Ginseng Peptide Improves the Cryopreservation Efficiency and Fertilization Potential of Yak Semen via FOXO1/PI3K/AKT Axis

TL;DR

Ginseng peptides improve yak semen cryopreservation and fertility by reducing stress and enhancing cell function.

Contribution

Ginseng peptide (GFREH) at 0.75 mg/mL optimizes yak sperm cryopreservation via the FOXO1/PI3K/AKT pathway.

Findings

GFREH improved sperm motility, membrane integrity, and reduced oxidative stress in frozen-thawed yak sperm.

GFREH increased in vitro fertilization and blastocyst formation rates at 0.5–1.0 mg/mL concentrations.

Proteomic analysis showed GFREH modulates PI3K/AKT signaling and downregulates FOXO1 expression.

Abstract

Semen cryopreservation is a critical biotechnological approach for preserving superior genetic resources in livestock. Spermatozoa are particularly vulnerable to cryogenic stress during the freeze–thaw process, resulting in impaired structure and function. Therefore, the development of effective cryoprotective additives is essential for improving yak semen cryopreservation. In this study, ginseng peptide (GFREH) was incorporated into the freezing extender at different concentrations (0, 0.25, 0.5, 0.75, and 1.0 mg/mL) to evaluate its effects on post-thaw sperm quality, in vitro fertilization (IVF) capacity, and the underlying regulatory mechanisms. Semen samples treated with 0 and 0.75 mg/mL GFREH were further subjected to proteomic analysis to elucidate the molecular basis of its cryoprotective action. The results demonstrated that GFREH significantly increased total motility (TM), progressive motility (PM), straight-line velocity (VSL), curvilinear velocity (VCL), average path velocity (VAP), as well as plasma membrane and acrosome integrity of frozen–thawed yak spermatozoa (p < 0.05). GFREH also significantly reduced malondialdehyde (MDA) levels while enhancing antioxidant enzyme activities, mitochondrial membrane potential (MMP), and ATP content (p < 0.05). Moreover, GFREH at concentrations of 0.5, 0.75, and 1.0 mg/mL significantly improved IVF and blastocyst formation rates compared with the control (p < 0.05), with the 0.75 mg/mL group exhibiting the highest fertilization and blastocyst rates. Proteomic analysis further revealed that GFREH modulated the PI3K/AKT signaling pathway and downregulated FOXO1 expression. Collectively, these findings indicate that ginseng peptides enhance yak sperm cryotolerance by coordinating oxidative balance, mitochondrial energy metabolism, and survival-related signaling, with 0.75 mg/mL representing an optimal effective concentration within the functional dose range tested.

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1. Introduction

Yaks represent an important livestock resource in the Qinghai–Tibet Plateau and surrounding regions [1]. Compared to ordinary cattle, yak possess remarkable adaptability to high-altitude environments [2]. Their unique physiological and metabolic regulatory mechanisms enable them to maintain normal growth and reproduction, under extreme high-altitude conditions such as hypoxia, cold temperatures, and intense ultraviolet radiation [3,4]. However, due to their inherently slow reproductive rate and limitations in production performance, overall yak productivity remains low [5]. Semen cryopreservation enables the global dissemination of superior genetic material through artificial insemination (AI), providing substantial benefits to the livestock industry, particularly for ruminants [6]. Artificial insemination not only effectively increases reproduction rates but also improves the genetic quality of yak populations affected by long-term inbreeding. To be effective for AI, semen must undergo both short-term and long-term cryopreservation [7]. Sperm quality after freeze–thaw cycles is also critical for normal fertilization. Accordingly, research on semen cryopreservation has long focused on improving the efficiency and success rate of sperm freezing [8].

During the freezing process, spermatozoa are highly sensitive to multiple stressors. Protein denaturation and intracellular ice crystal formation can directly impair sperm physiology, while excessive production of reactive oxygen species (ROS), cytoplasmic instability, membrane phase transitions, ion imbalance, and aberrant protease activation or inactivation may collectively induce oxidative stress [9,10]. These disruptions compromise mitochondrial integrity, plasma membrane stability, and acrosomal structure, ultimately resulting in sperm functional deterioration. To mitigate such freeze-induced damage, specific cryoprotectants must be incorporated into semen extenders to preserve sperm viability and function [11]. Numerous studies have demonstrated that enrichment of cryoprotectants with antioxidants can significantly attenuate cryo-induced oxidative stress [12]. For instance, glutathione [13], melatonin [14], and resveratrol effectively neutralize excess ROS [15], enhance antioxidant enzyme activities, and reduce lipid peroxidation products such as malondialdehyde, thereby safeguarding the structural integrity and functional competence of spermatozoa.

In recent years, increasing attention has been directed toward natural antioxidants due to the potential safety concerns associated with synthetic compounds. Among these, bioactive peptides have attracted considerable interest owing to their high biosafety, low immunogenicity, ease of synthesis, and potent antioxidant properties. The major bioactive constituents of ginseng include saponins, polysaccharides, and peptides [16]. Ginseng peptide (GFREH) is an enzymatic hydrolysate purified from ginseng roots using alkaline protease. GFREH exhibits diverse biological activities, including antioxidant effects [17], anti-inflammatory and immunomodulatory actions [18], glucose-lowering and hepatoprotective functions, as well as anti-aging and anti-fatigue effects [19]. Previous studies have shown that, in neuronal cells, ginseng peptides effectively inhibit L-Glu–induced oxidative damage, suppress Ca^2+^ influx, and prevent apoptosis, thereby protecting overall cellular function [20]. Moreover, they can restore nitric oxide (NO) signaling and bioavailability, attenuate oxidative stress, and protect endothelial cells from dysfunction [21].

This study investigated the effects of supplementing sperm cryoprotectant with various concentrations of ginseng peptides on the post-thaw structural integrity, motility, antioxidant capacity, and fertilization potential of yak sperm. Employing proteomics, we further elucidated the molecular mechanisms through which ginseng peptides enhance cryo-survival and identified key signaling pathways involved. The findings provide a theoretical basis for improving the cryopreservation system for yak semen.

2. Materials and Methods

2.1. Sperm Collection and Vitality Analysis

The semen samples used in this study were collected from the Longri Livestock Farm in Hongyuan County, Sichuan Province, China. Semen was collected from six sexually mature, healthy male yaks, with three ejaculates obtained from each male (total n = 18). Semen was collected individually using standard electroejaculation, and no pooling was performed. Immediately after collection, semen quality was assessed using a computer-assisted sperm analysis (CASA) system according to standardized protocols. Samples with sperm motility >90% were selected for subsequent experiments. Biological replicates corresponded to samples from different individual males, whereas repeated collections from the same male were treated as technical replicates.

2.2. Semen Freezing-Thawing Treatment

Freshly collected semen was immediately diluted (1:1, v/v) with a Tris–citric acid–fructose based transport extender containing egg yolk (R23022, Yuanye, Shanghai, China) to protect spermatozoa against cold shock, and then transported to the laboratory at 4 °C in insulated containers under dark conditions. Upon arrival, the samples were kept in the dark and allowed to equilibrate at room temperature for 30 min before further processing. GFREH powder was weighed, dissolved in distilled water, and incorporated into the thawed freezing extender to obtain final concentrations of 0.25, 0.5, 0.75, and 1.0 mg/mL. Spermatozoa equilibrated at room temperature were mixed with the freezing extender at a 1:1 ratio, after which the GFREH-supplemented extenders were added according to the respective treatment groups. The control group received the same extender without GFREH. After thorough mixing, the samples were allowed to stand at room temperature for 3 min. The semen mixtures were then loaded into 0.25 mL cryovials, sealed, and placed in prepared gauze bags. The samples were equilibrated at 4 °C for 1 h. A foam container was pre-cooled by filling it with liquid nitrogen for 5–7 min, and the cryovials were positioned 12–15 cm above the liquid nitrogen surface for 15 min to allow vapor freezing. After cool-down, the samples were submerged and stored in liquid nitrogen for one week. For thawing, the cryovials were rapidly removed from liquid nitrogen and placed in a 37 °C water bath for 1 min. The seals were then cut open to release the thawed semen into centrifuge tubes for subsequent analyses.

2.3. Sperm Motility Measurement

After thawing, semen samples were immediately analyzed using a computer-assisted sperm analysis (CASA) system (AndroVision, Minitube, Tiefenbach, Germany) with the following settings: frame rate, 60 Hz; minimum sperm size, 4 μm^2^; VAP threshold, 20 μm/s; VSL threshold, 30 μm/s; progressive motility was defined as VAP > 50 μm/s and STR > 70%. A microscope slide and coverslip were prepared in advance, and 2 μL of semen was pipetted onto the slide. The coverslip was gently placed over the droplet using tweezers, taking care to avoid air bubbles and without applying pressure. The sample was examined under the microscope, and 10 randomly selected fields per treatment group were analyzed to assess total motility and progressive motility. Sperm kinematics, including linear velocity (VSL), curvilinear velocity (VCL), and average path velocity (VAP), were recorded and statistically analyzed. All procedures were performed under minimal light exposure to prevent photo-induced sperm damage.

2.4. Sperm Membrane Integrity

Twenty-five microliters of thawed semen were placed into a brown centrifuge tube, and an equal volume of the Hoechst 33342/PI (Meilunbio, Dalian, Liaoning, China) mixture was added and thoroughly mixed. One microliter of the dye mixture was then added to the 25 μL semen sample and mixed gently. The samples were wrapped in aluminum foil and maintained in a 37 °C incubator in the dark for 15 min. After incubation, 10 μL of the stained semen was pipetted onto a glass slide and covered with a coverslip. Membrane integrity was assessed under a fluorescence microscope by analyzing 10 randomly selected fields per sample.

2.5. Sperm Acrosome Integrity

FITC-PNA (Sigma-Aldrich, St. Louis, MO, USA) and Hoechst 33342 were mixed at a 3:1 ratio, and 8 μL of this mixture was added to the 25 μL semen sample and mixed thoroughly. Samples were wrapped in aluminum foil and incubated in the dark at 37 °C for 20 min. After incubation, 10 μL of the stained semen was pipetted onto a glass slide and covered with a coverslip. 10 randomly selected fields per sample were analyzed under a fluorescence microscope to assess membrane and acrosome integrity.

2.6. Evaluation of ROS Level

ROS levels were measured using a commercial ROS detection kit (R6033, Uland, Nanjing, Jiangsu, China) according to the manufacturer’s protocol. Briefly, semen samples were incubated with the dye, washed to remove unbound probes, and then prepared into smears for analysis. Sections were observed under a fluorescence microscope (Olympus, Tokyo, Japan) at 630× magnification to assess overall ROS generation within sperm cells. Fluorescence intensity density was measured using a multimode microplate reader (SpectraMax iD3, Molecular Devices, San Jose, CA, USA). The relative fluorescence density for each sample was normalized based on the average fluorescence intensity of the 0 h control group.

2.7. MDA Content Detection

MDA content was measured using a commercial kit (A003-1-2, Jiancheng, Jiangsu, China). After thawing, semen samples were diluted with PBS at a ratio of 1:1 and transferred to centrifuge tubes. Reagents were added sequentially, mixed with a vortex mixer, and incubated in a 95 °C water bath for 40 min. After incubation, immediately place the samples under running water to cooled. Centrifuge at 12,000 rpm for 10 min at 4 °C. Subsequently, transfer 200 μL of supernatant to a 96-well plate. Measure absorbance at 523 nm using a microplate reader, with distilled water as the blank control.

2.8. Sperm Antioxidant Indicators

Commercial kits were used to detect the primary components of the antioxidant system in thawed yak sperm to evaluate its antioxidant capacity. The detection indicators primarily included superoxide dismutase (SOD; A001-3-2, Jiancheng, Nanjing, Jiangsu, China), catalase (CAT; A007-1-1, Jiancheng, Nanjing, Jiangsu, China), total antioxidant capacity (T-AOC; A015-3-1, Jiancheng, Nanjing, Jiangsu, China), and glutathione peroxidase (GSH-Px; S0056, Shanghai Bio-Tech, Shanghai, China). The specific operational procedures followed the manufacturers’ protocols. In brief, spermatozoa were resuspended in phosphate-buffered saline (PBS) after centrifugation. Cells were then disrupted using a homogenizer (SCIENTZIID, Ningbo, Zhejiang, China). Subsequently, samples were centrifuged at 12,000 rpm for 5 min at 4 °C, and the supernatant was collected for analysis. Each assay was performed according to the respective kit instructions. Absorbance was measured at the recommended wavelength using a spectrophotometer (Multiskan Sky, Thermo Scientific, Shanghai, China). Antioxidant levels were determined based on the standard curve provided with the kit.

2.9. Detection of Mitochondrial Membrane Potential

The commercial JC-1 mitochondrial membrane potential assay kit (J6004L, Uland, Nanjing, Jiangsu, China) was used to assess sperm MMP. Briefly, JC-1 working solution was added to each semen-containing centrifuge tube, mixed by gentle inversion several times, and incubated at 37 °C for 20 min. After incubation, samples were centrifuged at 1000 rpm for 5 min to remove the supernatant. Pellets were resuspended in pre-cooled 1× Assay Buffer, centrifuged at 1000 rpm for 5 min, and the supernatant was removed. This step was repeated once. The pellet was then resuspended in pre-cooled 1× Assay Buffer, the tube was covered, mixed using a vortex mixer, and 200 μL of supernatant was transferred to a 96-well plate for measurement using a fluorescence microplate reader. A portion of the sample was placed on a glass slide, covered with a coverslip, and observed and imaged under a fluorescence microscope.

2.10. ATP Content Assay

ATP content in sperm was measured using a commercial kit (A095-1-1, Jiancheng, Nanjing, Jiangsu, China). Place the thawed sperm sample into a centrifuge tube and centrifuge at 5000 rpm for 5 min at 4 °C. Discard the supernatant, leaving the pellet at the bottom of the tube. Resuspend the pellet in PBS, wash three times, and disrupt using an ultrasonic cell disruptor. Then centrifuge at 4 °C at 10,000 rpm for 3 min, collect the supernatant for detection.

2.11. In Vitro Fertilization Capability Assessment

Collected yak ovaries were washed three times with physiological saline containing 2% dual antibiotics. Follicular fluid was aspirated with a 10 mL syringe and transferred to cell culture dishes. Cumulus–oocyte complexes (COCs) with homogeneous cytoplasm, intact zona pellucida, and at least three layers of compact cumulus cells were selected under a stereomicroscope using egg-picking needles. Each treatment group contained 30 COCs per replicate. Selected COCs were washed two to three times and cultured in pre-equilibrated oocyte maturation medium (BO-HEPES-IVM™, HEPES-buffered oocyte maturation medium, IVF Bioscience, Falmouth, UK) at 38.5 °C in 5% CO_2_ for 22–24 h. Frozen yak semen was thawed in a 37 °C water bath for 30 s, transferred to capacitation medium (modified Tyrode’s medium containing 10 μg/mL heparin and 3 mg/mL BSA), and incubated at 37 °C for 1 h. During this period, mature oocytes were washed twice and placed in fresh fertilization medium (BO-IVF™, Fertilization medium, IVF Bioscience, Falmouth, UK). Fully capacitated sperm were added to the culture dishes at a final concentration of 1 × 10^6^ sperm/mL and co-incubated with mature oocytes at 38.5 °C in 5% CO_2_ for 20–24 h. Presumptive zygotes were treated with 0.1% (w/v) hyaluronidase to remove cumulus cells and transferred into embryo culture medium (BO-IVC™, Embryo culture medium, IVF Bioscience, Falmouth, UK) for further development. Cleavage and blastocyst formation rates were recorded.

2.12. Labeled Quantitative Proteomics

The group without GFREH added to the sperm cryoprotectant was defined as the control group, while the group with 0.75 mg/mL GFREH added to the sperm cryoprotectant was defined as the experimental group. After thawing frozen semen from control and experimental yaks, samples were centrifuged at 800× g for 5 min in a pre-cooled high-speed refrigerated centrifuge to remove the cryodiluent. The pellet was resuspended in pre-cooled PBS, centrifuged at 800× g for 5 min, and the supernatant was removed. This procedure was repeated three times. The purified sperm was sent to Shanghai Majorbio Biomedical Technology Co, Ltd. (Shanghai, China) for proteomic experiment.

2.13. Real-Time Quantitative PCR

Total RNA was isolated using the Total RNA Isolation Kit (R1017, Zymo Research Corporation, Irvine, CA, USA) according to the manufacturer’s instructions. Reverse transcription of 1 µg of RNA was performed with the 2 × SYBR Green qRCR Mixture (Vazyme, Nanjing, Jiansu, China, R233-01). The quantitative PCR was performed using cDNA templates diluted in nuclease-free water, with amplification and detection carried out on the LightCycler^®^96 Real-Time PCR System (Roche Diagnostics, Basel, Switzerland). All samples were analyzed in triplicate. Using standardized β-actin as an internal reference, expression analyses of selected target genes were performed using the 2^−ΔΔCT^ method [22].

2.14. Western Blot

After thawing yak semen samples, centrifuge to remove the supernatant. Subsequently, extract sperm proteins using the Total Protein Extraction Kit (BC3710, Solarbio, Beijing, China). Determine protein concentration with the BCA Kit (PC0020, Solarbio, Beijing, China). Total protein was separated by SDS-PAGE and transferred onto a PVDF membrane (FFP22, Beyotime, Shanghai, China), which was blocked with 5% skim milk at room temperature for 2 h. The membrane was incubated overnight at 4 °C. After three washes with TBST, the membrane was incubated with HRP-conjugated goat anti-rabbit IgG secondary antibody at room temperature for 2 h. The membrane was washed three times with TBST, visualized using an ECL kit (P0018AS, Beyotime, Shanghai, China), and images were captured using an imaging system. The primary antibody information used in the study is as follows: PI3K (1: 1000, AF6241, Affinity, Beijing, China), AKT (1: 1000, AF4691, Cell Signaling, Shanghai, China), FOXO1 (1: 3000, Huaan Biotechnology, Hongzhou, China), β-actin (1: 10,000, AF7018, Affinity, Liyang, Jiangsu, China), IgG (H + L) (1: 5000, S0001, Affinity, Liyang, Jiangsu, China). The gray values of each protein band were calculated using ImageJ software(Verson 1.48).

2.15. Data Statistical Analysis

Raw data were analyzed using SPSS 26 (SPSS Inc., Chicago, IL, USA), and two-way analysis of variance (ANOVA) was performed using GraphPad Prism (version: 8.01) to compare mean values among treatment groups. Each experiment was conducted in triplicate, and the mean value of the replicates was calculated. Results are presented as mean ± standard error of the mean (SEM). Differences were considered statistically significant at p < 0.05.

3. Results

3.1. Effects of GFREH on Sperm Vitality

The effect of GFREH on the motility of frozen-thawed yak sperm is shown in Figure 1. The result of frozen–thawed sperm (Figure 1A) revealed that compared with the control group, total motility was significantly increased in sperm treated with 0.25, 0.5, 0.75, and 1 mg/mL GFREH (Figure 1B, p < 0.05). Among these, sperm treated with 0.75 mg/mL GFREH exhibited the highest total motility (p < 0.05), whereas the 1 mg/mL group showed lower motility than the 0.75 mg/mL group (p < 0.05). The forward motility (Figure 1C) showed that sperm treated with 0.5, 0.75, and 1 mg/mL GFREH had significantly higher forward motility compared with the control group (p < 0.05). Among these, the 0.75 mg/mL group exhibited the highest forward motility (p < 0.05). Compared with the control, sperm treated with 0.25, 0.5, 0.75, and 1 mg/mL GFREH exhibited significantly higher VSL, VCL and VAP (Figure 1D–F), with the 0.75 mg/mL group showing the highest VSL, VCL and VAP, whereas the 1 mg/mL group displayed lower VSL than the 0.75 mg/mL group (p < 0.05).

3.2. Effects of GFREH Supplementation on Membrane and Acrosome Integrity of Frozen Sperm

The fluorescence-based assessment of plasma membrane integrity in frozen–thawed yak sperm treated with different concentrations of GFREH is shown in Figure 2A. The plasma membrane integrity revealed that, compared with the control, sperm treated with 0.25, 0.5, 0.75, and 1 mg/mL GFREH exhibited reduced membrane damage. Among these, the 0.75 mg/mL group showed the largest reduction in membrane damage (p < 0.05), whereas no significant difference was observed between the 0.25 and 1 mg/mL groups (Figure 2B, p > 0.05). The fluorescence-based assessment of acrosome integrity in yak sperm treated with different concentrations of GFREH is shown in Figure 2C. Following Hoechst 33342 staining, the sperm nucleus emits blue fluorescence. Concurrently, if the acrosome is intact, FITC-PNA staining produces crescent-shaped green fluorescence that overlays the blue nuclear signal. Conversely, if only blue fluorescence is observed without overlapping green fluorescence, the acrosome is considered damaged. The acrosome integrity analysis showed that sperm treated with 0.5, 0.75, and 1 mg/mL GFREH exhibited significantly reduced acrosome damage compared to the control (p < 0.05). Although the 0.75 mg/mL group had the numerically lowest damage rate, the differences among the three GFREH treated groups were not statistically significant (Figure 2D).

3.3. The Effect of GFREH on Sperm Antioxidant Indicators

ROS levels in sperm treated with ROS detection reagents are shown in Figure 3A. Green fluorescence intensity indicates ROS content, with higher fluorescence corresponding to increased ROS levels. Compared to the control group, adding 0.5, 0.75, and 1 mg/mL GFREH to the semen dilution medium significantly reduced ROS levels in freeze-thawed yak sperm (p < 0.05). Among these treatment groups, the 0.75 mg/mL GFREH group exhibited the lowest ROS content (Figure 3B). Similarly, malondialdehyde (MDA) levels were significantly decreased in all GFREH treated groups compared with the control (Figure 3C, p < 0.05), with the 0.75 mg/mL group showing the lowest MDA content. Treatment with 0.5, 0.75, and 1 mg/mL GFREH significantly increased superoxide dismutase (SOD) and CAT levels (Figure 3E,F, p < 0.05), with the 0.75 mg/mL group exhibiting the highest SOD content. Moreover, total antioxidant capacity (T-AOC) and GSH-Px was significantly increased in all treatment groups (Figure 3D,G, p < 0.05), with the 0.75 mg/mL group exhibiting the strongest antioxidant capacity.

3.4. Effects of GFREH on Sperm Mitochondrial Function

Mitochondrial membrane potential (MMP) in yak sperm was assessed (Figure 4A). Addition of GFREH to the sperm diluent significantly increased MMP (p < 0.05), with the 0.75 mg/mL group exhibiting the highest MMP. In contrast, the 1 mg/mL GFREH group showed a lower MMP than the 0.75 mg/mL group (p < 0.05). ATP levels in yak sperm were measured (Figure 4B). Compared with the control, the 0.25 mg/mL GFREH group showed no significant change in ATP content (p > 0.05), whereas the 0.5, 0.75, and 1 mg/mL GFREH groups exhibited significant increases (p < 0.05), with the 0.75 mg/mL group showing the highest ATP level (p < 0.05).

3.5. Effects of GFREH Treatment on the Fertilization Potential of Frozen Sperm

The yak sperm fertilization rates at different GFREH concentrations revealed that treatment with 0.5 mg/mL, 0.75 mg/mL, and 1 mg/mL GFREH significantly increased fertilization rates (p < 0.05), with the 0.75 mg/mL group showing the highest fertilization rate (Figure 5A, p < 0.05). Blastocyst rate analysis indicated that addition of 0.5 mg/mL, 0.75 mg/mL, and 1 mg/mL GFREH significantly increased the blastocyst rate compared with the control (p < 0.05). The 0.75 mg/mL group exhibited the highest blastocyst rate (p < 0.05), whereas the 1 mg/mL group showed a decrease compared with the control (Figure 5B, p < 0.05).

3.6. Identification and Analysis of Proteomics

Using a significance threshold of p ≤ 0.05 and FC ≥ 1.2 or ≤0.83, 85 differentially expressed proteins (DEPs) were identified, comprising 42 upregulated proteins and 43 downregulated proteins. Among these, FOXO1 exhibited the highest fold change among downregulated proteins in the experimental group compared to the control group (Figure 6A). KEGG enrichment analysis indicates that DEPs are primarily enriched in ECM-receptor interactions, ribosomes, focal adhesions, and the PI3K-AKT signaling pathway. The downregulated differential protein FOXO1 is also enriched in the PI3K-AKT signaling pathway (Figure 6B,D). Gene ontology (GO) analysis revealed that DEPs were primarily enriched in biological processes such as cellular processes, metabolism, biological regulation, localization, development, homeostasis, and immune system functions. In terms of cellular components, enrichment was observed in cell structures, organelles, and protein complexes. Regarding molecular functions, DEPs were mainly associated with binding, catalytic activity, transporter activity, molecular function regulation, and ATP-dependent activity (Figure 6C).

3.7. GFREH Reduces FOXO1 Expression Levels Through the PI3K/AKT Signaling Axis

Analysis of mRNA expression levels of PIK3R1, AKT1, and FOXO1 in the control and 0.75 mg/mL GFREH treatment groups revealed that 0.75 mg/mL GFREH significantly increased PIK3R1 and AKT1 expression (p < 0.05) while decreasing FOXO1 expression (p < 0.05). The mRNA levels in yak sperm treated with an AKT activator demonstrated that the 0.75 mg/mL GFREH group, 0 + AKT activator group, and 0.75 + AKT activator group all significantly upregulated PIK3R1 and AKT1 expression (p < 0.05) and downregulated FOXO1 expression (p < 0.05). In the 0 + PI3K inhibitor group, mRNA levels did not differ significantly from the control (p > 0.05). However, in the 0.75 + PI3K inhibitor group, PIK3R1 and AKT1 expression was significantly reduced while FOXO1 expression was increased compared with the 0.75 mg/mL GFREH group (Figure 7A–C, p < 0.05). Treatment with 0.75 mg/mL GFREH significantly increased PI3K and AKT protein expression (p < 0.05) and decreased FOXO1 expression (p < 0.05). Analysis of protein expression in sperm treated with an AKT activator showed that the 0.75 mg/mL GFREH group, 0 + AKT activator group, and 0.75 + AKT activator group all significantly upregulated PI3K and AKT expression (p < 0.05) and downregulated FOXO1 expression (p < 0.05). Protein expression analysis following PI3K inhibitor treatment indicated that the 0.75 mg/mL GFREH group significantly increased PI3K and AKT expression (p < 0.05) and decreased FOXO1 expression (p < 0.05). In the 0 + PI3K inhibitor group, protein levels did not differ significantly from the control (p > 0.05). However, in the 0.75 + PI3K inhibitor group, PI3K and AKT expression was significantly reduced, while FOXO1 expression was increased compared with the 0.75 mg/mL GFREH group (Figure 7D–F, p < 0.05).

4. Discussion

Semen freezing represents a major advancement in artificial insemination technology, facilitating the utilization of high-quality male livestock and accelerating the improvement of livestock breeds [23]. Simultaneously, it provides convenient channels for livestock introduction, promoting the exchange of high-quality semen across regions [24].

Sperm vitality and motility are key indicators of sperm quality and are widely used in evaluating sperm function [25,26,27]. Studies have demonstrated that at a concentration of 50 nM, progesterone significantly enhances the vitality of vitrified sperm and optimizes motility-related parameters of frozen sperm [28]. Adding 100 μM cysteine to buffalo semen enhances post-thaw sperm motility, antioxidant capacity, and DNA integrity [29]. Furthermore, during sperm capacitation, the plasma membrane participates in the acrosome reaction and plays a crucial role [30]. Intracellular calcium fine balance in the sperm cytoplasm is strictly dependent on sperm surface channels including the CatSper channel. Disruption or mutation of CatSper leads to impaired male fertility [31,32]. Supplementation with bovine serum albumin (BSA) in thawing diluents effectively enhances sperm plasma membrane integrity and acrosome status after thawing [33]. Adding melatonin (MLT) improves sperm cryopreservation outcomes by regulating receptors (MT1/MT2) on the sperm membrane, thereby influencing sperm capacitation, the acrosome reaction, lipid peroxidation, and DNA integrity [34]. Our results demonstrate that GFREH exerts a clear dose-dependent effect on yak sperm quality. While GFREH improved sperm quality and related parameters at 0.5 and 0.75 mg/mL, the effect was slightly reduced at 1 mg/mL. This may reflect a saturation effect or potential mild cytotoxicity at higher concentrations. Notably, the 0.75 mg/mL concentration produced the most stable and robust improvements, particularly in forward motility, linear velocity, and plasma membrane integrity. This pattern suggests that GFREH acts within an optimal concentration window, beyond which its beneficial effects may be attenuated due to saturation of cellular targets or the onset of mild cytotoxic or oxidative stress at higher doses.

ROS refers to the collective term for substances composed of oxygen and reactive oxygen species within the body or natural environment [35]. Elevated levels of ROS can adversely affect sperm motility, including damage to the sperm plasma membrane, mitochondria, DNA, and epigenetics [36]. Studies have shown that polysaccharides, used as protective agents, effectively reduce reactive oxygen species (ROS) levels during the freeze–thaw process [37]. In studies on rooster semen cryopreservation, supplementation with alpha-linolenic acid (ALA) significantly increased the activity of antioxidant enzymes, including superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px), while simultaneously reducing malondialdehyde (MDA) levels [38]. Moreover, ALA improved mitochondrial membrane potential (MMP) and decreased both early and late-stage sperm apoptosis [39]. In addition to small-molecule antioxidants, ginseng and its bioactive components have demonstrated significant protective effects on male reproductive function across multiple animal models. In avian models, supplementation of freezing media with Panax ginseng extract improved post-thaw sperm motility, membrane integrity, and antioxidant enzyme activity while reducing lipid peroxidation [40]. In ruminants, aqueous Panax ginseng extract enhanced the quality of chilled and cryopreserved bull sperm in a dose-dependent manner, maintaining motility, viability, and chromatin integrity, while pure ginseng improved testicular function in rams, as reflected in hormonal and histological parameters [41,42]. Although studies on ginseng peptides in yak or other ruminant semen are limited, these findings collectively support the antioxidant and cytoprotective potential of ginseng-derived compounds for improving sperm quality across taxa. The results of this study are consistent with these findings. Compared with the control group, GFREH supplementation significantly increased the levels of antioxidant enzymes, including SOD and T-AOC, in yak sperm, enhanced total antioxidant capacity, and reduced MDA content. These results indicate that GFREH supplementation enhances the antioxidant capacity of frozen yak sperm and mitigates freeze-induced oxidative stress. During cryopreservation, sperm mitochondria are highly susceptible to damage. Based on these findings, it is suggested that enhanced cryoprotectants may help preserve the structural integrity and functional stability of vulnerable sperm mitochondria during cryopreservation, thereby preventing the initiation of apoptosis [43]. These results suggest that GFREH exerts similar antioxidant effects and may function as an antioxidant in yak semen cryoprotectant diluents.

In the freeze-thawed cycle, freezing damage will lead to mRNA degradation, affecting the function and expression of fertility-related proteins [44]. To optimize and improve semen cryopreservation techniques, researchers have employed differential proteomics to investigate the mechanisms of freezing-induced injury across different species [45,46]. Bovine sperm proteomics reveal that antioxidant enzymes maintain sperm viability by regulating ROS generated during cryopreservation, thereby preventing oxidative stress and apoptosis in sperm [47]. Supplementing sperm cryoprotectants with 10 μM mitoquinone (MitoQ) enhances antioxidant capacity and glucose transporter abundance in boar sperm [48]. In this study, it was found that adding 0.75 mg/mL GFREH to sperm cryoprotectants resulted in DEPs primarily participating in biological processes such as transporter activity and ATP-dependent activity. Among these, FOXO1 protein exhibited the highest fold change among downregulated proteins in the experimental group compared to the control group. This indicates that FOXO1 plays a crucial regulatory role during sperm cryopreservation.

The forkhead box O1 (FOXO1) gene functions in multiple aspects including cell proliferation, apoptosis, inflammatory response, immune differentiation, and antioxidant stress [49]. In polycystic ovary syndrome (PCOS), FOXO1 is considered a key regulator of chronic inflammation, and knocking down FOXO1 can alleviate inflammatory and immune responses in a rat model of PCOS [49,50]. Studies have shown that non-esterified fatty acids (NEFA) significantly inhibit phosphatidylinositol 3-kinase (PI3K) and phosphorylated protein kinase B (p-AKT) activity, while increasing the expression of FOXO1 [51]. The mechanism of FOXO1 inhibition through the PI3K-AKT signaling pathway aligns with reports from other cellular systems AKT-mediated phosphorylation can lead to cytoplasmic retention of FOXO transcription factors, consequently suppressing the transcription of pro-apoptotic and oxidative stress-related genes [52,53]. This suggests that PI3K-AKT-FOXO1 regulation may be a conserved mechanism protecting sperm from cryopreservation-induced oxidative damage. In this study, activation of the PI3K-AKT signaling pathway inhibited the expression of the key protein FOXO1, thereby reducing oxidative stress in yak sperm, enhancing antioxidant defenses, and improving sperm quality. These results provide a theoretical foundation for use of GFREH as a cryoprotectant for yak semen.

5. Conclusions

This study demonstrates that ginseng peptides serve as an effective natural cryoprotectant for yak semen, improving post-thaw sperm quality by synergistically regulating oxidative balance, mitochondrial function, and survival-related signaling pathways. Within the tested concentration range (0.5–1.0 mg/mL), GFREH exhibited a pronounced dose-dependent protective pattern, with the 0.75 mg/mL concentration achieving an optimal balance between efficacy and cellular tolerance. Proteomics evidence further suggests that activation of the PI3K/AKT-FOXO1 axis may underlie its cytoprotective action, revealing the molecular mechanism by which GFREH promotes sperm survival under cryostress conditions. Collectively, these findings highlight the potential of ginseng peptides as bioactive additives for improving yak semen cryopreservation and support further evaluation of their optimal dosage and application in livestock reproductive biotechnology.