Carboxyfullerene C₆₀ preserves porcine sperm by enhancing antioxidant capacity and inhibiting apoptosis and harmful bacteria

Abstract

This study used a porcine model to systematically investigate whether carboxyfullerene C₆₀(CF-C₆₀) can be used for sperm preservation. The results indicated that CF-C₆₀ supplementation can preserve porcine sperm quality during storage at 17 °C. This effect was attributable to an improvement in the antioxidant capacity of sperm through a decrease in the reactive oxygen species (ROS) level. Additionally, CF-C₆₀ can maintain mitochondrial function, inhibit sperm apoptosis through the ROS/Cytochrome C (Cyt C)/Caspase 3 signaling pathway, and mediate suppression of bacterial growth through the effects of ROS. Finally, the results of artificial insemination experiments indicated that insemination with CF-C₆₀-treated sperm can increase the total number of offspring born and reduce the number of deformed piglets. Thus, CF-C₆₀ is safe for use as a component of semen diluent for sperm storage.

This research unveils that CF-C₆₀ improves porcine sperm quality by decreasing the levels of ROS to enhance antioxidation, antiapoptosis, and antibiosis during liquid storage at 17 °C. The findings provide a novel technical basis for expanding the application of carbon nano-materials as antioxidants in porcine reproduction.

Introduction

To date, preserved porcine sperm has been used on a global scale for artificial insemination (AI). However, there are still many limitations, mainly low-temperature intolerance, oxidative stress, and bacterial contamination. Spermatozoa integrity is necessary for successful fertilization (Liu et al., 2023). Due to the low cholesterol/phospholipid ratio in the cell membrane of porcine sperm, sperm exhibit poor tolerance to low temperatures (Hai et al., 2024). Therefore, the majority of pig farms choose the sperm stored at 17 °C for AI. Nonetheless, sperm preserved at 17 °C also faces challenges. Oxidative stress and bacterial contamination are accepted to be the main contributing factors, reducing the quality and quantity of porcine sperm and possibly even affecting the ability to fertilize and reproduce (Hussain et al., 2023). Efficient preservation of sperm high-quality and normal functioning sperm, which is an important basis for the preservation of fertility and the treatment of porcine subfertility. Currently, there is a need for effective semen diluents that can be used for the liquid preservation of porcine sperm.

Recent scientific advances in nanotechnology and materials have had implications for the development of novel sperm protectors for the preservation of porcine fertility. One of these materials is fullerene, which is the third carbon allotrope that was discovered after graphite and diamond. The surface of fullerene contains a large number of unsaturated double bonds that are able to accept electrons; as a result, it is able to react with free radicals and is known as a “free radical sponge” (Zhao et al., 2021). Of the fullerene family members, C₆₀ is the most common and stable one. Fullerene C₆₀ is a nonpolar molecule with a large π bond, and its special cage structure leads to its strong hydrophobic properties. It has been reported that underived fullerene C₆₀ has low solubility in polar solvents and a solubility of only 1.3 × 10⁻¹¹ g/L in water at 17 °C (Kamat et al., 2022). Therefore, chemical modification methods based on the amino, carboxyl, and hydroxyl groups are often used to modify its physical and chemical properties and improve its bioavailability.

Carboxyfullerene C₆₀ (CF-C₆₀), is one of the most important nanocarbon derivatives with an antioxidant capacity that is hundreds of times greater than that of other antioxidants (Monti et al., 2000; Rezaee et al., 2018). Previous reports have shown that fullerene derivatives act as superoxide dismutase (SOD) mimics; thus, they may have the ability to reduce mitochondrial superoxide production and improve mitochondrial function (Quick et al., 2008). Moreover, in recent years, CF-C₆₀ has been found to have neuroprotective, antiviral, antineoplastic (Huo et al., 2022), antiapoptotic, and drug delivery properties, which are mainly determined by its antioxidant capacity. In fact, researchers have found that CF-C₆₀ can reduce oxidative stress in the brains of mice, block the production of mitochondrial free radicals, and improve cognitive performance, thus significantly extending the lifespan of rodents (Ali et al., 2008). Another study demonstrated that CF-C₆₀ inhibited death receptor-mediated apoptosis by upregulating the expression of Hsp70 and interacting with lysosomal membranes (Li et al., 2011). Additionally, a number of studies have shown that some fullerene derivatives have bactericidal effects and can significantly inhibit Candida albicans, Bacillus subtilis, Enterococcus, and Mycobacterium avium (Bosi et al., 2000). As oxidative stress is known to reduce the quality and quantity of sperm and possibly even affect the porcine ability to fertilize and reproduce (Cattelan and Gasparini, 2021), CF-C₆₀ could be a potentially useful molecule in porcine sperm preservation methods. However, it is not clear whether the ability of CF-C₆₀ to reduce reactive oxygen species (ROS) levels and its antibacterial effects could also be useful for sperm preservation.

In this study, we have explored the protective effects of CF-C₆₀ in the liquid preservation of porcine sperm. We explored the effects of CF-C₆₀ on sperm quality and its antioxidant, antiapoptotic, and bactericidal abilities, as well as the underlying mechanisms. Furthermore, the safety and practical application of CF-C₆₀ were evaluated by AI experiments in pigs.

Materials and Methods

All experimental procedures involving animals were conducted in accordance with 125 the guidelines established by the Northwest A&F University Animal Care Committee and received approval (approval number NWAFU-20210517).

Synthesis of CF-C₆₀

CF-C₆₀ samples with a purity of 96% were obtained from Beijing Zhongke Leiming Technology Co. Ltd. The carboxylation process was carried out as previously reported and was based on the transfer of fullerene from the organic phase to the aqueous phase by physical mixing and ultrasonic treatment (Andrievsky et al., 2009). Briefly, the prepared crude product fullerene C₆₀ mixture was first subjected to a sulfuric acid/nitric acid reflux process for ultrasonic treatment. It was then extracted with benzene and filtered through a paper funnel to obtain the mixed solvent, which was finally purified by high-performance liquid chromatography to obtain CF-C₆₀.

Characterization of CF-C₆₀

In this study, a field emission scanning electron microscope (SEM; Nano SEM-450, FEI Company, USA) was used to analyze the surface morphology of CF-C₆₀. Briefly, C₆₀ was compacted on a conductive tape, and the vacuum gold spraying pretreatment was performed. To evaluate the stability of CF-C₆₀ in the aqueous phase, we measured zeta potential using a nanoparticle size potentiometric analyzer (Zetasizer Pro, Malvern Panalytical Ltd.). Briefly, we took 1 mL of the liquid, injected it into the sample pool at a slow and uniform rate, and finally inserted it into the sample tank for detection. The size distribution of CF-C₆₀ was measured with a laser particle size analyzer (Mastersizer 3,000).

Animals

The semen samples used in this study were from 8 healthy adult Duroc boars (aged 24 to 26 mo) that had been raised at Shaanxi Zhengneng Breeding Pig Gene Technology Co. Ltd. (China, Shaanxi). The experimental animals were strictly raised under standard feeding conditions (Supplementary Table S1). A specialist at the farm was responsible for feeding management, training, semen collection, and other related work based on the requirements of subsequent experiments.

Semen samples, processing, and preservation

The traditional hand-held method was used to collect boar semen, and only the semen in the middle of the ejaculation was collected in commercial bags (11022/0011, Minitube Biotechnology [Beijing] Co.; Zhu, 2015; Sancho and Vilagran, 2013). Near the pregnant sow and boar station, there is a sperm laboratory where the collected sperm can be swiftly transferred via a dedicated channel for immediate quality testing. The quality of the sperm was determined based on the following criteria: ≥5.0 × 10⁸/mL sperm concentration, ≥80% total motility sperm, and ≥80% normal morphology (Pavaneli et al., 2019). The qualified semen samples were mixed with Beltsville thawing solution (BTS, containing 37.15 g glucose, 6.00 g sodium citrate, 1.25 g/L EDTA, 1.25 g/L sodium bicarbonate, and 0.75 g/L potassium chloride) in a beaker and were stored in a constant temperature box (BC-95MN, KONKA Group Co., Ltd.) at 17 °C. The sperm samples were diluted with BTS at a ratio of 1: 3, where 1 mL of fresh sperm was mixed with 3 mL of BTS, resulting in a final concentration of approximately 50 million sperm cells per milliliter after dilution. The volume of the liquid used for sperm preservation is 8 mL, which is contained in a 10 mL centrifuge tube (C1010-P, Thomas Scientific, LLC). For AI, the liquid volume is 80 mL, and it is contained in an insemination bag (80 mL Boar Sperm Bag, Jiangs Animal Products Co., Ltd.). Then, different concentrations of CF-C₆₀, respectively, were added to the diluent, and the treatment concentrations were 1, 2, 3, 4, and 5 μg /mL. When the sperm was stored at 17 °C for 1, 3, 5, and 7 d, 100 µL sperm were randomly extracted from each treatment group at regular intervals in a 1.5 mL centrifuge tube and incubated in a 37 °C water bath for 10 ~ 20 min. Then 5 ~ 10 µL sperm drops were taken on a preheated special slide to detect sperm parameters.

Sperm motility

To explore the effect of CF-C₆₀ on sperm function, we detected changes in sperm quality parameters in groups with/without CF-C₆₀ supplementation during liquid storage at 17 °C. A computer-assisted sperm analysis system (CASA, V8.1, 400×, Fuzhou Hongye Software Technology Co. Ltd.) was used to measure sperm motility (total motility; Pavaneli et al., 2019, 2020), curvilinear velocity (VCL μm/s); straight linear velocity (VSL μm/s); average path velocity (VAP μm/s); linearity [LIN = (VSL/VCL) × 100%]; straightness[STR = (VSL/VAP) × 100%] and beat cross frequency [BCF (Hz)]. First, a 100 µL aliquot was taken from each treatment group at 1, 3, 5, and 7 d of storage and collected in a 1.5 mL centrifuge tube that was placed in a 37 °C water bath for 20 min. The sperm quality was then measured using the CASA system by placing a 10 µL semen drop on a preheated special slide (CASA, V8.1, 400×, Fuzhou Hongshiye Software Technology Co. Ltd.) onto a microscope (CX31RTSF). Each CASA video segment in our study spanned a duration of 5 min. This timeframe was deemed adequate for capturing the dynamic movements of sperm and was congruent with the range of image analysis computation time per group. We randomly selected 5 visual fields harboring more than 200 sperm for observation.

Plasma membrane integrity

Sperm plasma membrane integrity was assessed by SYBR14 and propidium iodide (PI, Gemme Gene, Shanghai) staining according to the manufacturer’s instructions. Briefly, 100 µL of semen was randomly selected for each treatment group at 1, 3, 5, and 7 d of storage and collected in a 1.5 mL centrifuge tube that was placed in a 37 °C water bath for 30 min. Later, the sperm samples were incubated with SYBR14 at 37 °C for 10 min and PI for 10 min. Sperm with intact plasma membrane exhibited green fluorescence when exposed to a wavelength of 520 nm, while sperm with damaged plasma membrane exhibited red fluorescence at 630 nm. Utilize fluorescence microscopy (40×, Cytation 5, United States BioTek) for observation and systematically select 5 distinct and visually clear fields of view, ensuring that each encompasses a minimum of 200 sperm cells for analysis.

Acrosomal integrity

The integrity of the sperm acrosome was evaluated by the fluorescein isothiocyanate-conjugated peanut agglutinin (PNA-FITC; Jemei Gene, Shanghai) dual-fluorescence staining technique. Briefly, 100 µL of semen was randomly selected for each treatment group at 1, 3, 5, and 7 d of storage in a 1.5-mL centrifuge tube that was placed in a 37 °C water bath for 30 min. Then, the sperm samples were incubated with PNA-FITC at room temperature for 5 min and DAPI for 20 min at 37 °C. Sperm with intact acrosomes emit green fluorescence at 533 nm and exhibit a complete cap structure. Sperm with damaged or detached acrosome cap structures do not exhibit fluorescence. The experiment was repeated 4 times and 5 fields of view were selected for observation in each group using fluorescence microscopy (40×, Cytation 5, United States BioTek), each containing at least 200 sperm.

Detection of T-AOC, MDA, SOD, and ROS

The total antioxidant capacity (T-AOC, A015-2-1, 405 nm, Nanjing Jiancheng Bioengineering Institute, Jiangsu, China; Liang et al., 2018), malondialdehyde (MDA, A003-1-1, 532 nm, Nanjing Jiancheng Bioengineering Institute, Jiangsu, China; Loh et al., 2010), SOD (A001-3-2, 450 nm, Nanjing Jiancheng Bioengineering Institute, Jiangsu, China; Loh et al., 2010), and ROS (E004-1-1, Jiangsu Jingmei Biological Technology Co. Ltd., Jiangsu, China, Optimal excitation wavelength 488, optimal emission wavelength 525; Zhang et al., 2020) of sperm stored for 1, 3, 5, and 7 d were detected according to the instructions provided with the assay kits, and all experiments in this study were conducted at least 4 times. Briefly, T-AOC and MDA content were evaluated using a spectrophotometer. T-AOC was expressed as U/mL, and the MDA content was expressed in nmol/mL. A double-antibody one-step sandwich enzyme-linked immunosorbent assay was used to detect ROS. Specimens, standards, and HRP-labeled detection antibodies were added to the coated micropores pre-coated with ROS antibodies and then incubated and thoroughly washed. Absorbance (based on the OD value) was measured by a spectrophotometer (RF-6000, Shimadzu Corporation, Japan) at a wavelength of 450 nm, and the sample concentration was calculated.

Mitochondrial membrane potential and ATP content

Mitochondrial membrane potential was assessed using the JC-1 kit (5,5ʹ,6,6ʹ-tetrachloro-1,1ʹ,3,3ʹ-tetraethylbenzimi-dazolylcarbocyanine iodide; Solarbio Science & Technology Co., Beijing, China) as described in a previous study (Kuwahara et al., 2016). Briefly, 1 × 10⁶ to 6 × 10⁷ cells were stained with 0.5 mL JC-1 working solution (50 μL JC-1 [200×], 8 mL ultra-pure water, and 2 mL JC-1 staining buffer [5×]), incubated in a cell incubator at 37 °C for 20 min. Finally, 1.25 mL dyeing buffer (1×) was re-suspended, and the solution was observed with a spectrophotometer. The ATP content of semen samples stored at 1, 3, 5, and 7 d was measured with an ATP assay kit (Nanjing Jian Cheng Institute of Biological Engineering, 636 nm) according to the manufacturer’s instructions.

Bacterial colony counts and compositions

To assess the antibacterial effect of CF-C₆₀, sperm bacterial concentrations during storage were measured using the Luria-Bertani (LB) agar plate method as described in the literature (Ngamdee et al., 2015). Briefly, 100 µL diluted bacterial solution was uniformly coated on LB solid medium and cultured in a constant temperature incubator at 37 °C for 12 h. Plates with 300 or more colonies or with 15 or fewer colonies were discarded. The number of colonies was expressed as CFU/mL. Semen samples stored at 7 d were sent to Novo Biotech Co. LTD. (Beijing, China) for microbial 16S rDNA gene sequencing and analysis.

Western blotting

Sperm samples that were stored for 7 d were selected to extract total proteins, and western blotting was performed as previously described (Kuwahara et al., 2016). The antibodies used are listed in Supplementary Table S2. Both goat anti-rabbit secondary antibody and goat anti-mouse secondary antibody was diluted by 1:10000. The enhanced chemiluminescence kit (New Cell & Molecular Biotech, Suzhou, China) was used for protein detection, and the gray levels of the protein bands were determined and quantified using the ImageJ software (Bio-Rad, USA).

Hydrogen peroxide(H₂O₂) oxidative stress test

The aim of this experiment was to further explore the antioxidant effects of CF-C₆₀ during sperm preservation. The experimental design consisted of 4 different groups: a treatment group with 3 μg/mL CF-C₆₀ alone, a treatment group that was preincubated with 3 μg/mL CF-C₆₀ for 30 min prior to exposure to 100 μM H₂O₂, a treatment group with 100 μM H₂O₂ only, and a CON group with an equal volume of deionized water. Sperm samples from each group were incubated at 37 °C for 3 h and then evaluated for various parameters.

AI experiments

In this experiment, 567 healthy sows of similar weight were selected from Shaanxi Zhengneng Liquan Sow Farm and sperm for AI comes from Duroc boars. The CON group was inseminated using sperm with the same volume of deionized water (n = 284); the CF-C₆₀ group was inseminated using sperm with CF-C₆₀ suspended in deionized water (n = 283). AI experiment was carried out using sperm stored on the 7 d in an insemination bag (80 mL Boar Semen Bag, Jiangs Animal Products Co., Ltd.) at a dose of 80 mL. Initially, both groups of diluted semen were placed in a 17 °C incubator (BC-95MN, KONKA Group Co., Ltd.) and shielded from light prior to 7 d to the AI test. To prevent sedimentation and death of sperm, the semen was gently shaken every 8 to 12 h. The program we have adopted for the number and time of inseminations is as follows. Use of boar odorants (Guangzhou Vbio Pharma Co., Ltd.) to induce estrus in sow. The first insemination is carried out 24 h after the detection of estrous behavior (standing reaction) in the sow herd, and the second insemination is carried out 8 to 12 h later. After 20 d of AI, a portable ultrasonic detector (ZXZ-618, Guangdong Zhuxianzi Breeding Technology Co., Ltd.) was used to assess and document the pregnancy status of the sows prior to parturition. During breeding, the sows in each group were provided the same nutrition and were subjected to uniform conditions (Supplementary Table S3). The reproductive performance of sows was assessed immediately after the 114 d gestation period, and the parameters recorded included the total number of piglets born, the number of piglets born alive, the number of healthy piglets (birth weight ≥ 1.2 kg), the number of weak piglets (birth weight < 1.2 kg), the number of deformed piglets, and the number of mummified piglets.

Statistical analysis

All data were first tested for normality (Shapiro–Wilk test). All the statistical analyses were performed with the GraphPad Prism software (version 8, GraphPad Software; San Diego, CA, USA). The student’s t-test was utilized to identify the statistical variances between 2 groups, and one-way analysis of variance (ANOVA) in combination with Tukey’s test was employed to assess the distinctions among multiple groups. All the results are presented as the mean ± standard error (SEM). P < 0.05 was considered to indicate statistical significance.

Results

Preparation and characterization of CF-C₆₀

Figure 1A shows a simple composite diagram of the structure of CF-C₆₀. CF-C₆₀ was verified by a SEM, a laser particle size analyzer, and a zeta potential measurement instrument. The SEM image represents CF-C₆₀ which was uniformly spherical (Figure 1B). The diameter of CF-C₆₀ was approximately 68.0 ± 5.5 nm (Figure 1C), which is in line with a previous report (Li et al., 2019). Further, zeta potential measurements revealed that CF-C₆₀ had a potential of approximately −27.67 mV, which suggests that it might be stable in the aqueous phase (Figure 1D). Based on the nanometer-scale size and the superior quality of CF-C₆₀ examined here, it was deemed that it could be used in subsequent experiments.

Figure 1.

Preparation and characterization of carboxyfullerene C₆₀ (CF-C₆₀). (A) Flow chart depicting the preparation of CF-C₆₀, by Figdraw. (B) Scanning electron microscope images of CF-C₆₀. Scale bar = 1 µm, 500 nm, 250 nm. (C) Size distribution of CF-C₆₀. (D) Electric potential of CF-C₆₀.

Ability of CF-C₆₀ to preserve sperm motility, plasma membrane, and acrosome integrity during liquid storage at 17 °C

We found that sperm motility gradually decreased during preservation. Moreover, a CF-C₆₀ concentration of 3 μg/mL was found to be the most beneficial for the preservation of sperm motility parameters on day 7 (Figure 2A and B; Supplementary Figure 1; Table S4). With the increase in CF-C₆₀ concentration, the plasma membrane integrity and acrosome integrity first increased and then decreased, adding the 3 μg/mL CF-C₆₀ supplementation exhibiting the highest value on day 7 (Figure 2C-F; Supplementary Figures 2 and 3). Although there was no significant difference between 2 and 3 μg/mL in terms of acrosome integrity, the combined effect of 3 μg/mL was significantly stronger than that of 2 μg/mL in terms of improving sperm motility and plasma membrane integrity. Therefore, 3 μg/mL CF-C₆₀ was considered the optimal concentration for the preservation of sperm quality and physiological function, and CF-C₆₀ was added to the semen diluent at this concentration for subsequent experiments.

Figure 2.

Preservation of sperm motility and plasma membrane and acrosome integrity during liquid storage with CF-C₆₀ supplementation at 17 °C. (A) Sperm motility was detected by the computer-aided analysis system (CASA). Scale bar = 100 μm. (B) Changes in sperm motility during preservation with different concentrations of CF-C₆₀. (C) Plasma membrane integrity was detected using SYBR14 and propidium iodide (PI) double staining. Scale bar = 100 μm. (D) Changes in the sperm plasma membrane during preservation with different concentrations of CF-C₆₀. (E) Acrosome integrity was detected using FITC-PNA and DAPI double staining. Scale bar = 100 μm. (F) Changes in sperm acrosome integrity during preservation with different concentrations of CF-C₆₀. The results are presented as mean ± SEM (n = 5 per group). The superscript letters (A to E) indicate significant differences (P < 0.05).

Ability of CF-C₆₀ to enhance the antioxidant capacity of sperm by decreasing the ROS level

Oxidative stress is one of the major obstacles to the in vitro preservation of sperm (Bisht et al., 2017). Therefore, the antioxidant capacity of sperm is an important index for its in vitro storage. Detection of the antioxidant capacity of sperm showed that T-AOC and SOD levels in the CF-C₆₀ group were significantly higher than those in the control (CON) group after 3 d of storage (Figure 3A and B). Conversely, the ROS level and MDA content in the CF-C₆₀ group were significantly lower than those in the CON group (Figure 3C and D). Additionally, the expression levels of the antioxidant proteins SOD1, SOD2, and CAT in the CF-C₆₀ group were significantly higher than those in the CON group (Figure 3E and F). These results indicate that CF-C₆₀ significantly improved the antioxidant capacity of sperm by reducing the ROS levels of sperm during in vitro storage.

Figure 3.

Increase in the antioxidant capacity of sperm preserved with CF-C₆₀ supplementation based on decreasing levels of reactive oxygen species (ROS). (A) ROS activities during sperm preservation. (B) MDA activities during sperm preservation. (C) T-AOC during sperm preservation. (D) SOD levels during sperm preservation. (E, F) Western blot analysis of antioxidant proteins in sperm after 7 d of storage. All data are presented as the mean ± SEM value calculated from 4 independent experiments. *P < 0.05, **P < 0.01.

To further validate the antioxidant effect of CF-C₆₀, we established an oxidative damage model by treating sperm with H₂O₂. As shown in Figure 4A-D, sperm motility, motor performance (based on curvilinear velocity, straight-line velocity, and average path velocity), and plasma membrane integrity in the CF-C₆₀ group (CF-C₆₀ + H₂O₂) were higher than those in the H₂O₂ group but lower than those in the CON group (Figure 4E and F). Furthermore, T-AOC in the CF-C₆₀ group (CF-C₆₀ + H₂O₂) was higher than that in the H₂O₂ group (Figure 4G), while the ROS content showed the opposite trend (Figure 4H). In addition, Western blotting results showed that CF-C₆₀ alleviated the degradation of CAT and SOD2 regardless of H₂O₂ treatment (Figure 4I and J). Thus, the overall results indicate that CF-C₆₀ could enhance the antioxidant capacity of sperm by eliminating ROS produced within sperm, thereby extending the storage time and boosting the efficacy of sperm preservation.

Figure 4.

Alleviation of oxidative damage in sperm preserved with CF-C₆₀ supplementation. (A) Sperm motility. (B) Straight-line velocity (VSL). (C) Curvilinear velocity (VCL). (D) Average path velocity (VAP). (E, F) Plasma membrane integrity. Scale bar = 100 µm, 25 µm. (G) T-AOC. (H) reactive oxygen species activities. (I, J) Western blot analysis. Protein activity of CAT, SOD2, GPX5, and β-tubulin. All data are presented as the mean ± SEM value from 4 independent experiments. Different superscript letters (A to E) indicate significant differences (P < 0.05).

Role of the ROS/cytochrome C (Cyt C)/caspase 3 signaling pathway in CF-C₆₀-mediated inhibition of sperm apoptosis

To clarify the energy-supplying role of mitochondria in sperm, mitochondrial membrane potential (ΔΨm) and ATP were detected. The results indicated a progressive decline in ATP content as storage duration increased. However, starting from day 5, the ATP content observed in the CF-C₆₀ group surpassed that of the CON group significantly (Figure 5A). Furthermore, ΔΨm within the CF-C₆₀ group was significantly higher than that in the CON group on day 7 of sperm preservation (Figure 5B and C). Furthermore, Western blotting results showed that CF-C₆₀ increased the expression of mitochondrial respiratory chain-related complex proteins (ATP5A, UQCRC2, UQCRC2, SDHB, and NDUFB8, Figure 5D and E). Subsequently, a transmission electron microscope (TEM) was used to evaluate the mitochondrial status, and the results showed that CF-C₆₀ significantly decreased mitochondrial vacuolation (Figure 5F and G).

Figure 5.

Inhibition of sperm apoptosis by CF-C₆₀ through the reactive oxygen species/Cytochrome C (Cyt C)/Caspase 3 signaling pathway. (A) Changes in sperm ATP levels during preservation in the control (CON) and CF-C₆₀ group. (B, C) Changes in sperm membrane potential (ΔΨm) were detected by flow cytometry in the CON and CF-C₆₀ group after 7 d of preservation. (D, E) Western blot analysis of mitochondrial proteins in sperm after 7 d of storage. (F, G) Transmission electron microscope (TEM) images of mitochondrial vacuolation in preserved sperm after 7 d of storage. (H-J) Changes in Cyt C levels in sperm mitochondria and cytoplasm. (K, L) Changes in sperm apoptosis after 7 d of storage were detected by flow cytometry. (M, N) Western blot analysis of apoptotic proteins in sperm after 7 d of storage. All data are presented as the mean ± SEM value from 4 independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

It has been reported that ROS can promote mitochondrial apoptosis signaling by regulating Cyt C (Xie et al., 2022). Hence, the content of Cyt C was also detected, and the results showed that CF-C₆₀ significantly increased the Cyt C content in mitochondria and decreased the Cyt C content in the cytoplasm (Figure 5H-J). Consistent with these results, the flow cytometry results showed that CF-C₆₀ decreased the apoptosis level of sperm at 7 d of storage (Figure 5K and L), and Western blot analysis revealed that CF-C₆₀ markedly reduced the levels of apoptosis-related proteins (Cleaved caspase 3) and upregulated the level of the antiapoptotic protein BCL-2 (Figure 5M and N). These findings demonstrate that CF-C₆₀ maintains the normal function of mitochondria and inhibits the release of Cyt C in mitochondria, subsequently inhibiting apoptosis through the ROS/Cyt C/Caspase 3 signaling pathway.

Antibacterial effect of CF-C₆₀ is mediated by its effect on ROS levels and apoptosis in sperm

We assessed the antibacterial effect of CF-C₆₀ by evaluating bacterial growth and composition in semen preserved with/without CF-C₆₀ supplementation. The results showed that CF-C₆₀ inhibited the bacterial content of semen (Figure 6A and B; Supplementary Figure 4). The bar plots show that the abundance of Yersiniaceae, Burkholderiales, Pseudomonadales, Ralstonia, and Stenotrophomonas in the CF-C₆₀ group was significantly lower than that in the CON group (Figure 6C-N). The results showed that Staphylococcus in semen was significantly positively correlated with T-AOC and negatively correlated with MDA and ROS; in contrast, Neisseria was significantly negatively correlated with T-AOC (Figure 6O). Notably, it was found that the semen bacteria detected in the CF-C₆₀ group might be related to peroxiredoxin, glutathione transferase, and Cyt C oxidase pathways (Figure 6P). Furthermore, analysis of microbial diversity and species association in the CON and CF-C₆₀ group semen (Supplementary Figure 5) revealed that the suppression of bacteria growth by CF-C₆₀ is related to ROS production and the apoptosis status in sperm. Thus, CF-C₆₀ may reduce damage to sperm caused by harmful bacteria via its antiapoptotic and ROS-lowering effects.

Figure 6.

Suppression of the growth of bacteria by CF-C₆₀ via reduction of reactive oxygen species levels and inhibition of apoptosis in sperm. (A, B) Changes in sperm bacterial concentrations during preservation in the CON and CF-C₆₀ group. (C–F) Bacterial compositions at the order level. (G-J) Bacterial compositions at the family level. (K-N) Bacterial compositions at the genus level. (O) Relationship between the microbiome and sperm quality. (P) Prediction of bacterial function in semen specimens of the CON and CF-C₆₀ group. The results are presented as mean ± SEM (n = 4 per group). *P < 0.05, **P < 0.01.

Localization of CF-C₆₀ on the surface of the middle piece of the sperm tail

To clarify the action pathway of CF-C₆₀, we detected its localization in sperm by TEM. The results showed that CF-C₆₀ was concentrated on the surface of the plasma membrane of sperm and was unable to enter the cell (Figure 7). Moreover, CF-C₆₀ was mainly localized on the surface of the middle piece of the sperm tail (Figure 7B) and was not found in the head or in the principal/end piece of the sperm tail (Figure 7A, C, and D). These results indicate that CF-C₆₀ might maintain the redox balance by absorbing excess ROS that escape from the cell.

Figure 7.

Localization of CF-C₆₀ on the surface of the middle piece of the porcine sperm tail. Spermatozoa of the CON and CF-C₆₀ group were collected after 7 d of storage. (A) TEM image of the sperm head. (B) TEM image of the middle piece of the sperm tail. (C) TEM image of the principal piece of the sperm tail. (D) TEM image of the end piece of the sperm tail. Scale bar = 200 nm.

Increase in litter size and decrease in the number of deformed piglets produced through AI with CF-C₆₀-preserved sperm

Therefore, we conducted an AI experiment to evaluate the safety and efficacy of CF-C₆₀ as a semen diluent. The schematic for the AI experiment is shown in Figure 8A. The results showed that sperm preservation with CF-C₆₀ supplementation resulted in an increase in the total number of piglets born per litter (Figure 8B), the number of piglets born alive per litter, and the number of healthy piglets per litter (Figure 8C and D), and reduced the number of deformed piglets (Figure 8F). Thus, the reproductive performance of sows was improved and no apparent toxicity of CF-C₆₀ was observed.

Figure 8.

Improvement in sow reproductive performance after artificial insemination (AI) with sperm preserved with CF-C₆₀ supplementation. (A) Schematic diagram of the procedure for AI, created with MedPeer (medpeer.cn). N indicates the number of experimental animals. (B) Total number of piglets born per litter. (C) Number of piglets born live per litter. (D) Number of healthy piglets per litter. (E) Number of weak piglets per litter. (F) Number of deformed piglets per litter. (G) Number of mummified piglets per litter. *P < 0.05, **P < 0.01.

That is, the results demonstrate that CF-C₆₀ is a safe component that can be added to porcine semen diluent for the preservation of sperm quality and function. We also delved into its potential mechanisms and found that it exerts antioxidant, antiapoptotic, and antibacterial effects that are attributable to its ability to reduce ROS levels, as shown in the schematic illustration in Figure 9.

Figure 9.

Schematic illustration of the pathways via which CF-C₆₀ preserves sperm quality.

As shown in the figure, CF-C₆₀ preserved sperm quality by enhancing its antioxidant capacity and inhibiting apoptosis and bacterial growth, and all these effects were related to the ability of CF-C₆₀ to lower the level of reactive oxygen species, by Figdraw and created with MedPeer (medpeer.cn).

Discussion

Sperm motility and structural integrity are important indices for the assessment of fertility (Lehti and Sironen, 2017). Previous in vivo and in vitro studies have revealed that CF-C₆₀ has important protective roles. For instance, CF-C₆₀ can shield cells and BALB/c mice from irradiation-induced damage by enhancing the level of endogenous antioxidants (Cui et al., 2013). CF-C₆₀ has also been reported to protect muscle cells against oxidative-induced stress, thereby preserving cell viability (Liu et al., 2013). Consistently, our results demonstrate that CF-C₆₀ preserves porcine sperm quality and fertility via maintenance of sperm structural and functional integrity. Additionally, our results suggest that CF-C₆₀ mainly attaches to the sperm cell membrane, this could mean that its position was related to its ability to maintain membrane function and integrity, which warrants further exploration.

ROS accumulation is considered the principal cause of porcine sperm motility decline during sperm preservation (Evans et al., 2021). It has been reported that CF-C₆₀ protected the gut from the toxic effects of deoxyniurenol by reducing ROS levels and that it significantly improved growth performance and immune function in mice (Liao et al., 2021). In addition, CF-C₆₀ was found to alleviate oxidative stress and acute liver injury after severe hemorrhagic shock in rats (Chen et al., 2018). Similarly, in our study, CF-C₆₀ enhanced sperm antioxidant capacity and preserved sperm quality by reducing ROS levels. A promising finding emerged was that CF-C₆₀ could alleviate sperm oxidative stress even in the presence of H₂O₂. Indeed, distinct from other nanocarbon materials such as single-layer graphene sheets, carbon nanotubes, and graphene quantum dots (Sajjadi et al., 2021), the globular CF-C₆₀ holds a peculiar cage structure (Zhang et al., 2021a), which endows it with the extremely stable physical and chemical properties as well as a mighty response to free radicals, being responsible for its strong antioxidant capability. Besides, ROS are mainly produced by sperm mitochondria, which may also explain why CF-C₆₀ was found to be primarily localized in the middle piece of the sperm tail, as this position would help it maximize the absorption of excess ROS to ensure the integrity of sperm function.

ROS-induced sperm apoptosis is also a key pathogenic factor leading to porcine sperm death (Boguenet et al., 2021). Studies have shown that ROS induces Cyt C, Cleaved caspase 3, and Cleaved caspase 9 to mediate cell apoptosis, resulting in a high incidence of broken single- or double-stranded DNA and, ultimately infertility (Yang et al., 2019). Cyt C, once released into the cytoplasm by mitochondria, can activate apoptosis-related pathways and cause the degradation of cell contents and cell apoptosis. Similarly, our study found that CF-C₆₀ reduced the release of Cyt C in sperm mitochondria and decreased the content of the apoptotic protein Cleaved caspase 3 by reducing ROS levels. Mitochondria are the chief source of ROS, and damage to mitochondria not only results in the failure of energy supply to the sperm, but also the release of a large number of proapoptotic factors to stimulate the apoptotic pathway, eventually triggering sperm cleavage and death (Treulen et al., 2016; Chianese and Pierantoni, 2021). Thus, it is plausible that mitochondrial oxidative damage and apoptosis are intertwined pathogenic molecular mechanisms. Accordingly, our results further demonstrate that supplementation of CF-C₆₀ during porcine sperm preservation protected the normal structure and the function of mitochondria and inhibited the release of mitochondrial Cyt C into the cytoplasm through the ROS/Cyt C/Caspase 3 signaling pathway, eventually inhibiting apoptosis.

It is generally accepted that the sperm of a healthy boar does not contain bacteria, and that bacteria principally come from the external environment, semen collection conditions, the dilution operation, and other factors (Ngo et al., 2023). Previous studies have shown the effects of detrimental bacteria, such as Xanthomonadales, Burkholderiaceae, and Ralstonia, which are commonly associated with low motility, a high number of agglutination events, low sperm concentrations, and even poor reproductive performance (Zhang et al., 2021b; Venneri et al., 2022). Because sperm are stored at 17 °C, a temperature that is generally conducive to bacterial growth (Tian et al., 2022), impaired sperm quality might occur as a result of competition between bacteria and sperm for energy substrates. Our study revealed that CF-C₆₀ protected sperm from the deleterious effects of pathogenic microorganisms when they were stored at 17 °C. Based on our findings, we have also deduced that there is a clear correlation between bacterial abundance and ROS levels, but further studies are needed to delve deeper into how these bacteria interact with sperm. Yet, results from previous and present studies suggest that CF-C₆₀ might achieve antibacterial effects by directly destroying bacterial structure or indirectly disrupting bacterial function (Tsao et al., 2002; Li et al., 2008), which points to its potential as a new antibacterial agent.

The safety of CF-C₆₀ is an important issue that cannot be ignored. Early studies have indicated that intravenous administration of nanocarbon PEGylated graphene (20 mg/kg body weight) to mice for 3 mo did not induce cytotoxicity (Yang et al., 2011). However, it has also been reported that endotracheal infusion of fullerene C₆₀ (1.0 mg/kg body weight) induced lung inflammation and morphological damage (Pinheiro et al., 2021). The underlying reasons for this discrepancy might be differences in the morphology of nanocarbon materials and in methods of fullerene derivation. At present, chemical modification of the amino, carboxyl, and hydroxyl groups is often applied to the globular fullerene molecule to boost its solubility, stability, and biosafety (Tang et al., 2007; Xu et al., 2022). In this study, we found that CF-C₆₀ mainly attached to the plasma membrane of the porcine sperm and could not enter the interior of the sperm. This is suggestive of its safety in sperm preservation. Reproductive performance is an important basis for the evaluation of sperm quality (Bertolla, 2020). Finally, we performed AI experiments and demonstrated that the use of CF-C₆₀ as a semen diluent supplement significantly improved the reproductive performance of sows without apparent side effects on the growth of piglets. Overall, CF-C₆₀ has considerable industrial value as a component of semen diluents and could be widely used for the preservation of porcine spermatozoa in the future.

Conclusions

To summarize, CF-C₆₀ improved the quality of porcine sperm preservation by decreasing the levels of ROS to enhance antioxidation, antiapoptosis, and antibiosis during liquid storage at 17 °C. The research results illustrate the efficiency and safety of using CF-C₆₀ as a component of semen diluent for porcine sperm preservation.

Glossary

Abbreviations

ΔΨm

mitochondrial membrane potential

AI

artificial insemination

CF-C₆₀

carboxyfullerene C₆₀

CON

control

CASA

computer-aided analysis system

Cyt C

cytochrome C

LB

luria-Bertani

MDA

malondialdehyde

PI

propidium iodide

SOD

superoxide dismutase

SEM

scanning electron microscope

SEM

mean standard error

T-AOC

total antioxidant capacity

TEM

transmission electron microscope

Conflict of interest statement

No conflicts of interest are declared by the authors.

Author contributions

Yuqing Li and Weijun Pang conceived the study and wrote the manuscript. Yuqing Li, Haoqi Xiao, Haize Zhang, and Xue Qin performed the experiments. Yuqing Li, Yi Zheng, Rui Cai, and Weijun Pang analyzed the data. Xue Qin was responsible for revising the manuscript. Weijun Pang administrated and provided the funding support. All authors read and approved the final submission.

Data availability

The data underlying this article are available in the article and in its online Supplementary Material.