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

doi:10.21203/rs.3.rs-3912181/v1

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

https://doi.org/10.21203/rs.3.rs-3912181/v1

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Background

The current approaches for the preservation of human sperm have several limitations, and there are a lack of effective non-freezing preservation methods. Recently, carboxyfullerene C₆₀ (CF-C₆₀) has gained attention as an important nanocarbon derivative with strong antioxidant and antibacterial activity. This study uses a porcine model to systematically investigate whether CF-C₆₀ can be used for the preservation of sperm.

Results

The results indicate that CF-C₆₀ supplementation can preserve sperm quality during storage at 17°C. This effect is attributable to improvement in the antioxidant capacity of sperm through a decrease in the ROS level. Additionally, CF-C₆₀ can maintain mitochondrial function, inhibit sperm apoptosis through the ROS/Cytochrome C/Caspase 3 signaling pathway, and mediate suppression of bacterial growth through the effects of ROS. Finally, the results of artificial insemination experiments indicate that insemination with CF-C₆₀-treated sperm can increase the total number of offspring born and reduce the number of deformed piglets.

Conclusions

Thus, CF-C₆₀ can preserve sperm quality by inhibition of apoptosis and bacterial growth via a reduction in ROS levels and is safe for use as a component of semen diluent for storage. These findings pave the way for the prospective clinical application of carbon nano-materials as antioxidants for non-freezing sperm preservation methods.

Cryopreservation

Artificial insemination

Sperm storage

Carboxyfullerene C60

Non-freezing storage

Antioxidants

Nanocarbons

Around 10–20% of couples of reproductive age experience infertility, which is defined as the incapability to conceive after one year of unprotected intercourse [1]. While the most important factor contributing to human fertility is the age of the female, in approximately half of all infertility cases, male subfertility/infertility is typically observed [2]. Male subfertility/infertility is characterized by oligozoospermia, asthenozoospermia, teratozoospermia or, the most extreme clinical symptom—azoospermia [3]. Currently, artificial reproductive technology (ART) serves as a core strategy to address the issues related to male subfertility/infertility [4]. The aim of ART is superior embryo production, which requires sperm with normal morphology and high motility [5]. A prerequisite for this is the efficient preservation of human sperm of high quality and normal function, which is an important basis for the preservation of male fertility and the treatment of male subfertility/infertility.

Human sperm cryopreservation has become an important element of ART for the treatment of male subfertility/infertility as well as for fertility preservation in male cancer survivors [6, 7]. It has been demonstrated that sperm can be cryopreserved in liquid nitrogen long term, and there are reports about live births after insemination with sperm that were cryopreserved for more than two decades [8, 9]. While the cryopreservation technique can, on the one hand, extend the storage of sperm, on the other hand, it may inevitably affect sperm quality and impair sperm structure and function [10]. Damage caused by cryopreservation, referred to as cryodamage, leads to impairment of the sperm plasma membrane, lower fertilization capacity, and even inferior embryo quality [11]. In addition, human sperm cryopreservation necessitates specific and costly equipment, which limits its clinical application to some extent. Non-freezing storage methods could be a viable option to effectively preserve sperm while ensuring sperm motility, plasma membrane integrity, fertilization capacity, and other sperm quality indices [12]. However, these methods are not without limitations, as they are vulnerable to oxidative stress [13]. Currently, there is a need for effective semen diluents that can be used for the non-freezing storage of human sperm.

Recent scientific advances in nanotechnology and materials have had implications for the development of novel sperm protectors for the preservation of male fertility. One of these materials is fullerene, which has appealed to scientists since its discovery in 1985 and has increasingly becoming one of the most widely used carbon-based materials [14]. It is the third carbon allotrope that was discovered after graphite and diamond. It is also referred to as soccerene because its appearance is similar to that of a soccer ball [15, 16]. 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” [17]. Due to its unique structure and chemical and physical properties, fullerene has been extensively applied in the optical, magnetic, electrocatalysis and cosmetics industries [18–20]. Of the fullerene family members, C₆₀ is the most common and stable one. It is relatively easy to obtain and is, therefore, becoming the most studied and applied fullerene at the moment. Fullerene C₆₀ is a non-polar molecule with a large π bond, and its special cage structure leads its strong hydrophobic properties. As a result, it is only soluble in benzene, toluene, and other aromatic solvents [21]. 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 [22]; these properties limit its application in the biomedical field. 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), as one of the most important nanocarbon derivatives, has been shown to be an effective free radical scavenger with antioxidant capacity that is hundreds of times greater than that of other antioxidants [23–25]. 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 [26]. Moreover, in recent years, CF-C₆₀ has been found to have neuroprotective [27], antiviral, antineoplastic [28], 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 [29]. Another study demonstrated that CF-C₆₀ inhibited death receptor-mediated apoptosis by upregulating the expression of Hsp70 and interacting with lysosomal membranes [30]. 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 [31]. CF-C₆₀ has also been found to inhibit Escherichia coli-induced meningitis and decrease the expression of TNF-α and IL-1, leukocyte inflammatory infiltration, and blood–brain barrier permeability [32]. As oxidative stress is known to reduce the quality and quantity of sperm and possibly even affect the male’s ability to fertilize and reproduce [33], CF-C₆₀ could be a potentially useful molecule in male fertility preservation methods. However, it is not clear whether the ability of CF-C₆₀ to reduce 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 non-freezing storage of sperm in a porcine model. This animal model was chosen because pigs are an increasingly prevailing animal model in translational studies based on their resemblance to humans in terms of anatomy, physiology [34], and reproductive maturation and the similarities between porcine and human sperm in terms of reproductive and functional capacity [35–37]. We explored the effects of CF-C₆₀ on sperm quality and its antioxidant, anti-apoptotic, and bactericidal abilities, as well as the underlying mechanisms. Further, the safety and practical application of CF-C₆₀ were evaluated by artificial insemination (AI) experiments in pigs. Thus, this study focuses on the issues related to male subfertility/infertility and sheds light on how ART can be optimized.

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 scanning electron microscope (SEM), a laser particle size analyzer, and a zeta potential measurement instrument. The SEM image represents CF-C₆₀ that was uniformly spherical (Fig. 1B). The diameter of CF-C₆₀ was approximately 68.0 ± 5.5 nm (Fig. 1C), which is in line with a previous report [38]. 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 (Fig. 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.

Ability of CF-C ₆₀ to preserve sperm motility, plasma membrane and acrosome integrity during liquid storage at 17°C

Sperm motility and integrity are necessary for successful fertilization [39]. 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. 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 (Fig. 2A, B and Additional fle 1: Fig. S1). With increase in CF-C₆₀ concentration, the plasma membrane integrity and acrosome integrity first increased and then decreased, with the 3 µg/mL CF-C₆₀ supplementation group exhibiting the highest value (Fig. 2C-F and Additional fle 1: Fig. S2, S3). Therefore, 3 µg/mL CF-C₆₀ was considered as the optimal concentration for preservation of sperm quality and physiological function, and CF-C₆₀ was added to the semen diluent at this concentration for subsequent experiments.

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 [40].Therefore, the antioxidant capacity of sperm is an important index for its in vitro storage. Detection of the antioxidant capacity of sperm showed that total antioxidant capacity (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 (Fig. 3A, B). Conversely, the ROS level and malondialdehyde (MDA) content in the CF-C₆₀ group were significantly lower than those in the CON group (Fig. 3C, 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 (Fig. 3E, 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, thereby alleviating oxidative damage and preserving sperm quality.

To further validate the antioxidant effect of CF-C₆₀, we established an oxidative damage model by treating sperm with H₂O₂. As shown in Fig. 4A–D, sperm motility and motor performance (based on curvilinear velocity, straight-line velocity, and average path velocity) in the H₂O₂ group were decreased, and this demonstrates the oxidative damage caused by H₂O₂. Sperm motility 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 (Fig. 4E, F). To probe the antioxidant protection mechanism of CF-C₆₀, T-AOC and ROS content were analyzed. The results showed that the addition of H₂O₂ reduced T-AOC. Further, T-AOC in the CF-C₆₀ group (CF-C₆₀ + H₂O₂) was higher than that in the H₂O₂ group (Fig. 4G), while the ROS content showed the opposite trend (Fig. 4H). In addition, Western blotting results showed that CF-C₆₀ alleviated the degradation of CAT and SOD2 regardless of H₂O₂ treatment (Fig. 4I, 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 in vitro sperm preservation.

Role of the ROS/Cytochrome C (Cyt C)/Caspase 3 signaling pathway in CF-C ₆₀ -mediated inhibition of sperm apoptosis

Mitochondria are the energy centers in sperm and are also largely responsible for ROS production [41]. To clarify the energy-supplying role of mitochondria in sperm, mitochondrial membrane potential (ΔΨm) and ATP were detected. The results showed that CF-C₆₀ increased the ATP content and ΔΨm of sperm after 7 d of storage (Fig. 5A–C). Further, Western blotting results showed that CF-C₆₀ increased the expression of mitochondrial respiratory chain-related complex proteins (ATP5A, UQCRC2, UQCRC2, SDHB, and NDUFB8, Fig. 5D, 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 (Fig. 5F, G).

It has been reported that ROS can promote mitochondrial apoptosis signaling by regulating Cyt C [42]. 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 (Fig. 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 (Fig. 5K, 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 anti-apoptotic protein BCL-2 (Fig. 5M, 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 mediated by its effect on ROS levels and apoptosis in Sperm

Bacterial contamination is an important factor contributing to the decline of sperm quality [43]. Therefore, we assessed the anti-bacterial effect by 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 (Fig. 6A, B and Additional fle 1: Fig. S4). 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 (Fig. 6C–N). To explore the relationship between sperm function and bacteria, the correlation of sperm antioxidant and antiapoptotic status with bacteria was studied, and the function of bacteria was predicted. 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 (Fig. 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 (Fig. 6P). Furthermore, analysis of microbial diversity and species association in the CON and CF-C₆₀ group semen (Additional fle 1: Fig. S5) 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 anti-apoptotic and ROS-lowering effects.

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 (Fig. 7). Moreover, CF-C₆₀ was mainly localized on the surface of the middle piece of the sperm tail (Fig. 7B) and was not found in the head or in the principal/end piece of the sperm tail (Fig. 7A, C, D). These results indicate that CF-C₆₀ might maintain the redox balance by absorbing excess ROS that escape from the cell.

Increase in litter size and decrease in the number of deformed piglets produced through artificial insemination (AI) with CF-C ₆₀ -preserved sperm

Reproductive performance is an important basis for evaluation of sperm quality [44]. 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 Fig. 8A. The results showed that sperm preservation with CF-C₆₀ supplementation resulted in an increase in the total number of piglets born per litter (Fig. 8B), the number of piglets born alive per litter, and the number of healthy piglets per litter (Fig. 8C, D), and reduced the number of deformed piglets (Fig. 8F). Thus, the reproductive performance of sows was improved and no apparent toxicity of CF-C₆₀ was observed. That is, the results demonstrate that CF-C₆₀ is a safe component that can be added to semen diluent for the preservation of sperm quality and function.

The present study demonstrates the potential applicability and safety of CF-C₆₀ as a semen component that can be used to preserve the quality and performance of sperm in a porcine model. 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 Fig. 9.

Sperm motility and structural integrity are important indices for assessment of male fertility [45]. Our first set of experiments on motility and sperm structure demonstrated that CF-C₆₀ was able to preserve sperm motility, plasma membrane and acrosome integrity. 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 [46], suggestive of its utility as a safe and effective anti-radiation agent. CF-C₆₀ has also been reported to protect muscle cells against oxidative-induced stress, thereby preserving cell viability [47]. Consistently, our results demonstrate that CF-C₆₀ preserves sperm quality and male fertility via maintenance of sperm structural and functional integrity. Additionally, our results suggest that CF-C₆₀ mainly attaches to the sperm cell membrane. It has been reported that fullerene C₆₀ can cause changes in membrane-binding enzyme activity without damaging membrane integrity [48]. 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 as the principal cause of sperm motility decline during semen preservation [49]. In this regard, CF-C₆₀ has been shown to act as a powerful antioxidant that exerts important protective effects in cells [50–52]. For example, 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 [53]. In addition, CF-C₆₀ was found to alleviate oxidative stress and acute liver injury after severe hemorrhagic shock in rats [54]. 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. This protective effect might be attributed to the powerful ability of CF-C₆₀ to eliminate free radicals. Since carbon-based nanomaterials, which are morphologically distinct, have differential physiological roles in cells, we presume that this antioxidant capacity of CF-C₆₀ might be largely related to its unique cage structure [55, 56]. Indeed, distinct from other nanocarbon materials such as single layer graphene sheets, carbon nanotubes and graphene quantum dots [57], the globular CF-C₆₀ holds a peculiar cage structure, 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 [58, 59] .As a result, CF-C₆₀ can continuously and steadily react with free radicals and eliminate them completely [60]. 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. Further, as CF-C₆₀ was found to mainly attach to the sperm cell membrane, this might imply that the effect of CF-C₆₀ on the oxidation-antioxidant balance system of sperm is mediated by its adsorption of excessive ROS overflowing from the cell.

ROS-induced sperm apoptosis is also a key pathogenic factor leading to male infertility [61]. Sperm apoptosis is an intricate physiological process, which is orchestrated by a host of factors and considered to be an important physiological mechanism in the regulation of the number of sperm [62]. Studies have shown that ROS induce 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, male infertility [63] .Cyt C, a soluble protein located in the mitochondria, is reported to be one of the essential regulators of programmed apoptosis [64]. Once released into the cytoplasm by mitochondria, it can activate apoptosis-related pathways and cause the degradation of cell contents and cell apoptosis. Additionally, it has been reported that Cleaved caspase 3 is a key promoter of apoptosis receptors, and its activation is related to the concentration of Cyt C in cytoplasm [65]. 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. Some other consequences of excessive ROS are mitochondrial respiratory chain metabolism disorder and mitochondrial vacuolation, both of which contribute to mitochondrial dysfunction [66, 67]. Mitochondria are the chief source of ROS, and damage to mitochondria not only results in failure of energy supply to the sperm, but also the release of a large number of pro-apoptotic factors to stimulate the apoptotic pathway, eventually triggering sperm cleavage and death [68, 69]. 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 semen 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 semen of healthy individuals does not contain bacteria [70], and that bacteria principally come from the external environment, semen collection conditions, the dilution operation, and other factors [71]. 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 [72–74]. Because sperm are stored at 17°C, a temperature that is generally conducive to bacterial growth [75], 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. Studies have shown that CF-C₆₀ can inhibit E. coli-induced meningitis, and its antibacterial activity was attributable to its ability to enhance the antibacterial activity of neutrophils [76]. In fact, it is known that granulocytes (of which neutrophils are a subset) are the most important white blood cells that play a role in defense against bacterial and viral pathogens to kill Gram-negative and Gram-positive bacteria [77]. 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 the present study suggest that CF-C₆₀ might achieve antibacterial effects by directly destroying bacterial structure or indirectly disrupting bacterial function [78, 79], 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 months did not induce cytotoxicity [80]. However, it has also been reported that endotracheal infusion of fullerene C₆₀ (1.0 mg/kg body weight) induced lung inflammation and morphological damage [81]. The underlying reasons for this discrepancy might be differences in 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 [82, 83]. In this study, we found that CF-C₆₀ mainly attached to the plasma membrane of the sperm and could not enter the interior of the sperm. This is suggestive of its safety in sperm preservation. 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, thus demonstrating its safety as a sperm protector and shedding light on its prospective clinical application in the future.

To summarize, CF-C₆₀ preserved sperm quality during liquid storage at 17°C through its antioxidant, antiapoptotic, and antibacterial mechanisms, which was attributable to its ROS-lowering effects. This was confirmed through AI results, which showed that CF-C₆₀ increased the litter size and reduced the number of deformed piglets. Thus, the present findings illustrate the efficiency and safety of using CF-C₆₀ as a component of semen diluent for sperm preservation. Our findings not only pave the way for the prospective clinical application of carbon nano-materials as antioxidants in sperm preservation techniques, but also have important implications for optimization of ART to preserve/enhance male fertility.

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 reportedand is based on the transfer of fullerene from the organic phase to the aqueous phase by physical mixing and ultrasonic treatment [84]. In this study, the arc method was used to prepare CF-C₆₀. 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 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 nano particle 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 3000).

Animals

All experiments involving animals were conducted in accordance with the guidelines and regulations of the Chinese Code of Animal Welfare and Ethic. The semen samples used in this study were from eight healthy adult Duroc boars (aged 24–26 months) 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 (Additional fle 1: Table S1). A specialist at the farm was responsible for the feeding management, training, semen collection, and other related work based on the requirements of subsequent experiments. The Northwest A&F University Animal Protection Association approved all the procedures (NWAFU No. 20210517).

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. Impurities in the semen were filtered by passing it through three-layer filter papers. The samples were immediately sent to the laboratory to determine the quality of the original semen specimen. The quality of the semen was determined based on the following criteria: ≥5.0 × 10⁸/mL sperm concentration, ≥ 80% motile sperm, and ≥ 80% normal morphology [85]. To avoid the effect of individual differences on experimental outcomes, the qualified semen samples were mixed with Beltsville thawing solution (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 at 37°C. 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. In addition, the sample was protected from light and gently shaken by turning it upside down every 12 h.

Sperm motility

A computer-assisted sperm analysis system (CASA, Fuzhou Hongye Software Technology Co. Ltd.) was used to measure sperm motility, including its progressive motility. First, 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 20 min. The semen quality was then measured using the CASA system by placing a 10 µL semen drop on a preheated special slide onto. We randomly selected five visual fields harboring more than 200 sperm for observation.

Plasma membrane integrity

Sperm plasma membrane integrity was assessed by SYBR-14 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 exhibit red fluorescence at 630 nm. Five clear visual fields harboring at least 200 sperms each were randomly selected for observation.

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 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 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 PI 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 structure do not exhibit fluorescence. Five clear visual fields harboring at least 200 sperms each were observed.

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

T-AOC (Nanjing Jiancheng Bioengineering Institute, Jiangsu, China), MDA (Nanjing Jiancheng Bioengineering Institute, Jiangsu, China), SOD (Nanjing Jiancheng Bioengineering Institute, Jiangsu, China), and ROS (Jiangsu Jingmei Biological Technology Co. Ltd., Jiangsu, China) of sperm stored for 1, 3, 5 and 7 d were detected according to the instructions provided with the assay kits. 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 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 (5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimi-dazolylcarbocyanine iodide; Solarbio Science & Technology Co., Beijing, China) as described in a previous study [86]. 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, and then washed twice with JC-1 dyeing buffer (1×) (1 ml JC-1 dyeing buffer (5×) and 4 mL distilled water). Finally, the appropriate amount of JC-1 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, Jiangsu, China) according to the manufacturer’s instructions, and the results are expressed in µmol/gprot.

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 [87]. 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. Three replicates were set for each concentration gradient of CF-C₆₀, and the experiment was conducted under sterile conditions. 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 [66].

Western Blotting

Sperm samples that were stored for 7 d were selected to extract total proteins, and western blotting was performed as previously described [86]. The antibodies used are listed in Table S2 (Additional fle 1). Both goat anti-rabbit secondary antibody and goat anti-mouse secondary antibody were 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).

AI Experiments

In this experiment, 567 healthy sows of similar weight were selected from Shaanxi Zhengneng Liquan Sow Farm and divided into two groups: one group was inseminated with semen that was stored without the addition of CF-C₆₀ (n = 284), and the other group was inseminated with semen that was stored with CF-C₆₀ (n = 283). We recorded the gestation rate of the sows after 21 d. During breeding, the sows in each group were provided the same nutrition and were subjected to uniform conditions (Additional fle 1: 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 two 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.

CF-C₆₀Carboxyfullerene C₆₀

ART Artificial reproductive technology

SOD Superoxide dismutase

AI Artificial insemination

SEM Scanning electron microscope

T-AOC Total antioxidant capacity

CON Control

MDA Malondialdehyde

Cyt C Cytochrome C

ΔΨm Mitochondrial membrane potential

TEM Transmission electron microscope

PI Propidium iodide

LB Luria-Bertani

SEM Standard error

CASA Computer-aided analysis system

Supplementary Information

Additional fle 1: Table S1: Composition and nutrient analysis of boar basal diet. Table S2: List of antibodies used for Western blot analysis. Table S3: Composition and nutritional level of the basal diets provided to gestating sows. Figure S1: CF-C₆₀ preserved boar sperm motility during liquid stored at 17 °C. CASA was used to detect the motility of boar sperm at 1, 3, 5, and 7 d of storage. Five fields of view were randomly selected for each observation, and the results are expressed as mean ± SEM. Green marks the sperm head and its movement is shown in red. Scale bar = 100 μm. Figure S2: CF-C₆₀ preserved sperm plasma membrane during storage. Sperms were stained with SYBR-14/PI fluorescent dye. Five fields of view were randomly selected for each observation to calculate the ratio of the intact plasma membrane sperm count to the total sperm count. The results are expressed as mean ± SEM. Red: sperm with damaged plasma membrane; Green: sperm with intact plasma membrane. Scale bar = 100 μm. Figure S3: CF-C₆₀ preserved sperm acrosome integrity during storage. Sperm were stained with FITC-PNA fluorescent dye. Sperm with an intact acrosome emitted green fluorescence and showed a complete cap structure. Sperm with a damaged or exfoliated acrosome showed an incomplete cap structure or no fluorescence. Five fields of view were randomly selected for each observation, and the results are expressed as mean ± SEM. Blue: sperm with damaged acrosome; Green: sperm with intact acrosome. Scale bar = 100 μm. Figure S4: Inhibition of bacterial growth by CF-C₆₀ during sperm preservation. Boar semen samples were diluted with Beltsville thawing solution containing different concentrations of CF-C₆₀ (0, 1, 2, 3, 4, and 5 μg/mL) during liquid storage at 17 °C. The bacterial contents in the semen specimens of each group after 1, 3, 5, and 7 d of storage were detected by the LB agar plate method. The results are presented as mean ± SEM (n = 3 per group). Different superscript letters (a to e) indicate significant differences (P < 0.05). Figure S5: Microbial diversity and species association analysis of semen specimens. (A) Genus-level species evolutionary tree. The abundance distribution information of the bacterium in different samples is shown. (B-D) Chao1, Shannon, and Simpson indexes calculated from the rarefaction curve, which is used to present sample diversity within a group. (E) Hierarchical clustering curve. Different samples are represented by broken lines of different colors. (F) Correlation analysis. Different nodes represent different genera, and the node size represents the average relative abundance of the genus.

Author Contributions

YL, HZ, HX, XQ, and WP conceived the study and wrote the manuscript. YL, HZ, HX,and XQ performed the experiments. YL, BH, MY, CC, XW, JC, LG, GC, YZ and WP analyzed the data. YZ, RC and WP supervised the study and WP administrated and provided the funding support. All authors read and approved the final submission.

Funding

The authors would like to extend the gratitude to the following individuals and institutions for their invaluable contributions to this study. The authors thank Yihui Wang at Zhengneng Co. LTD for their assistance with the feeding of experimental animals. The authors also acknowledge Lei Chen at the Life Science Research Core Services of Northwest A&F University for their support in carrying out imaging. This work was supported by the China Agriculture Research System of MOF and MARA (CARS-35-PIG to W.P.) and the Key Research and Development Program of Shaanxi Province (2022ZDLNY01-04 to W.P.).

Availability of data and materials

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Ethics approval and consent to participate

The Northwest A&F University Animal Protection Association approved all the procedures (NWAFU No. 20210517).

Consent for publication

All authors were aware of this submission and approved for publication.

Conflict of Interest

The authors declare no conflict of interest.

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No competing interests reported.

Graphicalabstract.jpg
Schematic illustration of the pathways via which CF-C₆₀ preserves sperm quality
Additionalfle1.pdf
Additional fle 1: Table S1: Composition and nutrient analysis of boar basal diet. Table S2: List of antibodies used for Western blot analysis. Table S3: Composition and nutritional level of the basal diets provided to gestating sows. Figure S1: CF-C₆₀ preserved boar sperm motility during liquid stored at 17 °C. CASA was used to detect the motility of boar sperm at 1, 3, 5, and 7 d of storage. Five fields of view were randomly selected for each observation, and the results are expressed as mean ± SEM. Green marks the sperm head and its movement is shown in red. Scale bar = 100 μm. Figure S2: CF-C₆₀ preserved sperm plasma membrane during storage. Sperms were stained with SYBR-14/PI fluorescent dye. Five fields of view were randomly selected for each observation to calculate the ratio of the intact plasma membrane sperm count to the total sperm count. The results are expressed as mean ± SEM. Red: sperm with damaged plasma membrane; Green: sperm with intact plasma membrane. Scale bar = 100 μm. Figure S3: CF-C₆₀ preserved sperm acrosome integrity during storage. Sperm were stained with FITC-PNA fluorescent dye. Sperm with an intact acrosome emitted green fluorescence and showed a complete cap structure. Sperm with a damaged or exfoliated acrosome showed an incomplete cap structure or no fluorescence. Five fields of view were randomly selected for each observation, and the results are expressed as mean ± SEM. Blue: sperm with damaged acrosome; Green: sperm with intact acrosome. Scale bar = 100 μm. Figure S4: Inhibition of bacterial growth by CF-C₆₀ during sperm preservation. Boar semen samples were diluted with Beltsville thawing solution containing different concentrations of CF-C₆₀ (0, 1, 2, 3, 4, and 5 μg/mL) during liquid storage at 17 °C. The bacterial contents in the semen specimens of each group after 1, 3, 5, and 7 d of storage were detected by the LB agar plate method. The results are presented as mean ± SEM (n = 3 per group). Different superscript letters (a to e) indicate significant differences (P < 0.05). Figure S5: Microbial diversity and species association analysis of semen specimens. (A) Genus-level species evolutionary tree. The abundance distribution information of the bacterium in different samples is shown. (B-D) Chao1, Shannon, and Simpson indexes calculated from the rarefaction curve, which is used to present sample diversity within a group. (E) Hierarchical clustering curve. Different samples are represented by broken lines of different colors. (F) Correlation analysis. Different nodes represent different genera, and the node size represents the average relative abundance of the genus.

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

Status:

Version 1

Abstract

Background

Results

Conclusions

Figures

Background

Results

Preparation and characterization of CF-C₆₀

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

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

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

Discussion

Conclusions

Methods

Synthesis of CF-C₆₀

Characterization of CF-C₆₀

Animals

Semen samples, processing and preservation

Sperm motility

Plasma membrane integrity

Acrosomal integrity

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

Mitochondrial membrane potential and ATP content

Bacterial colony counts and compositions

Western Blotting

AI Experiments

Statistical analysis

Abbreviations

Declarations

References

Additional Declarations

Supplementary Files

Status:

Version 1

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

Status:

Version 1

Abstract

Background

Results

Conclusions

Figures

Background

Results

Preparation and characterization of CF-C60

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

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

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

Discussion

Conclusions

Methods

Synthesis of CF-C60

Characterization of CF-C60

Animals

Semen samples, processing and preservation

Sperm motility

Plasma membrane integrity

Acrosomal integrity

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

Mitochondrial membrane potential and ATP content

Bacterial colony counts and compositions

Western Blotting

AI Experiments

Statistical analysis

Abbreviations

Declarations

References

Additional Declarations

Supplementary Files

Status:

Version 1

Preparation and characterization of CF-C₆₀

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

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

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

Synthesis of CF-C₆₀

Characterization of CF-C₆₀