Lip-1 (>98% HPLC) was obtained from Chengdu Preferred Biotechnology Co., Ltd. (Chengdu, China), while the kits for malondialdehyde (MDA), lipid peroxidation (LPO), GSH, and total antioxidant capacity (T-AOC) were sourced from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). Unless otherwise stated, all chemicals used in this study were purchased from Sigma-Aldrich (St. Louis, MO, USA). The modified Modena extender contains 46.64 mM Tris, 152.64 mM glucose, 15.09 mM citric acid, 26.74 mM sodium citrate, 6.98 mM EDTA-2Na·H₂O, 11.90 mM sodium bicarbonate, 1 million U/L penicillin, 1 million U/L streptomycin, and 4.00 g/L BSA, which is prepared prior to use and stored at 4°C.

In this experiment, six Large White boars aged 2 to 3 years were utilized. The boars were individually housed at a controlled temperature of 20 ± 2°C and were provided unrestricted access to food (Shenyang Hefeng Animal Husbandry, Shenyang, China) and water, ensuring no impact on the quality of boar semen. For each experiment, semen was collected from a boar using the gloved-hand method, and sperm motility was subsequently analyzed using the Computer Assisted Semen Analysis (CASA) system (Songjingtianlun Biotechnology, Nanning, China). The samples were transported to the laboratory within 2 h and were utilized for subsequent experiments only when sperm total motility exceeded 80%. Initially, various concentrations of Lip-1 were added to the modified Modena extender of boar semen, which was maintained in liquid form at 17°C. Sperm motility was assessed on days 1, 3, 5, and 7 of preservation to determine the optimal concentration of Lip-1. Additionally, sperm quality was evaluated at 0, 1, 3, 5, and 7 days, measuring parameters such as the integrity of the acrosome and plasma membrane, and mitochondrial membrane potential (MMP). The assessment included measures of antioxidant capacity, such as GSH and T-AOC; Lipid peroxidation index MDA, LPO, and ferroptosis indicators C11-bodipy. Additionally, we utilized the ferroptosis inducer erastin (Era, 1 μM) to investigate the mechanism by which Lip-1 enhances boar semen fluid preservation efficiency at 17°C.

Transfer 10 μL of the sample onto a glass slide and cover it with a coverslip. Analyze the motility and kinetic parameters of the sperm using the CASA system, which includes total motility, progressive motility, average path velocity (VAP), average straight-line velocity (VSL), and average curvilinear velocity (VCL). The standard parameter for CASA was set to 30 frames/s. Sperm with a straightness of path (STR) greater than 75% and VSL exceeding 25 μm/s were classified as motile. Each sample was randomly selected across five fields, with a minimum of 200 sperm measured using the accompanying software.

MMP was detected using JC-1 and propidium iodide (PI) as in the previous study with appropriate modifications (13). The JC-1 probe aggregates in the mitochondrial matrix to form a polymer when the MMP is high (hMMP), while it is excluded from the mitochondrial matrix in cases of low MMP. Total 50 μL sample was mixed with 200 μL of an isotonic buffer diluent containing 5 mM PI and 1 mM JC-1, and the mixture was incubated for 30 min at 37°C. Subsequently, a 10 μL aliquot of the semen sample was placed onto a coverslip with a glass slide and observed using an inverted fluorescence microscope (Mshot Optoelectronics Technology, Guangzhou, China), with analysis performed using the associated software. More than 200 sperm were examined per visual field. Red fluorescence on the head of the sperm indicates hMMP, while green fluorescence represents medium to low MMP.

The integrity of the sperm acrosome (14) was analyzed according to previous studies with appropriate modifications. 30 μL of the sample was placed on a slide, allowed to air dry, and then fixed with methanol for 10 min at 23°C. Each slide was treated with FITC-PNA solution (100 μg/mL) and incubated at 37°C for 10 min in the dark. After incubation, the slides were rinsed with phosphate-buffered saline (PBS) and mounted with an antifade solution to prevent fluorescence degradation. Finally, the slides were covered with a coverslip and photographed using an epifluorescence microscope to evaluate acrosome integrity.

Sperm plasma membrane staining method was adapted from a previous study with appropriate modifications (15). A 250 μL sample was centrifuged at 1200 r/min for 2 min. The supernatant was discarded, and 500 μL of HEPES buffer containing 10% BSA was added. Subsequently, 5 μL SYBR-14 dye working solution (0.1 mmol/mL) was added and incubated at 37°C for 5 min, followed by the addition of 5 μL of PI working solution (0.1 mg/mL) and incubation at 37°C for an additional 5 to 10 min. Observations were conducted using a fluorescence microscope in a dark room, examining 5 fields of view per group, with each field containing no fewer than 200 sperm.

LPO content, MDA content, GSH content, and T-AOC activity in sperm were assessed following the kit instructions and measured using a microplate reader. The parameters for the microplate reader were set to 450 nm, 532 nm, 412 nm, and 593 nm, respectively.

The semen samples from each treatment group were centrifuged and subsequently re-suspended in 500 μL of C11-bodipy working solution (1 μM), followed by incubation at 37°C in the dark for 1 h. After incubation, the semen samples were centrifuged again and washed three times with PBS. The stained sperm samples were re-suspended in PBS to achieve a density of 10⁶ sperm/mL and analyzed using a flow cytometer (Beckman Coulter, Brea, USA). The fluorescence signal intensity of the sperm was assessed through the red fluorescence channel at 488/510 nm, with a total of 10,000 sperm detected for each sample.

The expression of GPX4 protein in boar sperm was analyzed using immunofluorescence staining. Semen samples were washed three times with PBS, and the sperm density was adjusted to 10⁶/mL. A 30 μL aliquot of the semen sample suspension was prepared for smearing, fixed in 4% paraformaldehyde for 10 min, and subsequently permeabilized with 0.5% Triton® X-100 for 30 min. Following three washes with PBS, 200 μL of 5% BSA was added to block non-specific binding, and the samples were incubated at 37°C for 30 min. Subsequently, the samples were centrifuged, and the supernatant was carefully removed. Meanwhile, 200 μL of diluted GPX4 antibodies (1:1000) were added and incubated at 4°C overnight. Afterward, the relevant fluorescent secondary antibodies (1:1000) were applied for incubation at 37°C for 1 h, and sperm cell nuclei were stained with 4′,6-diamidine-2-phenylindole dihydrochloride (DAPI). The slides were sealed, and images were captured using a fluorescence microscope (Mshot Photoelectric Technology, Guangzhou, China), with data digitization performed using iMage software (Olympus, Tokyo, Japan).

Statistical analysis of experimental data was performed using SPSS 17.0. All data from each experiment were tested for normality with one-sample Kolmogorov–Smirnov’s test. If some data did not show normality, we arcsine-transformed the variable before the analysis and again checked the normality using one sample Kolmogorove-Smirnov’s test for this parameter. Furthermore, all data from each experiment were checked for the homogeneity of variances using Levene’s test. The test results showed that the replicated data from each experiment were homogeneous. The values are presented as mean ± standard deviation (SD), and multiple comparisons were analyzed using one-way ANOVA followed by Tukey’s multiple-comparison test. The differences between the two groups of data were assessed using an unpaired two-tailed Student t-test. Significant differences are represented by p < 0.05.

To investigate the protective effect of different concentrations Lip-1 on sperm damage during the liquid preservation of boar semen at 17°C, we examined sperm motility and kinetic parameters. The results indicated that, in comparison to the control group, treatments with 0.1 μM and 0.4 μM Lip-1 did not produce significantly effects on sperm total motility on days from 1 to 7 (Table 1, p > 0.05). In contrast, treatment with 0.2 μM Lip-1 significantly improved sperm total motility compared to the control group from days 3 to 7 (Table 1, p < 0.05). Additionally, 0.1 μM Lip-1 significantly improved the progressive motility of sperm relative to the control group on day 1 (Table 2, p < 0.05), it did not significantly affect sperm progressive motility on days from 3 to 7 (Table 2, p > 0.05). Meanwhile, compared to the control group, 0.4 μM Lip-1 showed no significant effects on sperm progressive motility on days from 1 to 7 day (Table 2, p > 0.05). In contrast, 0.2 μM Lip-1 significantly improved the sperm progressive motility from 1 to 7 day in comparison to the control group (Table 2, p < 0.05).

For sperm kinetic parameters, on day 1, 0.2 μM Lip-1 significantly increased sperm VAP (Table 3, p < 0.05) compared to the control group, while 0.1, 0.2 and 0.4 μM Lip-1 Lip significantly enhanced sperm VSL (Table 4, p < 0.05) and VCL (Table 5, p < 0.05). Furthermore, on day 3, concentrations of 0.1 μM and 0.4 μM Lip-1 did not demonstrate any significant effects on sperm VAP (Table 3, p > 0.05), VSL (Table 4, p > 0.05), and VCL (Table 5, p > 0.05); a concentration of 0.2 μM Lip-1 significantly improved all three parameters (Tables 3–5, p < 0.05). Moreover, on day 5, 0.2 μM Lip-1 significantly increased sperm VAP (Table 3, p < 0.05), VSL (Table 4, p < 0.05) and VCL (Table 5, p < 0.05). Importantly, on day 7, both 0.1 μM and 0.2 μM concentrations of Lip-1 significantly increased sperm VAP (Table 3, p < 0.05). Additionally, 0.2 μM Lip-1 notably enhanced sperm VSL (Table 4, p < 0.05), while both 0.2 μM and 0.4 μM concentrations of Lip-1 significantly improved sperm VCL (Table 5, p < 0.05). In summary, a concentration of 0.2 μM Lip-1 significantly enhances sperm motility and kinetic parameters during the liquid storage of pig semen at 17°C. Consequently, 0.2 μM Lip-1 was chosen for subsequent experiments.

The integrity of the acrosome in spermatozoa is a critical factor for the occurrence of the acrosomal reaction during fertilization and for the successful penetration of sperm into oocytes (16, 17). Consequently, we investigated the effect of 0.2 μM Lip-1 on the acrosomal integrity of sperm during liquid storage at 17°C. The result showed that, on days 1, 3, and 5, treatment with 0.2 μM Lip-1 did not significantly (Figure 1A, p > 0.05) effect the acrosome integrity of sperm compared to the control group; however, on day 7, it significantly enhanced the acrosome integrity (Figure 1A, p < 0.05).

Additionally, the integrity of the sperm plasma membrane serves as a crucial barrier between sperm cells and the external environment, significantly influencing several key reproductive processes, including sperm survival, capacitation, and acrosomal reaction (18). In this study, we utilized SYBR-14/PI staining to assess the integrity of the sperm cell membrane. Specifically, ‘a’ denotes intact sperm cell membranes, whereas ‘b’ signifies damaged sperm cell membranes (Figure 1B). The result showed that, on day 1, relative to the control group, treatment with 0.2 μM Lip-1 showed no significant (Figure 1C, p > 0.05) effect on plasma membrane integrity; however, on days 3, 5, and 7, it significantly (Figure 1C, p < 0.05) improved this effect.

Sperm with low MMP demonstrate a reduced capacity for ATP production, whereas those with hMMP are capable of generating greater amounts of ATP to facilitate sperm motility (19, 20). Consequently, we investigated the effect of 0.2 μM Lip-1 on the MMP of sperm. The result showed that, on day 1, compared to the control group, treatment with 0.2 μM Lip-1 had no significant (Figure 2, p > 0.05) effect on the hMMP of sperm; nonetheless, on days 3, 5, and 7, it significantly (Figure 2, p < 0.05) enhanced the hMMP.

During the liquid preservation of boar semen at room temperature, oxidative stress emerges as a significant factor contributing to sperm damage (6). Consequently, we measured the T-AOC activity and GSH content in sperm during liquid preservation. The results indicated that, on days from 1 to 7, compared to the control group, 0.2 μM Lip-1 significantly increased the activity of T-AOC (Figure 3A, p < 0.05) in sperm. Meanwhile, on days from 3 to 7, the content of GSH in 0.2 μM Lip-1 group was significantly higher than that of control group (Figure 3B, p < 0.05).

Since Lip-1 is a ferroptosis inhibitor that can protect cells by regulating lipid peroxidation (11), we examined lipid peroxide markers, specifically C11-bodipy, LPO, and MDA. The results showed that, compared to the control group, treatment with 0.2 μM Lip-1 significantly inhibited the levels of C11-bodipy in sperm on day 5 (Figures 4A,B, p < 0.05). Additionally, on days from 1 to 7, Lip-1 significantly reduced the content of MDA in sperm compared to the control group (Figure 4C, p < 0.05). Moreover, on days 3, 5, and 7, Lip-1 significantly decreased the content of LPO (Figure 4D, p < 0.05) in sperm relative to the control group. Furthermore, we examined ferroptosis-related protein GPX4 in sperm. The results indicated that, compared to the control group, Lip-1 significantly enhanced GPX4 protein levels in boar sperm during in vitro liquid preservation at room temperature on day 5 (Figure 5, p < 0.05).

To confirm that Lip-1 enhances boar sperm quality by regulating ferroptosis, we utilized the ferroptosis inducer, Era, to assess the protective effect of Lip-1 against Era-induced injury in boar sperm. The results showed that Era significantly decreased sperm total motility (Table 6, p < 0.05), progressive motility (Table 6, p < 0.05), VAP (Table 6, p < 0.05), VSL (Table 6, p < 0.05), VCL (Table 6, p < 0.05), and BCF (Table 6, p < 0.05) compared to the control group, but Lip-1 was significantly (Table 6, p < 0.05) recovered these changes.

Moreover, Lip-1 improved the integrity of sperm acrosome and plasma membrane, and hMMP in Era-treated sperm as shown in Figure 6. In comparison to the control group, Era significantly reduced boar sperm quality including the integrity of acrosome (Figure 6A, p < 0.05) and plasma membrane (Figure 6B, p < 0.05), and MMP (Figure 6C, p < 0.05), whereas 0.2 μM Lip-1 significantly restored these damages (Figures 6A–C, p < 0.05).

To further investigate the effect of Lip-1 on boar sperm quality by the regulation of ferroptosis, we assessed both the lipid peroxidation capacity and the antioxidant capacity of the sperm. The results demonstrated that Era significantly increased the levels of C11-bodipy (Figures 7A,B, p < 0.05), LPO (Figure 7C, p < 0.05), and MDA (Figure 7D, p < 0.05) compared with the control group, but 0.2 μM Lip-1 significantly restored these changes (Figures 7A–D, p < 0.05). Furthermore, in comparison to the control group, Era significantly reduced the antioxidant capacity of sperm, primarily including the activity of T-AOC (Figure 7E, p < 0.05) and the content of GSH (Figure 7F, p < 0.05), whereas the application of 0.2 μM Lip-1 notably ameliorated these alterations (Figures 7E,F, p < 0.05).