armonk, NY, USA) was used for statistical analysis. The normality of the raw data obtained from the study was assessed using the Shapiro-Wilk test. To compare differences between the control and PRP groups, an independent samples t-test was applied for parameters with normal distribution, whereas the non-parametric Mann-Whitney U test was used for non-normally distributed parameters. For normally distributed variables, results are presented as mean ± standard error (SE), while for non-normally distributed data, both the mean ± SE and median values are reported. A p-value of less than 0.05 was considered statistically significant.
Results
The effects of intraepidiymal PPR administration on the sperm quality parameters
Compared to the control group, PRP administration significantly increased the total motility (p < 0.01), progressive motility (p < 0.05), and rapid motility (p < 0.01) rates and HOS response (p < 0.05), as well as the kinetic parameters VCL (p < 0.05), VSL (p < 0.05), and VAP (p < 0.05) in frozen-thawed semen. Moreover, the proportions of static spermatozoa (p < 0.01) and acrosome-damaged spermatozoa (p < 0.05) were significantly reduced (Table 2).Table 2. The mean values of spermatological parameters measured in frozen-thawed semen samples from rams in the control and PRP groupsVariablenGroupt-valueP-valueControlPRPMean±SEMedian valueMean±SEMedian valueMotility(%)Total635.77 ± 2.1951.40 ± 2.31**−4.9110.001Progressive611.72 ± 1.2320.88 ± 3.25*−2.6360.025Rapid613.77 ± 2.5227.84 ± 2.55**−3.9270.003Medium67.53 ± 0.896.08 ± 0.631.3250.215Slow614.47 ± 1.0617.48 ± 1.15−1.9350.082KineticStatic664.23 ± 2.1948.60 ± 2.314.9110.001VCL (µm/s)677.62 ± 8.02101.97 ± 5.46*−2.5100.031VSL (µm/s)642.12 ± 4.5260.80 ± 6.98*−2.2480.048VAP (µm/s)650.37 ± 5.3569.57 ± 5.82*−2.4290.036LIN (%)654.20 ± 1.2154.0558.87 ± 4.2056.900.485STR (%)684.05 ± 1.9986.35 ± 2.82−0.6660.520WOB (%)664.72 ± 1.1165.5067.80 ± 2.6966.800.485ALH (µm)63.23 ± 0.223.63 ± 0.11−1.6300.134BCF (Hertz)67.40 ± 0.977.68 ± 0.57−0.2530.805Flow-cytometry(%)Live sperm646.45 ± 1.5746.4153.36 ± 3.7350.590.240Dead sperm652.50 ± 1.3752.7045.89 ± 3.6548.940.240Damaged acrosome644.25 ± 3.1145.3837.76 ± 3.91*41.450.041HMMP641.95 ± 1.4341.0445.74 ± 3.9841.990.589LMMP653.51 ± 1.9946.39 ± 2.792.0780.064Apoptosis619.59 ± 3.2514.66 ± 4.260.9200.379HOS (%)640.75 ± 1.8048.58 ± 2.70*−2.4130.036**Abnormality (%)Head64.92 ± 0.354.08 ± 0.461.4490.178Tail64.00 ± 0.434.253.42 ± 0.583.250.699Total68.92 ± 0.727.50 ± 0.681.4240.185-*P < 0.05 and **P < 0.01 different from control group- Median values are presented for non-normally distributed data, while t-values are presented for normally distributed data
The effects of intraepidiymal PPR administration on the oxidative stress parameters
The levels of MDA and GSH, along with the activities of GPx and CAT in frozen-thawed semen from the control and PRP groups, are presented in Fig. 2. Compared to the control group, PRP administration significantly reduced the MDA level (p < 0.01), while significantly increasing CAT activity (p < 0.01).Fig. 2. The mean values of oxidative stress parameters measured in frozen-thawed semen samples from rams in the control and PRP groups **P < 0.01 and ***P < 0.001 different from control group
The effects of intraepidiymal PPR administration on the lipid values
Table 3 shows the changes in lipid values of frozen-thawed semen. Intraepididymal PRP administration significantly increased the mean cholesterol level compared to the control group (p < 0.05). Among SFAs, palmitic acid (C16:0) was detected at the highest level, whereas margaric acid (C17:0) was found at the lowest level. A significant increase was observed in the mean myristic acid (C14:0, p < 0.05) level in the PRP group compared to the control.Table 3. The mean values of lipids measured in frozen-thawed semen samples from rams in the control and PRP groupsVariablenGroupt-valueP-valueControlPRPMean±SEMedian valueMean±SEMedian valueCholesterol (µg/ml)6111.690 ± 4.401142.383 ± 10.105*−2.7850.019SFAs (%)Myristic acid(C14:0)60.352 ± 0.0120.388 ± 0.011*−2.2580.048Palmitic acid(C16:0)625.216 ± 0.67325.332 ± 0.577−0.1300.899Margaric acid(C17:0)60.194 ± 0.0120.1880.189 ± 0.0190.1800.699Stearic acid(C18:0)611.578 ± 0.30611.938 ± 0.246−0.9200.379Heneicosanoic acid (C21:0)60.295 ± 0.0120.254 ± 0.0152.1170.060MUFAs (%)Pentadecanoic acid (C15:1)60.520 ± 0.0110.511 ± 0.0470.1850.857Palmitoleic acid (C16:1n7)62.330 ± 0.2132.147 ± 0.1320.7310.482Heptadecanoic acid (C17:1)60.060 ± 0.0040.0590.089 ± 0.0150.0840.180Oleic acid(18:1n9)636.660 ± 0.29637.188 ± 0.961−0.5250.611Eicosanoic acid(C20:1)60.244 ± 0.0260.263 ± 0.029−0.4810.641Nervonic acid(C24:1)60.231 ± 0.0110.248 ± 0.013−0.9520.364PUFAs (%)Linoleic acid(C18:2n6)614.974 ± 0.88214.866 ± 0.7070.0950.926Mead acid(C20:3)60.207 ± 0.0150.225 ± 0.016−0.8410.420Arachidonic acid(C20:4n6)62.861 ± 0.1403.141 ± 0.156−1.3390.210Docosapentaenoic acid (C22:5n6)61.330 ± 0.1231.414 ± 0.153−0.4310.675Docosahexaenoic acid (C22:6n3)61.237 ± 0.1611.1581.704 ± 0.2221.8050.132-*P < 0.05 different from control group- Median values are presented for non-normally distributed data, while t-values are presented for normally distributed data
Among MUFAs, oleic acid (C18:1n9) was the most abundant, while heptadecenoic acid (C17:1) was detected at the lowest concentration. Intraepididymal PRP administration had no significant effect on MUFA composition in frozen-thawed semen.
Among PUFAs, linoleic acid (C18:2n6) was present at the highest level, whereas mead acid (C20:3) was the least abundant. No significant effect of intraepididymal PRP administration on PUFA composition in frozen-thawed semen was observed.
The effects of intraepidiymal PPR administration on the values analyzed by ELISA
The findings obtained from the ELISA analysis of frozen-thawed semen samples from rams in the control and PRP groups are presented in Table 4.Table 4. The mean values of steroidogenic molecules, CatSper1 and growth factor levels measured by ELISA in frozen-thawed semen samples from rams in the control and PRP groupsVariablenGroupt-valueP-valueControlPRPMean±SE Median valueMean±SEMedian valueStAR (pg/ml)6136.32 ± 5.41156.16 ± 2.04**−3.4290.006HSD3β1 (ng/l)6632.06 ± 14.48789.95 ± 32.55**−4.4320.001CatSper1 (ng/ml)62.31 ± 0.042.92 ± 0.14**−4.1720.002IGF1(ng/ml)6107.20 ± 5.84112.44 ± 3.68−0.7590.465TGFβ(ng/l)6111.40 ± 7.96106.20 ± 6.030.5210.614VEGF (ng/l)6134.57 ± 3.06133.60123.42 ± 9.97127.220.310PDGF(pg/ml)6179.58 ± 3.45207.41 ± 3.82***−5.4060.000PDGFR (ng/ml)64.76 ± 0.156.04 ± 0.28**−4.0250.002FGF(ng/l)6150.43 ± 3.88148.37155.18 ± 12.66149.090.937-**P < 0.01 and ***P < 0.001 different from control group- Median values are presented for non-normally distributed data, while t-values are presented for normally distributed data
StAR and HSD3β1 Levels
The mean levels of StAR and HSD3β1 in the frozen-thawed spermatozoa of animals treated with intraepididymal PRP were significantly higher compared to the control group (p < 0.01).
CatSper1 level
The mean CatSper1 level in the frozen-thawed spermatozoa of animals treated with intraepididymal PRP was significantly higher than that of the control group (p < 0.01).
Growth factor levels
The mean levels of PDGF (p < 0.001) and PDGFR (p < 0.01) in the frozen-thawed spermatozoa of animals treated with intraepididymal PRP were significantly higher compared to the control group. However, no significant differences were observed between the groups in terms of IGF1, TGFβ, VEGF, and FGF1 levels.
The effects of intraepidiymal PPR administration on the values analyzed by qRT-PCR
The findings obtained from the qRT-PCR analysis of frozen-thawed semen samples from rams in the control and PRP groups are presented in Tables 5 and 6.Table 5. The mean fold-changes of ion channel genes analyzed by qRT-PCR in frozen-thawed semen samples from rams in the control and PRP groupsGene SymbolmRNA Fold-Changep-valueCatSper23.200.017CatSper35.740.008CatSper43.590.014ANO11.970.056TRPM33.580.014TRPV52.830.021-P < 0.05 and **P < 0.01 different from control group-GAPDH was used as control gene- The results are presented as fold increases or decreases relative to the control groupTable 6The mean fold-changes of miRNA genes analyzed by qRT-PCR in frozen-thawed semen samples from rams in the control and PRP groupsGene SymbolmRNA Fold-Changep*-valuehsa-miR-4100.800.405ppy-miR-161.150.569cfa-miR-1990.710.216hsa-miR-148a1.020.941hsa-miR-27a1.170.525hsa-miR-26a1.690.098mmu-miR-369-5p1.350.272bta-miR-1071.210.447hsa-miR-1500.520.056oar-miR-10b1.700.095bta-miR-22-3p0.210.008**rno-miR-4940.180.007hsa-miR-181a0.910.700oar-miR-3958-3p2.080.046oar-miR-1270.790.369oar-miR-23b1.220.433oar-miR-10a0.860.566hsa-miR-11971.000.993oar-miR-409-3p1.080.751bta-miR-4850.760.301oar-miR-200a0.760.311oar-miR-250.730.245oar-miR-376a-5p0.530.061oar-miR-211.800.077hsa-let-7a1.410.216hsa-miR-3741.720.092hsa-miR-1521.130.613cfa-miR-30a1.110.670hsa-miR-323-3p0.930.763oar-miR-125b2.620.025***P < 0.05 different from control group-hsa-miR-191 was used as control gene- The results are presented as fold increases or decreases relative to the control group
Ion channel genes
Compared to the control group, the PRP group showed a statistically significant increase in the presence of CatSper2 (p < 0.01), CatSper3 (p < 0.05), CatSper4 (p < 0.01), TRPM3 (p < 0.05), and TRPV5 (p < 0.05) genes. However, no significant difference was observed between the groups in the presence of the ANO1 gene (Table 5).
miRNA genes
Compared to the control group, PRP administration resulted in a significant decrease in the presence of bta-miR-22-3p and rno-miR-494 (p < 0.01), while causing a significant increase in the presence of oar-miR-3958-3p and oar-miR-125b (p < 0.05). PRP administration had no significant effect on the presence of the other miRNA genes analyzed (Table 6).
The effects of intraepidiymal PPR administration on the values analyzed by western blot
The findings obtained from the Western blot analysis of frozen-thawed semen samples from rams in the control and PRP groups are presented in Fig. 3.Fig. 3. Sperm CatSper3 (A), HSD3β2 (B), VEGFA (C), PDGFB (D), TGFβ1 (E), FGF10 (F) protein expressions, western blot band images (G) *P < 0.05, **P < 0.01, ***P < 0.001 different from control group
CatSper3 protein
The presence of CatSper3 protein in the frozen-thawed spermatozoa of animals treated with intraepididymal PRP was observed to be significantly higher compared to the control group (p < 0.05).
HSD3β2 protein
Intraepididymal PRP administration significantly increased the presence of HSD3β2 protein in frozen-thawed sperm compared to the control group (p < 0.05).
Growth factors proteins
Compared to the control group, the presence of VEGFA (p < 0.001) and TGFβ1 (p < 0.01) proteins in the frozen-thawed sperm of animals treated with intraepididymal PRP were significantly lower, whereas the level of PDGFA protein was significantly higher (p < 0.05). The differences observed in FGF10 protein presence were not statistically significant.
Discussion
As spermatozoa reach the elongated spermatid stage within the testis, their DNA undergoes condensation, leading to the cessation of transcriptional activity. In the caput region of the epididymis, the synthesis of new proteins is minimal; thus, sperm maturation largely relies on sequential interactions with region-specific intraluminal fluids (Dacheux et al. 2009). Throughout epididymal transit, spermatozoa experience a series of biochemical, functional and molecular modifications (Sullivan 1999; Müjica et al. 2003; Sahin et al. 2009). Therefore, intraluminal interventions targeting the epididymis may be reasonably considered a plausible approach to enhance reproductive success in males with fertility disorders. In this context, a previous study suggested that intraepididymal PRP administration exerts beneficial effects on the morpho-functional and biochemical properties of ram spermatozoa (Dayan Cinkara et al. 2025). Given the high cryosensitivity of ram sperm and the reported improvement effects of intraepididymal PRP on spermatozoa, the present study aimed to investigate the cryotolerance of sperm collected from PRP-treated rams. The results demonstrated that frozen-thawed sperm exhibited improved functional, biochemical and molecular characteristics compared to untreated controls.
Semen cryopreservation is a widely used technique for the long-term storage of genetically superior animals, transgenic lines, and endangered species (Bailey et al. 2000, 2003). It plays a key role in the global dissemination of genetic material via assisted reproductive technologies such as artificial insemination and in vitro fertilization (Kumar et al. 2019). However, cryopreservation induces structural and functional damage to spermatozoa, negatively affecting post-thaw quality (Bailey et al. 2000). Species-specific differences in sperm morphology, lipid and protein content limit the universal applicability of cryopreservation protocols (Lv et al. 2019). Farm animal spermatozoa (e.g., bull, ram, boar) are generally more sensitive to cryoinjury than those of humans, rabbits, cats, or dogs (Grötter et al. 2019). Additionally, factors such as cryoprotectant type, semen source (epididymal vs. ejaculated), extender composition, seasonal variation, and individual variability further influence cryopreservation outcomes (Lemma 2011; Yeste 2016).
In this study, semen from rams treated with intraepididymal PRP showed higher cholesterol and myristic acid levels after freezing and thawing compared to controls. Fatty acids and lipids are important for sperm membrane flexibility, motility, and viability, and cholesterol help sperm resist cold shock (Diaz et al. 2016). Cryopreservation changes the membrane lipid composition of ram sperm (Muller et al. 1999). Damage during freezing is mainly caused by ice crystals, high PUFA content, and increased reactive oxygen species (ROS) that lead to LPO (Peris-Frau et al. 2020). PRP contains many bioactive lipids, including PUFA-derived arachidonic acid. These lipids help cell growth and healing (Hoeferlin et al. 2015). Although no studies have examined intraepididymal PRP’s effects on cholesterol and fatty acids in frozen-thawed ram semen, it has been reported that it increases cholesterol levels and certain fatty acids in fresh ram semen, thereby enhancing lipid remodeling (Dayan Cinkara et al. 2025). In contrast, PRP has been shown to reduce cholesterol in other tissues (Hegab et al. 2019). Growth factors in PRP, like PDGF, can stimulate lipid production and regulate cholesterol metabolism in various cells (Demoulin et al. 2004). Other factors like VEGFB, IGF1, TGFβ, and FGF also influence lipid metabolism and cholesterol levels in the body (Burchardt et al. 2012; Yamane et al. 2016; Struik et al. 2019; Moessinger et al. 2020). While the exact reason for increased cholesterol and myristic acid in frozen-thawed semen after PRP treatment is unclear in this study, it is likely related to PRP’s rich bioactive lipids and the effects of its growth factors on lipid metabolism.
The intracellular redox state during cryopreservation affects cell viability. Under the stress conditions of freezing and thawing, antioxidants in semen maintain a balanced system with pro-oxidant metabolism to regulate intracellular redox status. While cryopreservation induces ROS production, it has been suggested that ROS levels increase further during the recovery phase after thawing (Dalton et al. 1999). Additionally, the rise in ROS post-thaw is reported to originate primarily from the mitochondrial electron transport chain (Brouwers and Gadella 2003) and plasma membrane NADPH oxidase (Agarwal et al. 2005). Spermatozoa are well-equipped with enzymatic and non-enzymatic antioxidants; however, removal of seminal plasma or dilution during cryopreservation significantly reduces antioxidant levels. LPO mainly occurs in the midpiece and tail of sperm, with mitochondria acting as a major endogenous ROS source. These organelles contribute to high levels of peroxidation, leading to ATP depletion, loss of motility, DNA damage, and membrane disruption (Brouwers and Gadella 2003; Tatone et al. 2010).
In this study, frozen-thawed semen from intraepididymal PRP-treated rams showed significantly lower mean MDA levels and higher CAT activity compared to controls. Although no previous studies have examined the effect of intraepididymal PRP on oxidative stress in frozen-thawed semen of rams or other species, Dayan Cinkara et al. (2025) reported that intraepididymal PRP reduces LPO and enhances antioxidant activity in native semen in rams. Furthermore, studies on frozen-thawed semen supplemented with in vitro PRP reported slight reductions in ROS levels in human semen (Yan et al. 2021), significant decreases in MDA levels in goat (Salama et al. 2024), ram (Alcay et al. 2022) and bull (El-Sherbiny et al. 2022) semen, increased total antioxidant capacity and superoxide dismutase (SOD) activity in bull semen (El-Sherbiny et al. 2022), as well as elevated GPx and CAT activities in goat (Salama et al. 2024) and bull semen (Almadaly et al. 2023). Although no studies have specifically investigated intraepididymal PRP application, the oxidative stress parameters observed in this study are consistent with previous findings from in vitro PRP treatments (Yan et al. 2021; Alcay et al. 2022; El-Sherbiny et al. 2022; Almadaly et al. 2023; Salama et al. 2024). The likely mechanism behind the observed reduction in MDA levels and increase in CAT activity following intraepididymal PRP treatment is its antioxidant content (Hashem 2020) and its ability to lower ROS levels in sperm (Bader et al. 2020; Yan et al. 2021). Reduced ROS decreases LPO in the sperm membrane, thus lowering MDA, a byproduct of LPO.
Growth factors, produced by nearly all animal cells, primarily function to regulate the cells that produce them and facilitate communication between cells. They help maintain tissue structure and function by modulating cellular behavior through continuous feedback and signaling (Bradshaw et al. 1994). Testicular growth factors can influence the epididymis through lumicrine signaling. Although the presence of a growth factor in a tissue does not always indicate local synthesis, mRNA expression of epidermal growth factor, FGF, VEGF, PDGF, IGF1, TGFβ, nerve growth factor and hepatocyte growth factor has been reported in the epididymis (Tomsig and Turner 2006).
There is no evidence on how intraepididymal PRP affects growth factor-related responses during cryopreservation of ram semen. However, some findings from other PRP applications suggest possible effects: intratesticular PRP reduced elevated TGFβ levels caused by testicular torsion (Gazia 2020); subscrotal PRP did not restore IGF1 mRNA expression in diabetic testes (Istiqamah et al. 2019); and in rabbits, PRP increased VEGF levels in semen collected at weeks 4 and 6, as well as IGF1 levels at week 4 (Abdulla et al. 2022b). On the other hand, cryopreservation reportedly does not affect VEGF expression in human mesenchymal stromal cells (Haack-Sorensen et al. 2007). Also, storing colostrum at −20 °C and − 80 °C did not alter TGFβ1 levels, although long-term storage (12 months) at these temperatures significantly reduced TGFβ1 (Ramirez-Santana et al. 2012). Additionally, in bucks, seminal plasma IGF1 levels positively correlated with sperm motility, viability, and membrane integrity after freezing and thawing (Kumar et al. 2024).
In this study, the levels of PDGF and PDGFR, as well as PDGFB protein expression, were significantly higher, while VEGFA and TGFβ1 protein expressions were significantly lower in the frozen-thawed semen of rams treated with intraepididymal PRP compared to the control group. Previous studies on human (Bogle et al. 2017), pig (Perez-Patino et al. 2019), bull (Arunkumar et al. 2022), ram (Yuxuan et al. 2016), rabbit (Rusco et al. 2022), rooster (Cheng et al. 2015), and sea bass (Zilli et al. 2005) sperm have shown that cryopreservation can lead to quantitative changes in the sperm proteome. These changes primarily affect proteins associated with membrane permeability, metabolism, flagellar structure, motility, intracellular signaling, capacitation, apoptosis, and fertilization. It is suggested that these alterations result from protein loss during the equilibration, freezing, and thawing phases of cryopreservation, mainly due to protein leakage, fragmentation, or degradation (Zilli et al. 2005; Arunkumar et al. 2022). In addition, elevated levels of ROS and other free radicals during cryopreservation can damage both lipids and proteins in spermatozoa (O’Flaherty and Matsushita-Fournier 2017). In this study, the higher levels of PDGF, PDGFR, and PDGFB expression observed in the PRP-treated group may reflect the PDGF-rich nature of PRP itself (Dayan Cinkara et al. 2025). Furthermore, the reduction in ROS levels induced by cryopreservation—evidenced by decreased MDA and increased catalase (CAT) activity in this study—suggests that antioxidants in PRP may help preserve the structural integrity of protein-based growth factors like PDGF. However, the reason for the decreased VEGFA and TGFβ1 expression levels in the PRP group remains unclear.
The StAR protein (Kallen et al. 1998) and the HSD3β enzyme (Lachance et al. 1990), which play a key role in testosterone synthesis from cholesterol, are expressed in Leydig cells, the adrenal cortex, theca cells, and other steroidogenic tissues. However, there is no available information regarding the expression of these two molecules in the epididymis, post-testicular tissues, or spermatozoa of rams. Although studies on tissues other than classical steroidogenic organs (testis, ovary, placenta, adrenal cortex) have shown that the StAR protein is not expressed in the epididymis of poultry (Iamsaard et al. 2016), StAR, HSD3β, and the cytochrome P450scc have been reported in normal human and mouse prostate and penile tissues (Hwang et al. 2011). In the present study, the detection of StAR and HSD3β1 in ram semen (seminal plasma + spermatozoa) by ELISA, and HSD3β2 protein expression by Western blot, suggests that—although not definitively proven—these molecules may either reach the sperm via a lumicrine route from the testis, or be synthesized by tissues of the genital tract or accessory sex glands during post-testicular transit. Cryopreservation is known to increase ROS levels (Dalton et al. 1999), and elevated ROS has been associated with decreased expression of steroidogenic genes (HSD3β, HSD17β, and steroidogenic factor-1) and antioxidant genes (SOD and CAT) in the testis (Saber et al. 2016). On the other hand, certain antioxidants have been shown to mitigate ROS-induced damage to StAR, HSD3β, and steroidogenesis (Glade and Smith 2015; Saber et al. 2016).
To date, no studies have reported how cryopreservation affects StAR and HSD3β levels or expression in ram or other species’ semen. However, a transient increase in StAR gene expression was observed after vitrification and thawing of immature lamb testes using a novel vitrification method (E.Vit) (Bebbere et al. 2019). In this study, significantly higher levels of StAR and HSD3β1, and higher expression of HSD3β2 protein, were observed in the frozen-thawed semen of rams treated with intraepididymal PRP compared to the control group. This suggests that cryopreservation-induced oxidative stress (Güngör et al. 2021) may reduce the levels and expression of StAR and HSD3β. Since ROS is known to damage both lipids and proteins in cells (O’Flaherty and Matsushita-Fournier 2017) and considering that enzymes are also composed of proteins (Robinson 2015), it is evident that both StAR and HSD3β—being protein-based molecules—are susceptible to such damage. Therefore, the elevated levels of these molecules in the PRP group may be explained by the antioxidant properties of PRP (Hashem 2020), which likely reduced ROS production associated with epididymal transit and aerobic sperm metabolism, as well as cryopreservation-induced oxidative stress (also demonstrated in this study), thereby protecting these protein-based molecules from degradation.
Ram spermatozoa contain a higher proportion of PUFAs and a lower cholesterol/phospholipid ratio compared to other species, making them particularly susceptible to damage from excessive ROS production (Holt et al. 1992). During cryopreservation, some of the most commonly observed morpho-functional damages include motility loss, increased proportions of dead and abnormal spermatozoa, and higher rates of spermatozoa with damaged plasma and acrosomal membranes, as well as reductions in kinetic parameters such as VCL, VSL, VAP, and STR (Güngör et al. 2021). Additionally, increased LPO—an indicator of oxidative imbalance—and decreased antioxidant activity have been reported after cryopreservation (Güngör et al. 2021). Numerous scientific studies have explored the supplementation of ram semen with enzymatic (e.g., SOD, GPx, CAT) and non-enzymatic (e.g., vitamins, minerals, fatty acids, amino acids) antioxidants to mitigate or prevent the harmful effects of cryopreservation-induced oxidative stress on semen quality (Ntemka et al. 2018).
In this study, frozen-thawed semen from rams treated with intraepididymal PRP showed higher values of HOS response, total, progressive, and rapid motility, as well as improved kinetic parameters (VCL, VSL, and VAP) compared to the control group, while the proportion of static and acrosome-damaged spermatozoa was lower. To date, no information has been found regarding how the quality of frozen-thawed semen obtained after intraepididymal PRP administration is affected in rams or other species. Nevertheless, several studies report that the addition of PRP to semen in humans (Bader et al. 2020; Yan et al. 2021), bucks (Salama et al. 2024), bulls (El-Sherbiny et al. 2022; Almadaly et al. 2023), rams (Alcay et al. 2022), and stallions (Pinaffi et al. 2021) results in improved post-thaw motility, viability, membrane integrity, antioxidant activity, fertilization capacity, and reduced morphological and acrosomal abnormalities, as well as lower ROS levels compared to controls. In this study, the reason for the better post-thaw semen quality observed in rams treated with intraepididymal PRP compared to the control group may be attributed to the antioxidant effect of PRP reducing the ROS generated during the aerobic metabolism of spermatozoa throughout epididymal transit, thereby protecting the membrane from ROS attacks and making it less vulnerable to cryopreservation-induced damage.
Mammalian spermatozoa are considered transcriptionally and translationally inert, at least at the nuclear level (Oliva 2006). Although transcriptional and translational activities are largely suppressed and most RNAs are lost during cytoplasmic extrusion, a complex and non-random population of small non-coding RNAs (sncRNAs) is retained in spermatozoa (Jodar et al. 2013; Godia et al. 2018). Given that spermatozoa are transcriptionally quiescent, the acquisition of new sncRNAs during post-testicular maturation is likely attributed to active interaction between spermatozoa and the epididymal fluid, particularly through the transfer of RNAs via epididymosomes (Trigg et al. 2019). Some sperm-borne RNAs may also originate from tissues other than the testis and epididymis, such as accessory sex glands, suggesting the involvement of exosomes from multiple sources (Jodar et al. 2013). Sperm-associated miRNAs are involved in the regulation of spermatogenesis (testis), sperm maturation (caput, corpus, and cauda epididymis), sperm–seminal plasma interactions (e.g., epididymosomes), and early embryo development (Alves et al. 2019). The dynamic nature of the non-coding RNA profile, including miRNAs, and its responsiveness to various environmental stressors is gaining increased attention (Trigg et al. 2019). Cryopreservation, a significant stressor on sperm cells, has been shown to affect mRNA expression in human, porcine, and bovine spermatozoa (Yeste 2016). In rams, cryopreservation has been reported to decrease let-7a, miR-485, and miR-29a expression, while increasing miR-127 expression (Güngör et al. 2021). Similarly, Govindaraju et al. (2012) identified several miRNAs in frozen-thawed bull sperm, seven of which showed differential expression related to fertility status, indicating their regulatory importance. miRNAs likely play a critical role in modulating mRNA expression during cryopreservation (Zhang et al. 2017). Specific miRNAs have been linked to key pathways such as ATP metabolism (Nishi et al. 2010), apoptosis (Shangguan et al. 2020), and ROS production (Ulker et al. 2022). Oxidative stress is a key mechanism underlying cryopreservation-induced sperm damage. This link between oxidative stress and miRNAs has prompted investigations into whether antioxidants can modulate miRNA expression during cryopreservation. For example, in rams, the antioxidant C_60_HyFn was shown to reduce miR-200a expression and increase miR-3958-3p levels in frozen-thawed sperm (Güngör et al. 2024a, b).
In the present study, frozen-thawed semen from rams treated with intraepididymal PRP showed decreased expression of bta-miR-22-3p and rno-miR-494, and increased expression of oar-miR-3958-3p and oar-miR-125b compared to controls. miR-22 is known to be upregulated in oligozoospermic men (Özsait Selçuk and Türkölmez 2016) suggesting that its downregulation may be associated with increased semen volume and concentration. Moreover, low miR-22-5p expression has been reported to regulate spermatogonial stem cell renewal via targeting enhancer of zeste homolog 2 (Lv et al. 2022). Although miR-494 upregulation suppresses apoptosis and autophagy in cardiomyocytes via the PI3K/AKT/mTOR pathway (Ning et al. 2020), its downregulation has also been shown to protect cells from TNF-α-induced apoptosis by targeting JunD (Wang et al. 2015). Therefore, both up- and down-regulation of miR-494 may be involved in anti-apoptotic mechanisms. miR-3958-3p has been associated with sustained motility in cryopreserved ram sperm (Güngör et al. 2024a, b b), while miR-125b expression has been reported to be higher in bulls with moderate to high fertility (Fagerlind et al. 2015).
Based on the literature, the downregulation of miR-22 and miR-494, and the upregulation of miR-3958-3p and miR-125b observed in this study appear to positively influence sperm cell function. These findings suggest that the molecular effects of intraepididymal PRP are associated with improvements in the morpho-functional integrity of frozen-thawed ram spermatozoa, consistent with previous studies. On the other hand, in this study, the observed downregulation of bta-miR-22-3p and rno-miR-494 and the upregulation of oar-miR-3958-3p and oar-miR-125b in frozen-thawed ram spermatozoa following intraepididymal PRP administration may be explained by the ability of miRNAs to: (a) activate or inhibit antioxidant enzymes, (b) target genes related to ROS, and (c) modulate specific transcription factors that regulate gene expression (Babu and Tay 2019; Carbonell and Gomes 2020; Lee and Im 2021; Ulker et al. 2022). In addition, the fact that PRP is rich in growth factors (as demonstrated in this study), and that growth factors can regulate miRNA expression—and vice versa, miRNAs can regulate growth factor expression (Kedmi et al. 2015; Li and Wang 2019; Guo et al. 2021)—may represent another underlying mechanism through which PRP influences the expression of these four miRNAs.
Ion channels are responsible for maintaining the intracellular and extracellular balance of ions such as Ca²⁺, K⁺, Cl⁻, Na⁺, and HCO₃⁻. Alterations in these channels can disrupt ion movement across the membrane, leading to cellular damage (Shukla et al. 2012). CatSper is a unique, sperm-specific, low-voltage-gated and pH-dependent ion channel located in the flagellum, responsible for the influx of Ca²⁺ ions in spermatozoa (Antonouli et al. 2024). Calcium-activated Cl⁻ channels, such as ANO1 (also known as TMEM16A), are anionic channels involved in regulating several physiological processes in somatic cells through Cl⁻ and Ca²⁺ transport (Segura-Covarrubias et al. 2020). TRP channels and their various subtypes, together with other ion channels, play a role in regulating Ca²⁺ homeostasis and are involved in the regulation of spermatozoa functions. Ion channels are essential for key sperm functions such as motility, hyperactivation, chemotaxis, capacitation, thermotaxis, and the acrosome reaction (Kumar et al. 2018). Biochemically, ion channels are composed of transmembrane proteins containing cysteine and methionine residues with sulfhydryl groups involved in gating, conduction, and associated signaling pathways. These structural features make ion channels particularly vulnerable to ROS and free radical attacks, which can alter their function (Ramirez et al. 2016). ROS and other reactive agents are known to modulate ion channel activity and disrupt their physiological functions (Kiselyov and Muallem 2016). Cryopreservation has been reported to affect the expression of ion channels such as CatSper family, ANO1, KCNJ11, TRPM, HVCN1, SLO1, and Hv1 in spermatozoa from humans, bulls, rams, buffalo, and macaques (Blasse et al. 2012; Chen et al. 2017; Alshawa et al. 2019; Dalal et al. 2020; Güngör et al. 2021; Delgado-Bermudez et al. 2022).
The use of antioxidant additives like epicatechin, low-density lipoproteins, MitoTEMPO, and C_60_HyFn has been shown to mitigate cryopreservation-induced ion channel damage (Dalal et al. 2020; Kumar et al. 2022; Banas et al. 2023; Güngör et al. 2024a, b b). In the present study, intraepididymal PRP administration significantly increased mRNA presence of CatSper2, CatSper3, CatSper4, TRPM3, and TRPV5 in frozen-thawed spermatozoa compared to the control group, while it had no significant effect on ANO1 presence. Given the known positive association between ion channel stability and sperm motility, these improvements in ion channel expression in the PRP group are consistent with the enhanced motility observed. To our knowledge, no previous studies have investigated the effect of intraepididymal PRP on ion channel expression in cryopreserved ram spermatozoa. However, a similar study by Abdulla et al. (2022a) in rabbits reported a significant increase in CatSper1 mRNA expression in frozen-thawed spermatozoa from animals treated with intratesticular PRP, suggesting a protective effect of PRP on ion channels. During cryopreservation, both the increase in ROS production and cholesterol efflux negatively affect sperm cryotolerance and post-thaw capacitation ability (Antonouli et al. 2024). In this context, PRP’s antioxidant capacity to reduce ROS and its ability to elevate cholesterol levels (both demonstrated in this study) make it a promising strategy for preventing or minimizing post-thaw degradation of sperm ion channels. The antioxidant molecules, growth factors, lipids, and ions released from PRP granules (McNicol et al. 1999; Marx 2004; Hoeferlin et al. 2015) may protect the sperm membrane from lipid and protein peroxidation, thereby preserving ion channel proteins during cryopreservation. These mechanisms support the hypothesis that intraepididymal PRP application helps protect sperm ion channels by mitigating ROS generation during both epididymal transport and cryopreservation.
Conclusion
The results of this study demonstrate that spermatozoa collected from rams treated with intraepididymal PRP are more resistant to cryopreservation-induced damage compared to the untreated group. This improved cryoresistance may be attributed to the multifaceted effects of PRP, which is rich in growth factors, lipids, and antioxidant molecules. These components appear to positively influence several critical parameters in frozen-thawed semen, including growth factors, cholesterol content, oxidative stress levels, the expression of specific ion channels and microRNAs, as well as steroidogenic molecules, StAR and HSD3β. Taken together, these findings suggest that intraepididymal PRP injection may represent a promising biotechnological intervention to enhance semen cryotolerance in mature rams.