Keywords: motility; boar; fatty acid; metabolism; β-oxidation
In mammals, sperm motility is essential for sperm migration from the ejaculation sites to the oviduct for fertilization in vivo [[
Previous studies reported that a diet supplemented with fish oil that was a rich source of long-chain fatty acids significantly improved sperm motility and semen production in boar [[
All reagents were purchased from Sigma Chemical (Shanghai, China), unless otherwise specified.
All animals and experimental procedures were approved by the Northwest A&F University Institutional Animal Care and Use Committee. Five mature and reproductively good duroc boars (aged 15–28 months) were used in this study. The boars were housed individually, maintained under natural daylight, fed basal diets, and provided free access to water. The sperm-rich ejaculate fraction was collected weekly from each boar using the gloved-hand method. Ejaculated semen was placed in flasks with isolated containers at 37 °C and processed in the laboratory within 30 min after collection. Only ejaculates containing more than 80% motile sperm (evaluated by phase-contrast microscopy) were used in this study. Fresh semen was diluted with the modified Modena solution according to Zhu et al. [[
Fatty acid analyses in sperm were performed using gas chromatography [[
The analysis was performed on a Varian CP-3900 Gas Chromatograph (Varian Inc., Palo Alto, CA, USA) equipped with a flame ionization detector, using a capillary column Model CP9205-VF-WAXms (Agilent Technologies Espana S.L., Madrid, Spain). The chromatograms with peak retention times and areas were produced on the recording integrator and were electronically transferred to the computer for analysis, storage, and report generation. Peak naming and column performance were achieved according to the calibration standard mix. Oleic acid and palmitic acid were identified by the order of elution and compared with known commercially prepared fatty acid standards GLC 566-C (Nu-Chek Prep Inc., Elysian, MN, USA).
According to Zhu et al. [[
The LIVE/DEAD Sperm Viability Kit (Leiden the Netherlands, L7011) and fluorescein isothiocyanate-peanut agglutinin (FITC-PNA) were used to evaluate sperm membrane integrity and acrosome integrity, respectively, according to Zhu et al. [[
The JC-1 (5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethyl benzimidazole-carbocyanine iodide) Mitochondrial Membrane Potential Detection Kit (Beyotime Institute of Biotechnology, China) was used to analyze sperm mitochondrial membrane potentials according to our previous study [[
The Annexin V-FITC/PI apoptosis detection kit (Sigma-Aldrich, St. Louis, MO, USA) was used to assess sperm apoptosis according to the manufacturer's instruction. The sperm sample was incubated with Annexin V-FITC/PI working solution at a concentration of 1 × 10
The ATP Assay Kit (Beyotime Institute of Biotechnology, China) was used to measure the sperm ATP level. According to the previous study, the sample was added to the luciferin/luciferase reagent in the 96 well plate. The luminescence at integration × 1000 ms was read using an Ascent Luminoskan luminometer (Thermo Scientific, Palm Beach, FL) with phosphate-buffered saline as a blank for each experiment. A standard curve was constructed using standards containing 0.01, 0.03, 0.1, 0.3, 1, 3, and 10 μM ATP. Analyses were performed in triplicate (n = 3).
The MDH and SDH activities were measured using a Malate Dehydrogenase assay kit and a Succinate Dehydrogenase assay kit (Nanjing Jiancheng Bioengineering Institute), respectively. According to Zhu et al. [[
Sperm samples were washed with PBS, fixed with 4% paraformaldehyde for 10 min, spread onto the poly-L-lysine slides, and air dried at room temperature. The samples were permeabilized with 0.5% Triton X-100 in PBS for 10 min. Nonspecific binding was blocked with PBS in the presence of 10% BSA (Sigma-Aldrich) for 30 min at room temperature. Samples were then incubated with anti-CPT1(sc-514555; 1:25) and anti-ACADVL (sc-271225; 1:50) overnight at 4 °C. The samples were rinsed with PBS 3 times and then incubated with biotinylated goat anti-mouse FITC-IgG (1:200) the next day. Sperm were washed and counterstained with DAPI (CWBIO). Fluorescent images were captured with fluorescence microscopy (80i, Nikon, Japan). Negative control immunostaining was also performed at the same time without the primary antibody.
The CPT1 and ACADVL activities were measured using a carnitine palmitoyltransferase 1 assay kit (Nanjing Jiancheng Bioengineering Institute) and a long-chain acyl-coenzyme A dehydrogenase assay kit (Oulu Biotechnology), respectively. Sperm samples were lysed ultrasonically (20 kHz, 750 W, operating at 40% power, 5 cycles of 3 s on and 5 s off) and centrifuged at 2000× g for 10 min at 4 °C. The supernatants were used to analyze the activities of CPT1 and ACADVL according to the manufacture's protocols. Analyses were performed in triplicate (n = 3).
Total proteins were extracted from sperm under different treatments with lysis buffer. The protein concentration was measured with a BCA assay kit (TaKaRa, Dalian, China). The proteins were then separated by 12.5% SDS-PAGE and transferred to a PVDF membrane. The membrane was blocked with 5% BSA for 2 h and then incubated with anti-cleave caspase3 (CST, 1:1,000), anti-cleave caspase 9 (CST, 1:1,000), anti-p53 (CST, 1:1,000), anti-Parp-1 (CST, 1:1,000), anti-CPT1 (Santa Cruz,1:1,000), anti-ACADVL (Santa Cruz,1:1,000), and anti-α-tubulin (Santa Cruz,1:1,000) at 4 °C overnight. HRP conjugated goat anti-rabbit and goat anti-mouse antibodies were used as the secondary antibody, respectively (1:2000 final dilution). The reagent for enhanced chemiluminescence (Bio-Rad) was used for detection and developed by X-ray film (Champchemi Top610, China).
Experiment 1 aimed to evaluate whether the exogenous oleic acid and palmitic acid were beneficial to boar sperm diluted with modified Modena solution during the liquid storage at 17 °C for 7 days. Firstly, the change of oleic acid and palmitic acid levels was measured at 0, 1, 3, 5, and 7 day points of the storage. Secondly, sperm were exposed to different concentrations of oleic acid (OA: 0, 50, 100, 150, 200 μM) and palmitic acid (PA: 0, 25, 50, 75, 100 μM) during liquid storage. Sperm motility, membrane integrity, acrosome integrity, and apoptosis were also assessed after 7 days of storage.
Experiment 2 was to investigate whether sperm metabolized oleic acid and palmitic acid via β-oxidation to generate ATP to maintain sperm quality. Firstly, the expression and localization of the CPT1 and ACADVL enzymes in boar sperm were identified by Western blotting and immunofluorescence, respectively. Secondly, the fresh semen was diluted with modified Modena solution at a concentration of 3.0 × 10
Experiment 3 was to confirm further that sperm utilize oleic acid and palmitic acid using the CPT1 selective inhibitor, etomoxir, to block sperm β-oxidation. Sperm were incubated with different concentrations (0, 1, 10, 100, 1000 μM) of etomoxir for 3 h, and the percentage of dead sperm, progressive motility, and straight-line velocity (VSL) and CPT1 activity were evaluated after 3 h of incubation. Secondly, one-hundred micromolar etomoxir was used for incubation with sperm in the following experiment: (
All data were tested for normality and variance of homogeneity prior to statistical analysis. If necessary, the data were transformed with arc-sin square root transformation. Data were analyzed by one-way ANOVA and analyzed with multiple comparisons with the Tukey test using SPSS Version 17.0 for Windows (SPSS Inc., Chicago, IL). All the values are presented as the mean ± standard deviation (SD). Treatments were considered statistically different from one another at p < 0.05.
As shown in Figure 1A, the OA level in sperm was not changed in the first day, but significantly decreased at 3 days, 5 days, and 7 days of storage compared to that of fresh sperm. The level of PA was also not changed after one day of storage, but significantly decreased after three days of storage (Figure 1B).
As the oleic acid and palmitic acid levels were decreased during liquid storage, different concentrations of OA (0, 50, 100, 150, 200 μM) and PA (0, 25, 50, 75, 100 μM) were added to the extender to elucidate whether they improved the sperm quality or not. It was observed that the addition of 100 and 150 μM OA significantly increased the total motility, progressive motility, and VSL of sperm, whereas the 50 and 200 μM OA treatments were similar to the control (Figure 2A–C). Interestingly, the 100 μM OA treatment showed the highest values of the sperm motility patterns among all the OA treatments (Figure 2A–C). The addition of PA at 25, 50, and 75 μM concentration also significantly improved sperm total motility, progressive motility, and VSL upon storage (Figure 2D–F); however, these motility patterns were decreased at 100 μM level (Figure 2D–F). The results of sperm motility patterns showed that 75 μM was the most effective concentration for palmitic acid improving sperm quality (Figure 2D–F).
In terms of sperm membrane integrity, the addition of both OA (100 μM) and PA (75 μM) to the extender significantly increased sperm membrane integrity after seven days of storage (Figure 3A,B). Moreover, the percentage of sperm with intact acrosome was increased by the addition of different concentrations of OA (100 and 150 μM) and PA (50 and 75 μM) to the extender (Figure 3C,D).
As shown in Figure 4A, the sperm stained with Annexin V-FITC/PI was classified into four groups: live sperm (AN-/PI-; blue arrow); early apoptotic sperm (AN+/PI-; white arrow); late apoptotic sperm (AN+/PI+; yellow arrow); and non-viable necrotic sperm (AN-/PI+; black arrow). It was observed that the addition of either oleic acid or palmitic acid significantly decreased the apoptosis of sperm (AN+/PI- and AN+/PI+ groups), where 100 μM OA and 75 μM PA treatments showed the lowest percentage of sperm with apoptosis (Figure 4B,C). In the results of Western blotting detection, it was observed that the expression of apoptotic factor proteins (Parp-1, cleaved-caspase 3, cleaved-caspase 9, and p53) was also significantly decreased by either the 100 μM OA or 75 μM PA treatment, compared to the control. (Figure 4D,E).
To test the hypothesis that boar sperm may utilize oleic acid and palmitic acid for ATP production via β-oxidation to maintain sperm motility, firstly, the CPT1 and ACADVL enzymes that were involved in β-oxidation were detected in boar sperm using Western blotting and immunofluorescence. CPT1 is an enzyme necessary for the transport of long-chain fatty acids into the mitochondrial matrix and hence for mitochondrial β-oxidation. The CPT1 protein was expressed in fresh boar sperm and mainly localized at the midpiece, where the mitochondria are distributed (Figure 5A,C). Moreover, ACADVL, an enzyme involved in the first step of fatty acid β-oxidation, was also expressed and localized in the midpiece, especially in the junction between sperm head and midpiece (Figure 5B,D).
The addition of either OA (100 and 150 μM) or PA (
As shown in Figure 8A–C, the motility tracks generated by CASA revealed that sperm linear motility was also improved by the addition of oleic acid and palmitic acid during the 3 h incubation. Moreover, higher sperm progressive motility and VSL were observed in both OA- and PA-treated groups than those in the control (Figure 8D,E), whereas the total motility did not change (Figure 8F).
Etomoxir, an inhibitor of CPT1, was used to re-evaluate the β-oxidation of the oleic acid and palmitic acid that sperm utilized for ATP production. It was observed that the percentage of dead sperm in the 1000 μM etomoxir treatment was the highest, while those in the other treatments were similar to the control (Figure 9A,D), indicating that 1000 μM etomoxir was toxic to sperm. Meanwhile, sperm progressive motility and VSL were decreased in 100 and 1000 μM etomoxir treatments, but the other treatment was similar to the control (Figure 9B,C). Moreover, the CPT1 activity was decreased only in 100 and 1000 μM etomoxir treatments (Figure 9E). Thus, 100 μM etomoxir was used to incubate with sperm in the presence of either 100 μM OA or 75 μM PA subsequently. It was observed that etomoxir significantly decreased the sperm CPT1 activity, mitochondrial activity, and ATP level (Figure 10A–C). Moreover, the addition of etomoxir also decreased the sperm progressive motility and VSL (Figure 10D,E).
In this study, we found that CPT1 and ACADVL, which are involved in mitochondrial β-oxidation, were expressed and localized at the midpiece of boar sperm. During the liquid storage, the addition of oleic acid and palmitic acid significantly increased sperm motility parameters, membrane integrity, and acrosome integrity, while decreasing apoptosis. Moreover, the activity of CPT1 and ACADVL, ATP level, mitochondrial activity, the activity of the TCA cycle enzymes (SDH and MDH), as well as the sperm motility patterns were significantly increased by adding oleic acid and palmitic acid during the 3 h of incubation. Interestingly, the addition of the mitochondrial β-oxidation inhibitor (etomoxir) counteracted the beneficial roles. Based on those data, it was likely that boar sperm might utilize fatty acid for maintaining the movement, and the addition of oleic acid and palmitic acid might improve sperm quality via the mitochondrial β-oxidation metabolic pathway to provide ATP (Figure 11).
The mission for sperm is the delivery of the male genetic information to the offspring [[
Interestingly, in the present study, exposure of boar sperm to 100 μM oleic acid or 75 μM palmitic acid resulted in an increase of motility parameters, membrane integrity, and acrosome integrity and a reduction of apoptosis. Those data suggested that boar sperm might consume fatty acids, which was in accordance with human sperm using endogenous components to keep alive for several days in culture medium without any exogenous substrates [[
Fatty acids are metabolized by mitochondrial β-oxidation [[
The artificial insemination (AI) technique is widely applied in the pig industry. However, the technique is still not efficient due to the large sperm dose required to obtain high reproductive performance [[
Take together, the mitochondrial β-oxidation metabolic pathway works in boar sperm. Sperm could utilization of oleic acid and palmitic acid as the energy substrates for ATP production. Addition of fatty acids to the extender would increase boar sperm quality during the liquid storage.
Graph: Figure 1 The levels of OA (A) and PA (B) in sperm were decreased during the liquid storage at 17 °C. Values are specified as the mean ± standard deviation (SD). Columns with different lowercase letters differ significantly (p < 0.05). OA: oleic acid. PA: palmitic acid.
Graph: Figure 2 (A–D) Dynamic changes in the sperm parameters by the addition of OA and PA to the diluted medium after seven days of preservation: (A,D) progressive motility, (B,E) VSL, and (C,F) total motility. Values are specified as the mean ± standard deviation (SD). Columns with different lowercase letters differ significantly (p < 0.05). VSL: straight-line velocity.
Graph: Figure 3 Effects of OA and PA on sperm membrane integrity and acrosome integrity after seven days of preservation: (A,B) membrane integrity; (C,D) acrosome integrity. Values are specified as the mean ± standard deviation (SD). Columns with different lowercase letters differ significantly (p < 0.05).
Graph: Figure 4 (A) Photomicrographs of the boar sperm stained with the Annexin V-FITC/PI assay kit: live sperm (AN-/PI-; blue arrow); early apoptotic sperm (AN+/PI-; white arrow); late apoptotic sperm (AN+/PI+; yellow arrow); and non-viable necrotic sperm (AN-/PI+; black arrow). Effects of different concentrations of OA (B) and PA (C) on boar sperm apoptosis. The Western blotting image shows the expression of apoptosis proteins in boar sperm after being treated with OA and PA (D). (E) Quantitative expression of the Parp-1, cleaved caspase-3, cleaved caspase-9, and p53 over α-tubulin generated from Western blotting (D). Data are the mean ± standard deviation (SD). Columns with different lowercase letters differ significantly (p < 0.05). Bars = 30 μm.
Graph: Figure 5 Detection and immunofluorescent localization of CPT1 (A) and ACADVL (B) in boar sperm. Western blotting analysis of CPT1 (C) and ACADVL (D) in fresh boar sperm. Bars = 30 μm. CPT1: carnitine palmitoyltransferase 1. ACADVL: long-chain acyl-coenzyme A dehydrogenase. S1-S5: five different boar sperm samples.
Graph: Figure 6 (A–D) Dynamic changes of CPT1 activity, ACADVL activity, and mitochondrial activity by the addition of OA and PA to the diluted medium at the 3h point of incubation: (A,B) CPT1 activity, (C,D) ACADVL activity, and (E,F) mitochondrial activity. Data are the mean ± standard deviation (SD). Columns with different lowercase letters differ significantly (p < 0.05).
Graph: Figure 7 Addition of OA and PA increased sperm MDH activity (A), SDH activity (B), and ATP level (C) after 3h of incubation. Data are the mean ± standard deviation (SD). Columns with different lowercase letters differ significantly (p < 0.05). MDH: malate dehydrogenase. SDH: succinate dehydrogenase.
Graph: Figure 8 (A–C) Computer-assisted sperm motility analysis (CASA) derived changes in sperm motility track by the addition of OA and PA to the diluted medium. (D–F) Dynamic changes in the sperm parameters: (D) progressive motility, (E) VSL, and (F) total motility. Data are the mean ± standard deviation (SD). Columns with different lowercase letters differ significantly (p < 0.05).
Graph: Figure 9 (A–C) Effects of different concentrations of etomoxir, an inhibitor of β-oxidation, on sperm membrane integrity (A), progressive motility (B), VSL (C), total motility (D), and CPT1 activity (E). Data are the mean ± standard deviation (SD). Columns with different lowercase letters differ significantly (p < 0.05).
Graph: Figure 10 Sperm CPT1 activity (A), mitochondrial activity (B), ATP level (C), progressive motility (D), and VSL (E) were decreased by the addition of etomoxir to the medium in the presence of OA or PA. Data are the mean ± standard deviation (SD). Columns with different lowercase letters differ significantly (p < 0.05).
Graph: Figure 11 Mechanisms of β-oxidation involved in regulating boar sperm motility. Boar sperm could utilize the fatty acids to generate ATP via β-oxidation, thus maintaining sperm motility. MDH: malate dehydrogenase. SDH: succinate dehydrogenase. ATP: adenosine triphosphate.
W.Z., Z.Z., and T.Z. conceived and designed the experiments; Z.Z. and R.L. (Rongnan Li) performed the experiments; Z.Z., R.L. (Ruifang Liu), and H.L. analyzed the data; C.F., Y.Z., D.W., and S.A.M.H. contributed reagents/materials/analysis tools; Z.Z. and W.Z. wrote the manuscript. All authors have read and agreed to the published version of the manuscript.
This study was supported in part by the National Key R&D Program of China (No. 2018YFD0501000) to W. Zeng.
The authors declare no conflict of interest.
By Zhendong Zhu; Rongnan Li; Chengwen Feng; Ruifang Liu; Yi Zheng; S. A. Masudul Hoque; De Wu; Hongzhao Lu; Tao Zhang and Wenxian Zeng
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