Dietary resveratrol improves semen quality by promoting mitochondrial function-related genes expression in aging boars

Abstract

Boar semen quality is a significant factor affecting reproductive efficiency in modern farms, and resveratrol (RES) has the potential to reduce mouse sperm mortality in our previous studies. Here, we found that dietary supplementation of RES (500 mg/kg) delayed the decline in semen quality by increasing sperm motility and survivability in aging boars (P < 0.01, n = 6) by a 12-wk trial. RES increased the testosterone level and total antioxidant capacity (P < 0.01), while decreasing the cortisol (P < 0.05) and malondialdehyde (MDA, P < 0.01) in serum. Immunofluorescence assay and electron microscope scanning revealed that RES reduced the levels of reactive oxygen species, 8-hydroxy-2′-deoxyguanosine, and MDA (P < 0.01) to alleviate oxidative damage in semen. Analysis of mitochondrial function-related genes indicated that RES improved mitochondrial structural integrity and mitochondrial membrane potential (P < 0.01) by increasing the mRNA expression of PGC-1α, TFB1M, and TFB2M (P < 0.01). These results demonstrated that RES can enhance semen quality and antioxidant capacity potentially through improving sperm mitochondrial function in aging boars.

Supplemental resveratrol showed significant beneficial effects on sperm motility and survivability, hormone levels, and inflammation in aging boars. Further analysis showed that resveratrol may improve semen quality by improving sperm antioxidant capacity and mitochondrial function.

Introduction

Boars are crucial elements within the breeding herds of contemporary pig farms and perform critical functions throughout the entire pig industry chain, as boar semen can directly impact the pregnancy rate, litter size, and offspring health of sows (Lopez Rodriguez et al., 2017). However, boars have a short service life and experience a high elimination rate after reaching 2 yr of age, while the rate of elimination caused by poor semen quality can account for 18.4% to 26.4% (Knox et al., 2008; Koketsu and Sasaki, 2009). Various challenges such as changes in ambient temperature, frequent semen collection, invasions of pathogenic microorganisms, and obesity can increase the production of reactive oxygen species (ROS) (Hao et al., 2021), which can cause lipid peroxidation of the plasma membrane, oxidative damage, and mitochondrial dysfunction in sperm (Tremellen, 2008; Hamada et al., 2012). Mitochondrial biogenesis is essential for maintaining boar sperm motility, which requires coordinated expression of multiple genes. Specifically, as a core subunit of mitochondrial Complex I, ND1 participates in electron transport chain activity, while mitochondrial transcription factor A (TFAM) regulates mitochondrial DNA replication and transcription during spermatogenesis. However, elevated ROS levels may induce oxidative damage to these critical components, consequently compromising ATP production and disrupting the mitochondrial transcriptional activity. Therefore, improving antioxidant capacity and mitochondrial function may be the key to improving boar semen quality.

Resveratrol (3,4′,5-trihydroxystilbene, RES) is a polyphenol that is naturally found in plants such as berries and grapes (Pezzuto, 2019) and has been approved as a food supplement with no side effects by the European Food Safety Authority (European-Commission, 2016; Pang et al., 2023). It can scavenge ROS via its phenolic hydroxyl structure (Mongioì et al., 2021) and activate antioxidant pathways (Xia et al., 2017). In addition, RES has been reported to enhance the antioxidant capacity of frozen boar semen, preserve the mitochondrial function and structural integrity of sperm, and reduce sperm mortality (He et al., 2020; Torres et al., 2021). We hypothesized that dietary supplementation with RES can alleviate the decline of semen quality in aging boars. To further explore the effect and mechanisms of dietary RES on the semen quality, a 12-wk trial was conducted in aging boars focusing on hormones, redox status, inflammatory factors, and sperm mitochondrial function.

Materia and Methods

All experimental procedures involving animals were approved by the Hunan Agricultural University Institutional Animal Care and Use Committee (No. 2023073) and were developed by the Guide for the Care and Use of Laboratory Animals, 8th edition (NRC, 2011).

Experimental diets

The basal diet for the boars was formulated in accordance with the Nutrient Requirements of Swine (NRC, 2012), and the composition and nutritional components are shown in Table 1. The RES group was fed a diet containing 500 mg/kg RES (≥ 98%, provided by the Co-Innovation Center of Education Ministry for Utilization of Botanical Functional Ingredients, Changsha, China) based on our previous studies.

Table 1.

Basal diet composition and nutritional components of boars (as-fed basis, %)

Item	Content
Ingredients
Corn	47.00
Flour	20.00
Soybean meal, 43% crude protein	9.00
Fermented soybean meal	5.00
Extruded soybean	3.00
Fish meal	3.00
Wheat bran	10.00
Soybean oil	1.00
Premix1	2.00
Calculated nutrient composition2
Digestible energy, Kcal/kg	3,360
Net energy, Kcal/kg	2,400
Crude protein	17.00
Ether extract	4.50
Crude fiber	3.65
Neutral detergent fiber	11.80
SID lysine	0.96
SID methionine	0.32
SID threonine	0.54
SID serine	0.67
Total phosphorus	0.65
Digestible phosphorus	0.34
Calcium	0.65

¹Premix provided per kilogram of diet: Cu, 15 mg; Fe, 250 mg; Zn, 60 mg; Mn, 70 mg; I, 0.6 mg; vitamin A, 10,000 IU; vitamin D, 3,000 IU; riboflavin, 4.0 mg; pantothenic acid, 40 mg; nicotinic acid, 40 mg; biotin, 0.3 mg; folic acid, 1.0 mg; vitamin B_12, 0.04 mg.

²Values of digestible energy, net energy, SID amino acids, and digestible P were calculated, while the others were measured.

Abbreviation: SID = standardized ileal digestibility.

Animal management and experimental design

The animal experiment was conducted in Hunan Xinguang’an Agriculture Husbandry Co., Ltd (Hengyang, China). A total of 12 28-mo-old PIC line 337 boars (initial body weight 250.08 ± 12.59 kg, PIC China, Pig Improvement Co., Ltd, Shanghai, China) were housed in individual boar stalls (0.75 m width × 2.40 m length × 1.20 m height) within the same swine barn for 12 wk. The boars were randomly assigned to two dietary groups: a basal diet (CTL) and a basal diet supplemented with 500 mg/kg RES (RES), with six replicates each (one boar per pen). Semen volume, sperm density, survivability, and counts were recorded at each sperm collection by the boars during the experiment, and semen and plasma samples were collected immediately after the experiment. The daily breeding management procedures for all boars were consistent and as follows: daily feed intake and refusals were recorded to ensure that each boar was fed 3 kg/day (twice daily with 1.5 kg at 07:00 and 14:00) and had ad libitum access to water. Throughout the entire period, the room temperature was kept at 21 °C, with humidity at 50%, and the environment was well-ventilated and clean.

Boar semen collection

Boar semen was collected at 5-day intervals by a single technician following the same sequence after the morning feeding. The semen collection pen is located within the swine barn, where stall-confined boars cannot observe the collection process. The boar was guided onto the platform, wore double gloves, and disinfected the boar’s foreskin before semen collection. All semen ejected from boars was collected in a 37 °C thermos cup, filter paper was removed, and the semen was then sent to the semen processing room.

Boar plasma collection

Immediately after the experiment, the ear vein collected 10 mL of plasma samples into centrifuge tubes without anticoagulants (Needle size: 16 × 50 mm). After standing at room temperature for 30 min, the plasma samples were centrifuged at 1,500 × g for 10 min to collect the serum, which was then stored at −80 °C for subsequent analysis.

Semen quality parameter assays

Semen volume was determined by weighing the collected ejaculate (1 g of semen is equivalent to 1 mL), and sperm survivability was evaluated microscopically at a magnification of 400× (SAGA thermostatic microscope, SAGA Optics Co., Ltd, Suzhou, Zhejiang, China). Sperm density was measured using the Bovine Accuread photometer (IMV Technologies Group, France). Sperm count was calculated by multiplying semen volume by semen density.

Computer-assisted sperm assay (CASA)

The semen collected at the last time of the experiment was diluted to 2 billion sperm/mL using PRIMXcell Ultra (IMV Technologies Group, France) after detecting the semen volume, density, and survivability. Subsequently, computer-assisted sperm analysis (CASA) was used to evaluate sperm motility parameters (ML-CASA10-4, Song Jing Tian Lun Bio-Technology Co., Ltd, Nanning, Guangxi, China). A 10 μL semen sample was incubated at 37 °C and loaded into disposable analysis chambers (ML-CASA10-4, Song Jing Tian Lun Bio-Technology Co., Ltd, Nanning, Guangxi, China) for evaluation on a 37 °C heated stage. CASA evaluation included five randomly selected fields, recording 2-s sperm motility videos at 25 fps, followed by immediate analysis at an identical frame rate. Analyzed parameters included the percentage of progressively motile sperm (straightness > 75%), curvilinear velocity (VCL, μm/s), straight-line velocity (VSL, μm/s), average path velocity (VAP, μm/s), straightness (STR = VSL/VAP × 100), linearity (LIN = VSL/VCL × 100), the wobble of the curvilinear trajectory (WOB = VAP/VCL × 100), amplitude head displacement (ALH, μm), beat cross frequency (BCF, Hz). The sperm motility index was calculated as: (VSL × 0.895) + (VAP × 0.686) + (VCL × 0.895) + (ALH × 0.592) × 100 %. Sperm with VAP > 10 μm/s were considered motile.

Serum biochemical parameter assays

All serum assays were done in accordance with the manufacturer’s instructions (Supplementary material). The levels of testosterone (T, #MM-0410O2, MEIMIAN, Inc., Yancheng, China), lipopolysaccharide (LPS, #DY870-05, R&D Systems Inc., Minneapolis, MN), cortisol (#E-OSEL-P0002, Elabscience Biotechnology Co., Ltd, Wuhan, China), interleukin-6 (IL-6, #E-EL-P3008, Elabscience Biotechnology Co., Ltd, Wuhan, China), and tumor necrosis factor-α ( #E-EL-P0010, Elabscience Biotechnology Co., Ltd, Wuhan, China) were determined in serum using enzyme-linked immunosorbent assay (ELISA) assay kits. Total antioxidant capacity (T-AOC, #A015-1-1) and malondialdehyde (MDA, #A003-1-1) levels were determined in serum using assay kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China).

Semen oxidative damage assays

The last ejaculate was assessed for oxidative stress biomarkers, with all semen assays performed according to the manufacturer’s instructions (Supplementary Materials). Lipid peroxidation expressed through MDA (#A003-1-1) production was assessed using assay kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China), and DNA oxidative damage expressed through 8-hydroxy-2′-deoxyguanosine (8-OHdG, #E-EL-0028) production was assessed using ELISA assay kits (Elabscience Biotechnology Co., Ltd, Wuhan, Hubei, China).

Semen ROS levels

As described previously (Hu et al., 2024), the ROS levels of sperm were determined by OxiSelect Intracellular ROS Assay Kit (#STA-342, Cell Biolabs, Inc., San Diego, CA) and operated strictly following the manufacturer’s instructions (Supplementary Material). Median fluorescence intensity was quantified using the NIH ImageJ software and observed under a fluorescence microscope to capture photographs (Axio Vert.A1, ZEISS Group, Oberkochen, Germany).

Semen ATP content

The ATP content in semen samples was determined using the ATP content assay kit (#AKOP004M, Boxbio, Beijing, China). ATP standard solution, control working solution, and determination working solution were prepared according to the instructions. Semen samples (1 × 10⁹ sperm/mL) were washed with phosphate-buffered saline (PBS) and centrifuged at 10,000 × g for 10 min, then homogenized, and the supernatant was collected after centrifugation for chloroform extraction. The sample to be tested and the control working solution were incubated with the determination working solution, respectively, and absorbance at 340 nm was measured at 10 s and 190 s after incubation. Finally, the ATP concentration was determined based on the ATP standard curve (expressed as μmol/10⁹).

Transmission electron microscopy of mitochondrial ultrastructure

The sperm samples were rinsed three times with 0.1 mol/L PBS (pH 7.4), treated with 2% osmic acid, fixed at 4 °C in the dark for 60 min, and then rinsed again with PBS. Subsequently, the sample was dehydrated by the acetone gradient dehydration method and then embedded in the embedding agent after an additional 15 min in anhydrous acetone. Once the temperature naturally decreased to room temperature, the embedded block was removed from the oven and sliced. The sections were stained with 2% uranyl acetate and lead citrate and then air-dried. Finally, transmission electron microscopy was used to capture images of mitochondria (H-7700, Hitachi High Technologies Corp., Japan). When taking an image, select at least five clear views.

Sperm mitochondrial membrane potential (MMP)

The MMP was evaluated using the JC-1 fluorescence assay. In brief, sperm samples were washed with PBS, and then 10 μL of sperm sample was mixed with 6.8 μL of JC-1 staining solution and incubated in an incubator at 37 °C for 30 min in the dark. After incubation, 10 μL of propidium iodide solution was added to the mixture and incubated for an additional 5 min. After centrifugation, the supernatant was collected, and the semen concentration was diluted to 5 × 10⁶ sperm/mL. Finally, the Varioskan Flash spectral scanning multimode reader (Thermo Fisher Scientific Inc., USA) was used to detect the fluorescence intensity of the JC-1 monomer (excitation wavelength of 490 nm, emission wavelength of 530 nm) and aggregates (excitation wavelength of 525 nm and an emission wavelength of 590 nm).

Sperm mitochondrial DNA quantification

As described previously (Hu et al., 2024), the quantity of mitochondria was determined by measuring mitochondrial DNA through real-time quantitative polymerase chain reaction (RT-PCR). Sperm mitochondrial DNA was extracted using the Qiagen DNA extraction kit (Qiagen, Hilden, Germany), and mitochondria-encoded NADH dehydrogenase 1 (ND1) and glucagon gene (GCG) were employed as mitochondrial marker genes and internal reference genes, respectively (Supplementary Table S1).

Real-time quantitative polymerase chain reaction (qPCR)

As previously described (Hu et al., 2024), the expression of mRNA in sperm samples was detected by qPCR. Sperm RNA was extracted using the Trizol method (# AG21101, Accurate Biotechnology Co., Changsha, China), and reverse transcription was performed with the Evo M-MLV reverse transcription kit (# AG11705, Accurate Biotechnology Co., Changsha, China). The experimental procedures were strictly adhered to according to the kit instructions, and fluorescence quantification was performed using a 10 µL reaction system. Detailed parameters for the reaction conditions are presented in Supplementary Table S2, while the primers used are listed in Supplementary Table S1. Each sample underwent three technical replicates, and the results were calculated using the 2^−ΔΔCt method, with normalization to the CTL group.

Statistical analysis

The results were presented as means ± SD. All data were tested for normality and variance homogeneity before analysis. Longitudinal measurements (12-wk data) were analyzed using two-way repeated measures analysis of variance (ANOVA), while endpoint measurements were compared using independent t-tests. Analyses were conducted using SPSS 22.0 (IBM SPSS Statistics, Armonk, NY). Pearson correlation analysis was used to examine the correlation between sperm quality parameters and mitochondrial functional gene expression. Differences were considered highly significant at P < 0.01 and significant at P < 0.05.

Results

The effect of RES on the decline of semen quality in aging boars

The weekly semen quality parameters of each boar are shown in Figure 1. Throughout the experimental period, significant differences were observed in semen density (Figure 1C), sperm survivability (Figure 1D), and count (Figure 1E) between the CTL and RES groups (treatment: P < 0.05). While all three parameters showed elevated values in the RES group at multiple time points, only sperm survivability reached statistically significant between-group differences by week 12 (P < 0.05). However, dietary RES showed no significant effect on semen volume (P > 0.05, Figure 1B). Notably, no significant time effects or treatment-time interactions were detected for any parameters, indicating that the treatment effects remained consistent throughout the 12 wk without being influenced by temporal variations. The sperm motility parameters are shown in Table 2. Dietary RES enhanced sperm motility (P < 0.01) but showed a limited effect on kinematic parameters, including ALH, WOB, VSL, VAP, VCL, BCF, MAD, and STR (P > 0.05).

Figure 1.

The effect of RES on the decline of semen quality in aging boars. (A) The diagram shows experimental procedures. The changes of (B) semen volume, (C) semen density, (D) sperm survivability, and (E) sperm count of boars in 12 wk. *P < 0.05 and **P < 0.01. CTL, basal diet; RES, dietary supplemented with 500 mg/kg resveratrol. Mean values are based on six replicates, and SDs are represented by vertical bars.

Table 2.

The effect of RES on the decline of semen quality in aging boars (assessed by CASA)

Sperm parameters	CTL group	RES group	P-value
Sperm motility, %	66.67 ± 2.96	77.36 ± 6.23	0.003
Sperm survivability, %	80.12 ± 2.64	87.83 ± 4.04	0.003
Sperm mortality, %	19.88 ± 2.64	12.17 ± 4.04	0.003
Active sperm count, 108/mL	842.67 ± 1,024.22	736.33 ± 848.06	0.849
Dead sperm count, 108/mL	222.50 ± 255.16	115.17 ± 131.41	0.381
Straight-line velocity, μm/s	198.14 ± 10.76	200.42 ± 30.21	0.865
Average path velocity, μm/s	169.34 ± 7.26	168.72 ± 29.40	0.961
Curvilinear velocity, μm/s	241.21 ± 10.34	270.90 ± 85.82	0.420
Amplitude head displacement, μm/s	71.87 ± 3.08	80.72 ± 25.57	0.420
Wobble of the curvilinear trajectory, %	0.17 ± 0.12	0.22 ± 0.22	0.608
Beat cross frequency, Hz	47.33 ± 11.01	48.52 ± 17.47	0.891
Linearity/LIN, %	0.84 ± 0.07	0.82 ± 0.16	0.789
Mean angular displacement, degree	58.23 ± 14.61	66.37 ± 36.50	0.623
Straightness, %	1.20 ± 0.10	1.17 ± 0.23	0.777

CTL, a basal diet; RES, a basal diet supplemented with 500 mg/kg resveratrol. Mean values are based on six replicates, and the results were presented as means ± SD.

Effects of RES on hormone, antioxidant capacity, and inflammatory factors

Testosterone levels are essential for maintaining semen quality. As indicated in Table 3, supplementation of 500 mg/kg RES increased serum testosterone levels (P < 0.01) and decreased cortisol levels (P < 0.05). In addition, RES enhanced the T-AOC of aging boars (P < 0.001) and reduced the concentration of MDA (P < 0.01), LPS (P < 0.01), and IL-6 (P < 0.05) in aging boar serum.

Table 3.

Effects of RES on hormone, antioxidant capacity, and inflammatory factors in aging boars

Serum indicators	CTL group	RES group	P-value
Testosterone, nmol/L	0.63 ± 0.12	0.95 ± 0.09	<0.001
Cortisol, μg/L	17.94 ± 2.74	12.08 ± 4.81	0.027
Total antioxidant capacity, μmol/L	37.73 ± 2.18	51.70 ± 6.21	<0.001
Malondialdehyde, nmol/mL	3.93 ± 0.47	2.75 ± 0.65	0.005
Lipopolysaccharide, pg/mL	47.58 ± 6.51	36.27 ± 3.64	0.004
Tumor necrosis factor-α, pg/mL	190.51 ± 19.90	188.58 ± 19.99	0.870
Interleukin-6, ng/mL	57.74 ± 2.92	53.78 ± 1.71	0.017

CTL, a basal diet; RES, a basal diet supplemented with 500 mg/kg resveratrol. Mean values are based on six replicates, and the results were presented as means ± SD.

The effect of RES on sperm oxidative stress

The immunofluorescence image of boar sperm is presented in Figure 2. RES reduced the ROS levels in sperm (P < 0.01, Figure 2A), as well as the levels of 8-OHdG (P < 0.01, Figure 2B) and MDA (P < 0.01, Figure 2C), two important factors that can evaluate sperm oxidative damage.

Figure 2.

The effect of RES on sperm oxidative stress. (A) Representative pictures of sperm immunofluorescence (DAPI: 4′,6-diamidino-2-phenylindole; ROS: reactive oxygen species). Semen levels of (B) 8-hydroxy-2′-deoxyguanosine (8-OHdG) and (C) malondialdehyde (MDA). CTL, basal diet; RES, dietary supplemented with 500 mg/kg resveratrol. Mean values are based on six replicates, and SDs are represented by vertical bars.

Effects of RES on sperm mitochondrial structure and function

Sperm mitochondrial function is a crucial factor influencing sperm motility. As shown in Figure 3, dietary RES had no significant effect on ND1 mtDNA expression (P > 0.05, Figure 3A) and ATP levels (P > 0.05, Figure 3B) in sperm. The transmission electron microscopy images of sperm mitochondria (Figure 3C) revealed a significant gap between the mitochondrial membrane and the plasma membrane, which appeared loose and incomplete in the CTL group. In contrast, the sperm mitochondria of the RES group appeared oval-shaped and were neatly arranged, with an intact plasma membrane closely attached to the mitochondria. The fluorescence intensity of the JC-1 monomer in the RES group was reduced (P < 0.01, Figure 3D), while that of the aggregates was increased (P < 0.01, Figure 3E), indicating that RES enhances MMP.

Figure 3.

Effects of RES on sperm mitochondrial structure and function. (A) Relative mitochondrial encoded NADH dehydrogenase 1 (ND1) DNA expression. (B) Semen adenosine triphosphate (ATP) levels. (C) Transmission electron micrographs (80 K) of mitochondria of sperm. Fluorescence intensity of JC-1 (D) monomer and (E) aggregates. CTL, basal diet; RES, dietary supplemented with 500 mg/kg resveratrol. Mean values are based on six replicates, and SDs are represented by vertical bars.

Modulation of RES on sperm mitochondrial function-related genes

The expression of genes related to sperm mitochondrial biogenesis such as Nrf1, peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α), TFAM, TFB1M, and TFB2M was measured by RT-PCR. Dietary RES increased the mRNA expression levels of PGC-1α (P < 0.01, Figure 4B), TFB1M (P < 0.01, Figure 4D), and TFB2M (P < 0.01, Figure 4E) in sperm. However, there was no significant difference in Nrf1 (Figure 3A) and TFAM (Figure 4C) mRNA expression levels. The correlation between sperm quality parameters and the regulation of mitochondrial functional genes was further analyzed. The results indicated that PGC-1α, TFB1M, and TFB2M were positively correlated with sperm motility and survivability (P < 0.05, Figure 4F).

Figure 4.

Modulation of RES on sperm mitochondrial function-related genes. The mRNA expression of (A) nuclear respiratory factor-1 (Nrf1), (B) peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α), (C) mitochondrial transcription factor A (TFAM), (D) mitochondrial transcription factor B1 (TFB1M), and (E) mitochondrial transcription factor B2 (TFB2M) was quantitated by real-time qPCR. (F) Pearson’s correlation analysis analyzed the correlation between sperm quality parameters and the regulation of mitochondrial functional genes. *P < 0.05 and **P < 0.01. CTL, basal diet; RES, dietary supplemented with 500 mg/kg resveratrol. Mean values are based on six replicates, and SDs are represented by vertical bars.

Discussion

Due to frequent collection of semen over an extended period, boars typically encounter a decline in semen quality after reaching 2 yr of age (Koketsu and Sasaki, 2009). Recent studies suggested that plant polyphenols with antioxidant capabilities may positively influence the semen quality of boars (Han et al., 2021). In this study, consistent improvements in semen quality parameters, particularly the significant enhancement of sperm survivability and motility, underscore the potential of RES as an effective nutritional strategy to mitigate the decline in aging boar fertility. Multiple studies have shown that testosterone levels are essential for maintaining high semen quality (Zhang et al., 2021; Maistrelli et al., 2022). This hormone interacts with the cells of the seminiferous epithelium to regulate and facilitate the differentiation and maturation of sperm (Grande et al., 2022). When the serum testosterone level is lower than normal, the meiotic process of spermatocytes is inhibited, resulting in a low number of mature sperm and an increased rate of malformations, leading to poor sperm function and even infertility (Walker, 2011). Furthermore, maintaining high levels of libido and frequent sperm collection over an extended period of time made boars highly susceptible to stress, which resulted in elevated cortisol levels (Rey-Salgueiro et al., 2018; Lightman et al., 2020). Although the negative effects of cortisol on sperm are unclear, studies have reported that increased cortisol levels in men can result in reduced testosterone levels and lower semen density (Sánchez González et al., 2023). At the same time, the frequent collection of artificial semen increases the risk of bacterial infection in boar testicular tissue and accessory glands (Kuster and Althouse, 2016). Upon activation by inflammation, white blood cells generate a significant amount of ROS through the nicotinamide adenine dinucleotide phosphate system and release proinflammatory factors, resulting in increased oxidative stress and inflammatory response (Fraczek et al., 2016; Zambrano et al., 2016). Increased levels of ROS caused by inflammation and oxidative stress inhibit the production of testicular steroids (Li et al., 2022). Our study showed that RES significantly increased testosterone levels while reducing the levels of cortisol, IL-6, and LPS in the serum of aging boars.

Oxidative stress caused by adverse factors in production has a serious negative impact on boar semen quality. Oxidative stress can cause excessive ROS levels in animals and inhibit the production of testicular steroid hormones (Li et al., 2022). Recent studies have shown that natural plant polyphenols such as epicatechin, lycopene, and curcumin can reduce ROS levels in semen, and play an antioxidant role in protecting sperm, thereby improving sperm motility and function (Tvrdá et al., 2022). In the present study, dietary RES significantly reduced the level of MDA in the serum of boars and increased the T-AOC of boars, which suggests that the positive effect of RES on boar hormone levels may be attributed to its antioxidant capacity. In addition, different from other cells, sperm are more sensitive to ROS due to their unique structure and function. ROS is essential for regulating sperm capacitation and acrosome reaction, but excessive ROS can have a detrimental effect on sperm function (Hall et al., 2017). The sperm plasma membrane comprises a rich proportion of polyunsaturated fatty acids and is liable to lipid peroxidation under the influence of high levels of ROS (Moazamian et al., 2015). On one hand, this will damage the integrity of the sperm plasma membrane structure. On the other hand, lipid peroxidation can also produce toxic substances such as MDA and 4-hydroxynonenol, which can lead to the formation of DNA adducts like 8-OHdG, causing oxidative DNA damage (Gogol et al., 2009; Lee et al., 2017). In this study, dietary RES was found to significantly reduce the levels of ROS, MDA, and 8-OHdG in semen, indicating that RES improved sperm’s antioxidant capacity and alleviated oxidative damage. Therefore, the positive effect of dietary RES supplementation might be due to the enhancement of aging boar antioxidant capacity.

Mitochondrial oxidative phosphorylation is crucial for sustaining the linear movement of sperm, but it is also the primary source of ROS, making mitochondria more susceptible to oxidative damage. ROS can hinder cell enzyme activity, thereby inhibiting ATP production, and ultimately leading to decreased sperm motility and even sperm death(Park and Pang, 2021; Escada-Rebelo et al., 2022). In the present study, we observed the ultrastructure of sperm mitochondria by transmission electron microscopy and evaluated the oxidative damage of sperm mitochondria. The results indicated that the sperm mitochondria in the RES group exhibited a consistent oval shape, were arranged in a more organized manner, and indicated that RES supplementation enhances the structural integrity of sperm mitochondria. In the process of aerobic respiration, the energy produced by mitochondria is stored in the mitochondrial inner membrane as electrochemical potential energy, resulting in an asymmetric distribution of protons and other ion concentrations on both sides of the inner membrane, creating the MMP. Normal MMP is a necessary condition for maintaining ATP production in mitochondria (Paoli et al., 2011). Although this study did not observe a significant increase in ATP levels in semen, we did find a notable enhancement in sperm MMP, suggesting that RES at least partially improves sperm mitochondrial biosynthesis.

We further explored the reasons why RES impacts sperm mitochondrial function, measuring the expression levels of mitochondrial function-related regulatory genes. Dietary RES did not significantly upregulate the expression of ND1 in sperm DNA, which explains why there was no significant increase in semen density and sperm count. Mitochondrial biogenesis requires the expression of multiple genes, including TFAM, PGC-1α, and Nrf1 among others (Bouitbir et al., 2012; Escada-Rebelo et al., 2022). This study found that dietary RES had no significant effect on the expression levels of TFAM, and Nrf1, but a significant increase in the expression levels of PGC-1α, TFB1M, and TFB2M in boar sperm. The PGC-1α coactivator plays a crucial role in regulating numerous genes that are essential for the expression and function of the mitochondrial respiratory chain (Bouitbir et al., 2012). An increasing body of evidence indicates that PGC-1α serves as a key target for RES in enhancing mitochondrial function. For example, it has been reported that RES can increase PGC-1α protein levels in the muscles of obese individuals and improve mitochondrial function, but it does not alter mitochondrial content (Timmers et al., 2011). It has also been reported that RES can regulate mitochondrial function and improve cardiac injury in diabetic rats through PGC-1α (Fang et al., 2018). In summary, although dietary supplementation of RES did not significantly impact sperm ATP levels, it improved mitochondrial structural integrity and MMP by upregulating the mRNA expression of PGC-1α, TFB1M, and TFB2M, which may be one of the factors that increase sperm motility in aging boars.

Conclusion

Dietary supplementation with 500 mg/kg RES can significantly enhance sperm motility and survivability in aging boars. The serum biochemical indexes of boars showed that RES significantly increased testosterone levels and T-AOC, while significantly decreasing cortisol, IL-6, LPS, and MDA levels. Moreover, RES reduced the levels of ROS, 8-OHdG, and MDA to alleviate oxidative damage in semen with improved mitochondrial structural integrity and MMP potentially by increasing the mRNA expression of PGC-1α, TFB1M, and TFB2M.

Glossary

Abbreviations:

ALH

amplitude head displacement

ATP

adenosine triphosphate

BCF

beat cross frequency

CASA

computer-assisted sperm testing system

CTL

control group

DAPI

4′,6-diamidino-2-phenylindole

MAD

mean angular displacement

MDA

malondialdehyde

MMP

mitochondrial membrane potential

ND1

mitochondrial encoded NADH dehydrogenase 1

Nrf1

nuclear respiratory factor-1

PGC-1α

peroxisome proliferator-activated receptor γ coactivator-1α

RES

resveratrol

ROS

reactive oxygen species

STR

straightness

T-AOC

total antioxidant capacity

TFAM

mitochondrial transcription factor A

TFB1M

mitochondrial transcription factor B1

TFB2M

mitochondrial transcription factor B2

VAP

average path velocity

VCL

curvilinear velocity

VSL

straight-line velocity

WOB

wobble of the curvilinear trajectory

Author contributions

Jing Lv (Data curation, Formal analysis, Investigation, Writing—original draft), Wentao Zhang (Formal analysis, Investigation), Ruizhi Hu (Conceptualization, Data curation, Investigation, Methodology), Xizi Yang (Formal analysis, Investigation, Methodology), Jiatai Gong (Formal analysis, Investigation), Siqi Ma (Formal analysis, Investigation), Hongkun Xiang (Formal analysis, Investigation), Xupeng Yuan (Project administration, Resources), Hongfu Zhang (Funding acquisition, Project administration, Resources, Supervision), Xi He (Funding acquisition, Project administration, Resources, Supervision), Jianhua He (Funding acquisition, Project administration, Resources, Supervision), Rejun Fang (Funding acquisition, Project administration, Resources, Supervision), and Shusong Wu (Conceptualization, Funding acquisition, Project administration, Resources, Writing— review & editing)

Conflict of interest statement

The authors declare no real or perceived conflicts of interest.

Data and model availability statement

The data supporting the findings of this study are available from the author upon reasonable request.

Declaration of generative AI and AI-assisted technologies in the writing process

The authors did not utilize any AI and AI-assisted technologies.