Dietary Lycopene Mitigates Reproductive Impairment in Heat-Stressed Rongchang Boars: Roles of Antioxidant, Anti-Inflammatory and Nrf2 Pathway

TL;DR

Adding lycopene to the diet of heat-stressed Rongchang boars improves sperm quality and reduces tissue damage by reducing oxidative stress and inflammation.

Contribution

This study identifies the testis and cauda epididymis as key tissues where lycopene mitigates heat stress through the Nrf2 pathway.

Findings

Heat stress increased sperm abnormalities and oxidative stress in multiple tissues, with the testis and cauda epididymis most affected.

Lycopene supplementation improved sperm motility, reduced inflammation, and upregulated Nrf2 pathway genes in heat-stressed boars.

Lycopene's protective effects are linked to its regulation of the Nrf2 antioxidant signaling pathway and reduction of inflammatory markers.

Abstract

Heat stress (HS) severely impairs boar reproductive function by inducing oxidative stress and inflammatory responses, while lycopene (LYC), as a potent antioxidant, exerts a potential protective effect on the male reproductive system. This study aimed to clarify the mechanism underlying LYC-mediated alleviation of HS-induced decline in semen quality in Rongchang boars, identify the most affected tissues, and explore its regulatory role in the Nrf2 (Nuclear factor E2-related factor 2) pathway. A total of 18 Rongchang boars with an initial body weight of 15.81 ± 1.07 kg were randomly assigned to three groups (6 boars per group): the control group (CON, 26 ± 1 °C), the heat stress group (HS, exposed to 35 ± 1 °C for 8 h daily), and the heat stress + 100 mg/kg lycopene group (HS + LYC). After 28 days of adaptive feeding and 14 days of HS treatment, samples were collected for semen quality analysis, testicular histological analysis, antioxidant index detection, transcriptome analysis, Nrf2 pathway detection, and inflammatory index detection. The results showed that HS significantly increased the sperm abnormality rate (p < 0.05), damaged the testicular structure, and induced oxidative stress in serum, lung, liver, left ventricle, testis, and epididymis (caput epididymis, corpus epididymis, cauda epididymis), with varying degrees of oxidative stress observed in these samples. Among these tissues, the testis and cauda epididymis exhibited the most significant responses to HS and LYC, with the comprehensive impact magnitudes of 317% and 514%, respectively. Enrichment analysis of differentially expressed genes (DEGs) in these two tissues revealed that the pathways mediating oxidative stress response displayed distinct tissue specificity, and all of them were closely associated with the Nrf2 antioxidant signaling pathway. HS significantly downregulated the mRNA expressions of Nrf2, Quinone Oxidoreductase (NQO1), Heme Oxygenase 1 (HMOX1) and Glutamate-Cysteine Ligase Catalytic Subunit (GCLC) genes as well as the protein level of Nrf2 in the testis and cauda epididymis, increased the protein level of Keap1, and significantly elevated the levels of interleukin-6 (IL-6), interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) in these two tissues (p < 0.05). Compared with the HS group, dietary supplementation of LYC significantly improved sperm motility and the proportion of rapidly progressive sperm, reduced the proportion of immotile sperm and sperm abnormality rate (p < 0.05), alleviated testicular damage and oxidative stress in various tissues, upregulated the mRNA expressions of Nrf2 and HMOX1 genes in the testis as well as the mRNA expressions of Nrf2, NQO1, HMOX1 and GCLC genes in cauda epididymis (p < 0.05), significantly increased the Nrf2 protein level and decreased the Keap1 protein level in these two tissues, and simultaneously decreased the levels of the aforementioned inflammatory factors (p < 0.05). In conclusion, dietary supplementation with 100 mg/kg LYC can alleviate HS-induced decline in semen quality and testicular damage by regulating the oxidative status and inflammatory level of relevant tissues (e.g., testis and cauda epididymis) in boars, and this protective effect may be associated with the regulation of the Nrf2 signaling pathway.

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Full text

1. Introduction

With global climate change, frequent occurrences of extreme high temperatures have made ambient temperatures more likely to exceed the physiological compensation limit of animal organisms, resulting in Heat stress (HS) [1]. Due to their low density of functional sweat glands and thick subcutaneous fat layer, pigs have limited thermoregulatory capacity and are more susceptible to HS, which exerts numerous adverse effects on their growth and development, physiological functions, and reproductive performance [2]. Spermatogenesis in male animals relies on a microenvironment 2–4 °C lower than body temperature; therefore, the testes are usually suspended outside the body in the scrotum to maintain this temperature difference [3]. Boars under HS typically exhibit reduced reproductive capacity, mainly characterized by decreased sperm motility, concentration, and volume, as well as abnormal sperm morphology [4,5], thereby affecting the conception rate and litter size of sows and causing substantial economic losses to the pig industry. Against the backdrop of the intensification and large-scale development of the swine industry, artificial insemination technology has been widely adopted, which has consequently improved the utilization efficiency of high-quality breeding boars. Rongchang pigs (Sus scrofa domesticus) are among the world’s eight outstanding pig breeds and have been included in the list of key protected pig genetic resources by the Food and Agriculture Organization (FAO) of the United Nations; their excellent genetic resources have been extended to many countries such as Vietnam and North Korea. Meanwhile, pigs serve as an ideal mammalian model for studying human reproductive physiology. Therefore, exploring strategies to alleviate HS-induced reproductive damage in boars is not only of great significance to the pig breeding industry but also provides an important reference for the prevention and treatment of related reproductive injuries in humans.

The mechanism by which high temperatures affect animals is closely associated with oxidative stress [6,7]. Under normal physiological conditions, the antioxidant defense system in organisms can counteract the excessive production of reactive oxygen species (ROS) [8]. However, HS increases ROS generation and reduces the activity of antioxidant enzymes [9,10,11]. When the antioxidant system is insufficient to scavenge excess ROS, the surplus ROS will attack lipids, proteins, and nucleic acids, triggering inflammatory responses [12]. Nuclear factor E2-related factor 2 (Nrf2) is an inducible transcription factor that plays a crucial role in maintaining redox signaling and combating oxidative stress [13,14]. Under normal circumstances, Nrf2 binds to Keap1 and remains in a low-activity state. When the organism is subjected to oxidative stimuli, Keap1 is inactivated, leading to the nuclear translocation of Nrf2, which in turn stimulates the transcription of downstream antioxidant genes [15]. Numerous studies have demonstrated that HS induces oxidative damage in various tissues, and activation of the Nrf2 pathway can effectively mitigate such oxidative damage [7,16,17,18,19]. Therefore, activating the Nrf2 antioxidant pathway is a viable strategy to alleviate HS-induced oxidative damage.

Lycopene (LYC) is a potent antioxidant among carotenoids, second only to astaxanthin, and is a major carotenoid in plasma and tissues [20]. In recent years, LYC has attracted widespread attention as a natural antioxidant feed supplement. When used as a pig feed additive, it can enhance the antioxidant capacity of tissues such as serum, muscle, and liver [21]. Various LYC supplementation studies in humans and rats have shown a positive correlation between LYC intake and semen quality. LYC can improve sperm count and motility by reducing sperm DNA fragmentation, decreasing lipid peroxidation, and regulating the antioxidant system [20]. However, the differential protective effects of LYC on multiple tissues of heat-stressed replacement boars have not been reported, and the key mechanism underlying its improvement of semen quality lacks research at the level of epididymal segmentation. Therefore, this study systematically investigated the differential protective effects of LYC on multiple tissues (including lung, left ventricle, liver, testis, and epididymal caput/corpus/cauda) of Rongchang replacement boars exposed to HS, further identified the tissues most responsive to LYC in alleviating HS, and thereby revealed the potential molecular mechanisms by which LYC improves sperm quality.

2. Materials and Methods

2.1. Experimental Diets and Feeding Management

All animal procedures were approved by the Animal Ethics Committee of Sichuan Agricultural University (Approval No.: SCAUAC202308-4) and conducted in accordance with the ARRIVE guidelines (https://arriveguidelines.org/, accessed on 19 May 2025) and the Guidelines for the Care and Use of Laboratory Animals.

The diet formulations during the trial were designed with reference to the nutritional requirements specified in the NRC (2012) [22] and Nutrient Requirements of Pigs in China (2020) [23]. The LYC diet was prepared by adding 0.01% lycopene (equivalent to 100 mg/kg) to the BD formulation, and the dosage of lycopene was determined based on the summary of [24,25]. The detailed diet formulations and nutritional levels are presented in Table 1.

Eighteen healthy Rongchang boars with similar initial body weight (15.81 ± 1.07 kg) were selected for the experiment. The trial was conducted at the Teaching and Research Base of the Institute of Animal Nutrition, Sichuan Agricultural University. All boars were initially housed in an environment with a thermoneutral temperature (26 ± 1 °C) and provided ad libitum access to feed and water. After a 4-week adaptation period, the boars were randomly divided into three groups (n = 6): the control group (CON) was maintained at the thermoneutral temperature for another 2 weeks, while the heat stress group (HS) and lycopene-supplemented heat stress group (HS + LYC) were exposed to a simulated natural high temperature (35 ± 1 °C) mimicking summer conditions for 2 weeks. Ambient temperature (T, °C) and relative humidity (RH, %) were recorded daily during the trial to calculate the temperature-humidity index (THI) using the formula THI = (1.8 × T + 32) − (0.55 − 0.55 × RH × 0.01) × (1.8 × T − 26).

2.2. Sample Collection

At the end of the trial, all boars were fasted for 12 h prior to anaesthesia and euthanasia. Blood was collected from the anterior vena cava and centrifuged to obtain serum (n = 6). Lung (L), Liver (Liv), left ventricle (LV), testis (Tes), caput epididymis (CE), corpus epididymis (CoE), and cauda epididymis (CaE) were harvested (n = 6). Spermatozoa from the left cauda epididymis were immediately subjected to semen quality analysis. Serum and all tissue samples were snap-frozen in liquid nitrogen and then stored at −80 °C for subsequent analyses of oxidative indices, inflammatory cytokines and gene expression.

2.3. Semen Quality Analysis

The caudal segment of the left epididymis was longitudinally incised with fine ophthalmic scissors, and the semen was completely extruded by gentle squeezing. The semen samples were placed on a Leja four-chamber counting slide (20 μm in thickness) and equilibrated on a thermostatic stage at 37 °C for 2 min. A Computer-Assisted Sperm Analysis (CASA) system (Model IVOS II, Hamilton Thorne Biosciences, Beverly, MA, USA) was used to automatically determine sperm viability, the percentage of rapidly progressive sperm, and the percentage of immotile sperm. Sperm kinematic parameters were defined according to the manufacturer-recommended porcine semen analysis protocol: sperm viability was the percentage of sperm with an average path velocity (VAP) ≥ 5 μm/s; rapidly progressive sperm referred to those with VAP ≥ 35 μm/s and distinct forward-moving trajectories; immotile sperm had a VAP < 5 μm/s (without obvious movement trajectories). For each sample, measurements were repeated across 4 fields of view and the mean value was calculated.

Then, 20 μL of semen was pipetted onto a glass slide and dispersed evenly. Each prepared slide was air-dried and fixed in Immunol Staining Fix Solution (Beyotime, Shanghai, China) for 15 min. After rinsing with ultrapure water and air-drying again, the slides were stained with Crystal Violet-Gentian Violet Staining Solution (Beyotime, Shanghai, China) for 15 min. Finally, the processed slides were rinsed and dried once more, placed in sealed bags for storage, and the sperm abnormality rate was to be determined by subsequent morphological observation. The criteria for sperm morphological abnormalities were strictly in accordance with the standards specified in the WHO Laboratory Manual for the Examination and Processing of Human Semen (6th Edition, 2021) [26].

2.4. Testicular Histological Analysis

Testicular tissue blocks fixed in 4% paraformaldehyde were processed for dehydration, clearing and wax infiltration, followed by paraffin embedding. Paraffin sections with a thickness of 5 μm were cut with a microtome and stained with hematoxylin-eosin (H&E). The stained sections were mounted with neutral balsam, then observed and photographed under a light microscope. A total of 50 seminiferous tubules were randomly selected from each group, and the Spermatogenic epithelium height and seminiferous tubule diameter were measured using ImageJ software (National Institutes of Health, Bethesda, MD, USA, Version 1.54f).

2.5. Measurement and Analysis of Oxidative Stress Indicators

2.5.1. Measurement of Oxidative Stress Indicators

Oxidative stress indicators in serum, liver, left ventricle, lung, testis, caput epididymis, corpus epididymis, and cauda epididymis were measured using commercial assay kits purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). The indicators included malondialdehyde (MDA, Cat. No. A003-1), protein carbonyl (PCO, Cat. No. A087-1), total antioxidant capacity (T-AOC, Cat. No. A015-2-1), catalase (CAT, Cat. No. A007-1-1), and superoxide dismutase (SOD, Cat. No. A001-3). The protein concentrations of various tissue samples were determined via the Bradford method using a commercially available assay kit (Cat. No. P0006, Beyotime Biotechnology, Shanghai, China).

2.5.2. Analysis of Impact Magnitudes of Oxidative Stress Indicators in Tissues

Calculation of Single-Indicator Impact Magnitudes

The impact magnitudes of heat stress and lycopene on individual oxidative stress indicators across different tissues were quantified using the following formulas:

Heat stress impact magnitude:

$$[eqn]$$

Lycopene mitigation magnitude:

$$[eqn]$$

Comprehensive Multi-Indicator Impact Assessment

Bar charts were constructed to visualize the impact magnitudes of each indicator (MDA, PCO, CAT, SOD, T-AOC) in each tissue. The sum of the absolute values of all indicators within each tissue was calculated, and the tissues most significantly affected by heat stress and lycopene were selected based on this sum value.

2.6. Transcriptome Analysis

Testis and cauda epididymis samples (n = 3) were randomly selected, snap-frozen in dry ice, and sent to LC Sciences (Hangzhou, China) for total RNA extraction, transcriptome sequencing on the Illumina platform, and basic bioinformatics analysis. The analytical workflow included raw data quality control, reference genome alignment, screening of differentially expressed genes (DEGs) (|log_2_FC| ≥ 1 and Padj < 0.05), Gene Ontology (GO) functional enrichment analysis, and Kyoto Encyclopedia of Genes and Genomes (KEGG) functional enrichment analysis.

2.7. Quantitative Real-Time PCR Analysis

Total RNA was isolated using the TIANGEN Animal Total RNA Extraction Kit (Cat. No. DP431), and its integrity was verified via spectrophotometry with a SuperMiro instrument (Model: SM-100, purchased from Shanghai Xituo Scientific Instruments Co., Ltd., Shanghai, China); samples with an OD_260_/OD_280_ ratio between 1.8 and 2.0 were considered acceptable for subsequent downstream processing, after which RNA was reverse-transcribed into complementary deoxyribonucleic acid (cDNA) using the HiScript III All-in-one RT SuperMix Perfect for qPCR (Cat. No. R333-01, purchased from Vazyme Biotech Co., Ltd., Nanjing, China). Each 10 μL polymerase chain reaction (pCR) mixture was prepared in the following proportions: 1 μL of cDNA, 5 μL of TB Green^®^ Premix Ex Taq™ II (containing Tli RNaseH Plus, Cat. No. RR820, purchased from TaKaRa, Beijing, China), 3 μL of sterile water, and 0.5 μL each of the forward and reverse primers, and the qPCR was performed with specific cycling parameters: initial denaturation at 95 °C for 30 s, followed by 40 cycles (each cycle including denaturation at 95 °C for 5 s and annealing/extension at 60 °C for 30 s). Primers were designed for the target sequences using the Primer-BLAST tool from the National Center for Biotechnology Information (NCBI) and commercially synthesized by Sangon Biotech Co., Ltd. (Shanghai, China), with detailed primer information provided in Table 2; the relative gene expression levels were calculated using the 2^−ΔΔCt^ method, with β-actin serving as the endogenous control.

2.8. ELISA Assay

The levels of inflammatory factors (interleukin-6 [IL-6], interleukin-1β [IL-1β], tumor necrosis factor-α [TNF-α]) and the protein levels of nuclear factor erythroid 2-related factor 2 (Nrf2) and Kelch-like ECH-associated protein 1 (Keap1) in the testis and cauda epididymis were determined using enzyme-linked immunosorbent assay (ELISA) kits purchased from Jiangsu Meimian Industrial Co., Ltd. (Yancheng, China)

2.9. Statistical Analysis

The normality of data and homogeneity of variances were evaluated using the Shapiro-Wilk test and Levene test, respectively. Based on the results of these preliminary tests,

When normality and homogeneity of variances were satisfied, one-way analysis of variance (One-way ANOVA) followed by Tukey’s post-hoc test was employed;When normality was satisfied but homogeneity of variances was violated, Welch’s ANOVA coupled with the Games-Howell post-hoc test was used;When normality was not satisfied, the Kruskal-Wallis H test followed by Dunn’s post-hoc test (with Bonferroni correction) was applied.

A p-value < 0.05 was considered statistically significant for all analyses. All statistical analyses were performed using SPSS version 27.0 (IBM Corp., Armonk, NY, USA).

3. Results

3.1. Establishment of Heat Stress Model and Evaluation of Semen Quality

The environmental temperature and humidity during the experimental period are illustrated in Figure 1. Throughout the trial, the average temperature, relative humidity, and temperature-humidity index (THI) recorded in weeks 1–4 were 26.78 °C, 83.27%, and 78.18, respectively. For weeks 5–6, the mean temperature, mean relative humidity, and mean THI were 36.35 °C, 62.06%, and 88.89, respectively (Figure 1a).

The semen quality parameters of different treatment groups are illustrated in Figure 1b–d. Compared with the CON group, the HS group exhibited a significantly higher abnormality rate (p < 0.05), while viability and the percentage of rapidly progressive sperm decreased and the percentage of immotile sperm increased without statistical significance (p > 0.05). In contrast, a marked improvement in semen quality was observed in the HS + LYC group. Specifically, viability and the percentage of rapidly progressive sperm were significantly increased (p < 0.05), whereas the percentage of immotile sperm and abnormality rate were significantly decreased compared with the HS group (p < 0.05).

3.2. Lycopene Ameliorates Heat Stress-Induced Testicular Structural Damage

Compared with the CON group, the HS group exhibited testicular structural damage in boars, and the spermatogenic epithelium height and seminiferous tubule diameter of testicular tissue were significantly decreased (p < 0.05). Compared with the HS group, lycopene supplementation significantly increased the spermatogenic epithelium height and seminiferous tubule diameter (p < 0.05; Figure 2a,b). Specifically, the HS group showed partial loss of spermatogenic cells and nuclear vacuolization relative to the CON group; lycopene supplementation ameliorated the vacuolization and increased the number of spermatogenic cells at all developmental stages (Figure 2c).

3.3. Lycopene Supplementation Alleviates Heat Stress-Induced Oxidative Stress in Boar Serum

Serum antioxidant indicators reflect the systemic oxidative stress status. The HS group exhibited obvious oxidative stress, as evidenced by increased PCO (p < 0.05) and decreased T-AOC (p < 0.05) compared with the CON group. After LYC intervention, MDA was decreased (p < 0.05), PCO was reduced (p < 0.05), and T-AOC was increased (p < 0.05). Additionally, no significant differences were observed in CAT and SOD activities (p > 0.05) (Figure 3).

3.4. Effects of Lycopene Supplementation on Heat Stress-Induced Oxidative Stress in Various Tissues of Boars

Compared with the CON group, HS significantly increased MDA content in the Liv, LV, Tes, CoE, and CaE of boars and significantly elevated PCO content in all tested tissues; meanwhile, it significantly reduced CAT activity in the Liv and Tes but abnormally increased CAT activity in the CE and CaE and significantly decreased SOD activity in all reproductive tissues, with T-AOC significantly reduced in all tissues except the CE (p < 0.05). Compared with the HS group, lycopene supplementation significantly reduced MDA levels in the L, CoE, and CaE and decreased PCO content in the Liv, LV, Tes, CoE, and CaE; it also significantly enhanced CAT activity in the L, Liv, Tes, and CE. However, lycopene failed to significantly improve SOD activity in the CE while effectively increasing it in the Liv, LV, Tes, CoE, and CaE and significantly enhanced T-AOC in the Liv, LV, Tes, CoE, and CaE under HS conditions (p < 0.05) (Figure 4a–e).

By quantifying the comprehensive impact of HS and LYC supplementation on various tissues, the following results were obtained: the total impact magnitudes on L, Liv, LV, Tes, CE, CoE, and CaE were 265%, 286%, 220%, 317%, 173%, 270%, and 514%, respectively. Among these tissues, Tes (317%) and CaE (514%) were the two tissues most significantly affected by HS and LYC, with their comprehensive response intensity far exceeding that of other tissues (Figure 4f).

3.5. Testicular Transcriptome Analysis Results

By comparing the testicular tissue gene expression profiles between the HS group and the CON group, we identified a total of 282 significantly differentially expressed genes (DEGs): 157 were significantly downregulated and 125 were significantly upregulated. Further analysis of the regulatory effect of the HS + LYC group on testicular gene expression revealed 694 DEGs between the HS + LYC and HS groups—326 downregulated and 368 upregulated—with a more pronounced magnitude of difference. Hierarchical clustering heatmap results showed that testicular samples from the CON, HS, and HS + LYC groups clustered into distinct, independent branches, reflecting clear divergence in their gene expression patterns. We then performed enrichment analysis of the DEGs using the KEGG database. Figure 5d presents the enrichment outcomes of oxidative stress-related signaling pathways, where Oxidative phosphorylation and Glutathione metabolism displayed relatively high enrichment significance. The involved DEGs include ND1, CYTB, COX1, ATP6, CHAC1, and GPX7, etc. (Table S1). These core functions were partially validated by Gene Ontology (GO) enrichment analysis. Notably, GO enrichment specifically detected the “DNA damage response” term, involving key genes such as ATM and RAD54B (Table S2).

3.6. Cauda Epididymis Transcriptome Analysis Results

A total of 854 DEGs were identified between the HS and CON groups, including 388 downregulated and 466 upregulated genes. This finding suggests that heat stress induces a milder disruption of gene expression in the cauda epididymis compared to testicular tissue. Comparative analysis between the HS + LYC and HS groups revealed 2365 DEGs, with 904 downregulated and 1461 upregulated. Hierarchical clustering heatmap analysis demonstrated that the cauda epididymis samples could be clearly divided into three discrete clusters. Enrichment analysis of DEGs in the cauda epididymis was performed using the KEGG database, and Figure 6d presents the enrichment profile of oxidative stress-related signaling pathways. Among these, the MAPK signaling pathway, Drug metabolism—cytochrome P450, and the PI3K-Akt signaling pathway exhibited relatively high enrichment significance, involving the DEGs MAPK10, MAPK13, JUN, GSTO2, AOX1, JAK1, and PIK3R6 (Table S3). Correspondingly, GO enrichment analysis for oxidative stress-related terms showed significant enrichment in processes such as oxidoreductase activity and reactive oxygen species metabolic process, which were associated with key genes including SOD3, GPX5, GPX6, and UCP2 (Table S4).

3.7. Lycopene Alleviates Oxidative Stress by Regulating the Nrf2 Pathway in the Testis and Cauda Epididymis and Its Correlation with Semen Quality

3.7.1. Changes in Nrf2 Pathway Gene Expression and Nrf2/Keap1 Protein Levels

In testicular tissues, compared with the CON group, the HS group showed a significant decrease in both Nrf2 gene expression and Nrf2 protein level, along with a significant increase in Keap1 protein level (p < 0.05); the expression levels of its downstream antioxidant genes NQO1, HMOX1 and GCLC also exhibited a significant downward trend (p < 0.05). Compared with the HS group, the HS + LYC group had a significant upregulation in Nrf2 gene expression, Nrf2 protein level and HMOX1 gene expression and a significant decrease in Keap1 protein level (p < 0.05). In the cauda epididymis, compared with the CON group, the HS group presented a significant reduction in Nrf2 gene expression and Nrf2 protein level as well as a significant elevation in Keap1 protein level (p < 0.05), and the expression levels of NQO1, HMOX1 and GCLC were also significantly downregulated (p < 0.05). Compared with the HS group, the HS + LYC group had a significant increase in Nrf2 protein level and a significant decrease in Keap1 protein level (p < 0.05), while the expression levels of Nrf2, NQO1, HMOX1 and GCLC were all significantly upregulated (p < 0.05) (Figure 7a–d).

3.7.2. Correlation Analysis Between Semen Quality and Key Genes in the Nrf2 Pathway

The correlation analysis between semen quality and key genes in the Nrf2 pathway revealed the following results: In the testes, NQO1 showed negligible to weak negative correlations with Viability Rate and Rapid Progressive Motility Rate (|r| = 0.08/0.21) and a negligible correlation with Immotility Rate (|r| = 0.08). By contrast, Nrf2, HMOX1, and GCLC in both the testes and cauda epididymis exhibited weak to moderate positive correlations with Viability Rate and Rapid Progressive Motility Rate (|r| = 0.13–0.44) and weak negative to moderate positive correlations with Immotility Rate (|r| = 0.13–0.40).

Key genes of the Nrf2 pathway displayed high coordination (|r| > 0.5) within the same tissue; additionally, their expression patterns showed certain consistency between the testes and cauda epididymis (|r| = 0.43–0.9), which reflects the overall regulatory trend of this pathway in the reproductive system (Figure 7e).

3.8. Effects of Lycopene on Inflammatory Cytokine Levels in Testes and Cauda Epididymis

Under heat stress, the levels of inflammatory cytokines (IL-6, IL-1β, and TNF-α) in both testes and cauda epididymis were significantly higher than those in the CON group (p < 0.05). Notably, lycopene supplementation exerted a marked alleviative effect in the HS + LYC group, as evidenced by significant reductions in the levels of these inflammatory cytokines (p < 0.05). However, the levels of these cytokines in the HS + LYC group remained significantly higher than those in the CON group (p < 0.05) (Figure 8).

4. Discussion

High temperature and humidity prevail in southern China during summer, exerting a significant impact on pig herds with poor thermoregulatory capacity. The optimal growth temperature for boars is 26 °C, while the average temperature during heat stress (HS) treatment in this experiment reached 36.35 °C, with a relative humidity of 62.06% and a temperature-humidity index (THI) as high as 88.89. A THI ≥ 82 indicates that animals are in a state of severe HS [27]; thus, all boars in this experiment were exposed to severe HS conditions.

High temperatures in summer can impair reproductive function and reduce semen quality in various male animals [28,29]. Our study found that the rate of sperm malformation in boars increased significantly under HS conditions; consistent with this, HE staining results of testicular tissue showed obvious structural damage in the HS group, characterized by partial loss of spermatogenic cells, nuclear vacuolization of spermatogenic epithelium, and significant decreases in spermatogenic epithelium height and seminiferous tubule diameter (p < 0.05) when compared with the CON group. Abnormal sperm directly affects the fertilization process and reduces the probability of conception, whereas dietary supplementation with LYC reversed the negative effects of HS on boar semen quality and significantly improved core indicators including sperm motility, the proportion of rapidly progressive motile sperm, the proportion of immotile sperm, and sperm malformation rate; meanwhile, LYC supplementation effectively ameliorated testicular structural damage induced by HS, alleviating the vacuolization of spermatogenic cell nuclei and significantly increasing the spermatogenic epithelium height and seminiferous tubule diameter (p < 0.05). In fact, the protective effect of lycopene on the male reproductive system has been verified in different species. Previous studies have reported that serum lycopene levels in infertile men are significantly lower than those in healthy men [30]. Gupta et al. demonstrated through clinical trials that oral administration of lycopene increased sperm concentration in 66% of infertile patients, improved sperm motility in 53% of patients, and normalized sperm morphology in 46% of patients [31]. Li et al. gavaged heat-stressed mouse models with different doses of lycopene, and the results showed increased sperm motility and sperm count in all dose groups, along with alleviated testicular damage in mice [32]. This study confirmed that dietary supplementation with 100 mg/kg lycopene effectively alleviated the decline in boar semen quality caused by HS and repaired the damaged testicular spermatogenic structure, further corroborating the positive role of lycopene in protecting male reproductive function against stress.

It is well established that HS enhances ROS production, inhibits the activity of antioxidant enzymes in the body, and causes oxidative damage to multiple tissues. MDA, a lipid peroxidation product, is widely recognized as a biomarker of tissue oxidative stress, while PCO is a characteristic product of protein oxidative damage under oxidative stress conditions. Studies have confirmed that high temperatures reduce the activity of antioxidant enzymes in in vitro cultured testicular tissues, significantly increase MDA content, and simultaneously disrupt testicular tissue morphology and impair testicular physiological functions [33]. In a heat-stressed rat model, the production rate of superoxide anion radicals in liver and heart tissues was significantly accelerated, and the activity of total superoxide dismutase (SOD) decreased, whereas catalase (CAT) activity showed no significant change [11]. The results of this study indicated that different tissues exhibit tissue-specific differences in antioxidant responses to HS. At the serum level, HS significantly increased PCO content and decreased total antioxidant capacity (T-AOC), reflecting a systemic state of oxidative stress in the body. The lung, liver, and left ventricular tissues all exhibited characteristics of exacerbated oxidative damage and reduced antioxidant capacity: increased PCO content and decreased T-AOC in lung tissue; increased PCO content, along with decreased CAT activity and T-AOC in liver tissue; and simultaneous increases in MDA and PCO content, coupled with a significant reduction in T-AOC in left ventricular tissue. Testicular tissue and all segments of the epididymis (caput epididymis, corpus epididymis, cauda epididymis) showed obvious oxidative damage under HS conditions, specifically manifested by increased MDA and PCO content and decreased SOD and T-AOC activity. Notably, the response patterns of CAT activity in different tissues were heterogeneous, showing three trends: increased (caput epididymis, cauda epididymis), decreased (liver, testis), or no significant change. Existing studies have confirmed that the response of the antioxidant enzyme system in different tissues to high-temperature environments is significantly heterogeneous. Chih-Ya Yang et al. [11] reported that in a high-temperature-treated rat model, CAT activity exhibited distinct trends in different brain regions: no significant change in the cerebral cortex, a significant decrease in the hippocampus and hypothalamus; liver CAT activity first decreased and then increased, while no obvious change was observed in heart tissue. Another study found that both SOD and CAT activities increased in broiler embryos under chronic HS conditions [34], reflecting differences in the tolerance and compensatory capacity of different tissues to HS-induced oxidative stress. Previous studies have reported that dietary supplementation with different doses of lycopene (12.5, 25, 37.5, 50 mg/kg) can improve the antioxidant status of the liver in finishing pigs [35]; adding 100~200 mg/kg lycopene to the diet of finishing pigs for 70 consecutive days significantly increased the activity of antioxidant enzymes in serum, liver, and muscle tissues, while reducing MDA content [25]; at the cellular level, lycopene can alleviate the toxic effects of zearalenone (ZEA) on porcine Sertoli cells, enhance cellular antioxidant capacity, and reduce intracellular MDA and ROS levels [36]. Similar conclusions were obtained in this study: dietary supplementation with 100 mg/kg lycopene reduced serum MDA and PCO content in heat-stressed boars and significantly increased T-AOC; meanwhile, lycopene exerted a significant reversing effect on HS-induced oxidative damage in various tissues, specifically by decreasing MDA and PCO content and enhancing the function of the antioxidant system in each tissue. Comprehensive effect analysis showed that the testis (317%) and cauda epididymis (514%) were the two tissues most responsive to HS and lycopene, with their comprehensive response intensities much higher than those of other tissues. Therefore, subsequent studies should focus on an in-depth analysis of these two tissues.

To further clarify the molecular mechanism by which lycopene (LYC) alleviates HS-induced reproductive damage in boars, transcriptome sequencing analysis was performed on testicular and cauda epididymal tissues. The results of differential comparisons showed that both HS and lycopene could regulate the gene expression profiles of boar testis and cauda epididymis. Hierarchical clustering analysis indicated that all samples were clearly divided into three independent clusters, corresponding to the control group (CON), HS group, and lycopene treatment group (HS + LYC), confirming the good stability of the transcriptomic responses induced by HS and lycopene intervention.

Notably, the enrichment profiles of antioxidant-related KEGG pathways for DEGs in the two tissues were inconsistent. In testicular tissue, DEGs were mainly enriched in the oxidative phosphorylation and glutathione metabolism pathways: oxidative phosphorylation represents the primary endogenous source of ROS [37]. The DEGs involved in this pathway, including ND1, COX1, CYTB and ATP6, are all core respiratory chain genes encoded by mitochondrial DNA, and their expression levels and functional status directly affect the efficiency of the mitochondrial electron transport chain, thereby regulating ROS production. Mitochondrial ROS, in turn, can activate Nrf2 through an indirect kinase-dependent mechanism. Meanwhile, as a core upstream transcriptional regulator of the glutathione metabolism pathway, Nrf2 can promote the de novo synthesis of glutathione (GSH) [38], whereas CHAC1, a DEG in this pathway, is responsible for GSH degradation [39]. HS and LYC can thus regulate oxidative stress by targeting this gene. In cauda epididymal tissue, the MAPK and PI3K-Akt signaling pathways, where DEGs were significantly enriched, are both involved in mediating oxidative stress responses and act as upstream regulatory pathways of Nrf2 [40]. The results of GO functional enrichment analysis showed that DEGs in testicular tissue were associated with the term “DNA damage response”, while those in cauda epididymal tissue were linked to “oxidoreductase activity” and “reactive oxygen species metabolic process”. These findings collectively indicate that HS and LYC interventions have altered the antioxidant status of the two tissues. As a core transcriptional pathway mediating cellular antioxidant stress, activation of the Nrf2 pathway can directly regulate the expression of various downstream antioxidant enzymes [41], serving as a key target for reflecting changes in tissue antioxidant capacity. Based on these observations, qPCR was subsequently performed to detect the expression characteristics of Nrf2 and its downstream target genes in testicular and cauda epididymal tissues, aiming to clarify the molecular mechanisms by which HS and LYC regulate the antioxidant capacity of these two tissues.

A variety of natural antioxidants usually alleviate oxidative stress-mediated tissue damage by regulating the activation of the Nrf2 signaling pathway. For example, under oxidative stress conditions, the plant extract resveratrol (RSV) can directly protect porcine intestinal epithelial cells (IPEC-J2) from oxidative damage by significantly activating the Nrf2 pathway [19]; another study on carotenoids found that astaxanthin can upregulate the mRNA levels and protein expression of Nrf2 downstream target genes (NQO1, GCLM, HO-1, GCLC), thereby protecting human retinal pigment epithelial cells (ARPE-19) from oxidative stress damage [42]; similarly, lycopene has been confirmed to alleviate ZEA-induced oxidative damage in porcine Sertoli cells through the Nrf2 signaling pathway [36]. Our experiments found that HS significantly downregulated the mRNA levels of the Nrf2 transcription factor in testicular and cauda epididymal tissues and simultaneously reduced Nrf2 protein abundance while increasing Keap1 protein levels in both tissues (p < 0.05), while also significantly reducing the mRNA expression of its downstream target genes NQO1, HMOX1, and GCLC. After lycopene intervention, the mRNA level of HMOX1 in testicular tissue was extremely significantly increased, whereas the regulatory effects on NQO1 and GCLC were not significant; in cauda epididymal tissue, lycopene significantly upregulated the mRNA levels of NQO1, HMOX1, and GCLC. Notably, Nrf2 protein expression was significantly upregulated and Keap1 protein level was significantly decreased in both tissues after lycopene intervention (p < 0.05). Among them, the downregulation trend of HMOX1, a key gene in the Nrf2 pathway, was more significant in testicular tissue; meanwhile, lycopene intervention only significantly increased the transcriptional level of this gene. Existing studies have reported inconsistent expression patterns of Nrf2 target genes following Nrf2 activator treatment, which aligns with the tissue-specific regulatory differences of Nrf2 downstream genes observed in the present study. Specifically, two studies analyzing whole-blood RNA from patients receiving Nrf2 activators showed conflicting results for NQO1 and HMOX1 levels: Hammer et al. [43] found that HMOX1 expression remained unchanged while NQO1 was significantly upregulated, whereas Doss et al. [44] observed a significant increase in HMOX1 expression (p < 0.05) but no significant differences in NQO1 mRNA levels across different treatments or doses. Additionally, Jakubowska et al. [45] further noted in their review that Nrf2 activation leads to inconsistent expression of Nrf2 target genes (including NQO1, GCLC, GCLM, HMOX1, TXNRD1, and SRXN1) in non-communicable diseases. Notably, they found that HMOX1 is the most frequently upregulated gene in respiratory diseases and the most consistently differentially expressed gene reported in cardiovascular disease-related studies, which provides strong evidence for the heterogeneous characteristics of Nrf2 target gene activation and offers a reasonable explanation for the phenomenon observed in the present study.

This study further analyzed the correlation between key genes of the Nrf2 pathway in testicular and cauda epididymal tissues and semen quality indicators, including sperm motility, progressive motility rate, and immotile sperm rate. The results showed that Nrf2 pathway genes in testicular and cauda epididymal tissues were generally weakly to moderately correlated with semen quality indicators. This result is consistent with the established role of the Nrf2 pathway in antioxidant protection of the reproductive system. For example, ginsenoside Rg3 can improve dibutyl phthalate (DBP)-induced decline in mouse semen quality through the Nrf2/ARE pathway [46]; Neopyropia yezoensis polysaccharide can also improve epididymal sperm quality via the Nrf2 pathway [47]. These studies have confirmed that activation of the Nrf2 pathway is indeed closely related to the maintenance of sperm function. Notably, the NQO1 gene in testicular tissue was weakly negatively correlated with sperm motility and progressive motility rate but had no correlation with immotile sperm rate. Based on existing studies, the potential mechanism may be that the NQO1 gene in testicular tissue is more inclined to participate in the regulation of cell proliferation and differentiation [48,49], rather than directly maintaining sperm motility-related functions. Future studies can further clarify the specific role of the NQO1 gene in testicular spermatogenesis through NQO1 gene silencing or overexpression experiments; meanwhile, combined with metabolomic analysis, research can explore the association between the NQO1 gene and metabolites related to sperm energy metabolism and membrane function, thereby revealing the molecular mechanism underlying this weak negative correlation.

The damage of HS to male reproductive organs in boars is not only reflected at the oxidative stress level but is also accompanied by obvious inflammatory damage. Oxidative stress promotes the secretion of proinflammatory cytokines, and the inflammatory response in turn exacerbates oxidative stress damage [50], thus forming a mutually reinforcing vicious cycle. The results of animal experiments in this study confirmed that HS significantly upregulated the expression of inflammatory factors IL-6, IL-1β, and TNF-α in testicular and cauda epididymal tissues, which is consistent with previous research reports: Du et al. found that excessive ROS in microglia of heatstroke mice activates the NLRP3 inflammasome, leading to increased levels of inflammatory factors IL-1β and IL-18 [51]; Guo et al. reported that HS reduces sperm quality in dairy goats and significantly increases the expression levels of TNF-α and IL-6 in testicular tissue [52]. Lycopene intervention significantly downregulated the expression levels of the above proinflammatory factors. Previous studies have confirmed that excessive production of proinflammatory cytokines disrupts cellular homeostasis and causes irreversible tissue damage; lycopene, however, can break the vicious cycle of oxidative stress and inflammation by inhibiting excessive inflammatory activation, thereby exerting a tissue-protective effect under HS conditions. For example, in a rat model of bisphenol A-induced lung tissue inflammatory injury, lycopene intervention reduced lung tissue hemorrhagic spots and inflammatory cell infiltration, and significantly downregulated the mRNA expression levels of IL-1β and IL-6 [53], confirming the anti-inflammatory and tissue-protective effects of lycopene.

5. Conclusions

Dietary supplementation with 100 mg/kg lycopene can upregulate the expression of key genes in the Nrf2 pathway, thereby targeting and protecting the testes and cauda epididymis (the most affected tissues) of Rongchang replacement boars from heat stress-induced oxidative damage and inflammatory responses, alleviating heat stress-induced testicular structural damage, and ultimately alleviating the decline in semen quality caused by heat stress. However, this study has several limitations, including the unclear specific molecular targets underlying lycopene’s protective effects. Future research needs further refinement to support the large-scale application of lycopene in pig production.