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. 2024 Mar 7;103(5):103632. doi: 10.1016/j.psj.2024.103632

Alpha-lipoic acid improves cryopreservation of rooster semen by reducing oxidative stress

Xiaoxin Chen 1, Jianqiang Liu 1, Yi Liu 1, Xu Li 1, Dingjie An 1, Xiaohui Liu 1, Lichun Zhang 1,1
PMCID: PMC10978532  PMID: 38518670

Abstract

Inhibiting oxidative stress is key for ensuring sperm motility during semen cryopreservation. The aim of this study was to investigate the effect of adding alpha-lipoic acid (ALA) as an extender in rooster semen cryopreservation. Different concentrations of ALA were added to the frozen diluent of rooster semen; subsequently, computer-aided semen analysis was used to determine membrane functional integrity, acrosome integrity, antioxidant capacity (based on T-AOC, GSH-Px, SOD, CAT, and MDA contents), and mitochondrial integrity. The frozen sperm ultrastructure was observed using transmission electron microscopy. The results showed that the addition of different concentrations of ALA partially to greatly improved the quality of frozen sperm; in particular, 8 μg/mL ALA significantly improved multiple parameters of sperm quality, including sperm motility and antioxidant enzyme activity, after freeze–thaw. The results of this study provide empirical and theoretical support for effective rooster semen cryopreservation and can inform the development of new protective agents in the field of livestock reproduction.

Key words: alpha-lipoic acid, antioxidant capacity, semen freezing, rooster sperm ultramicrostructure

INTRODUCTION

Semen cryopreservation represents a major breakthrough in artificial insemination technology. In particular, it accelerates the breeding and improvement of high-quality breeding males and facilitates the introduction of livestock and exchange of high-quality semen between regions, which is crucial in the development of contemporary animal husbandry (Sasaki et al., 2010; Abouelezz et al., 2015; Rakha et al., 2017; Gangwar et al., 2020). Frozen semen technology for cattle has developed rapidly in recent years, with a practically complete and standardized workflow (Long et al., 2014; Moghbeli et al., 2016; Partyka et al., 2017). In the process of poultry semen cryopreservation, researchers improved the freezing efficiency by adjusting the cooling rate and by adding antifreeze and antioxidants, but there is still room for improvement. Olexikova et al. (2019) compared fresh and frozen semen by transmission electron microscopy (TEM) and found that only approx. 25% of high-quality sperm remained active after freeze–thaw. Cryopreservation can damage the sperm plasma membrane and acrosome, resulting in decreased fertilization ability. Therefore, the application of appropriate cryoprotectants and thawing methods during freezing or low-temperature preservation can improve the outcomes of assisted reproductive technology.

Rooster sperm plasma membranes contain various polyunsaturated fatty acids, which interfere with mitochondrial function and cause excessive reactive oxygen species (ROS) production during sperm cryopreservation (Gürler et al., 2016). Excessive ROS production can further lead to DNA double-strand breaks (DSB) and cell apoptosis (Tao et al., 2019). Sperm contains antioxidant enzymes (Zanganeh et al., 2013; Najafi et al., 2014; Sharafi et al., 2015); however, their activity gradually decreases with semen thawing (Fattah et al., 2017). Auxiliary protective substances can greatly improve chicken sperm functional integrity (Awda et al., 2009).

Alpha-lipoic acid (ALA) is a common coenzyme factor in mitochondria, containing a disulfide 5 ring structure with high electron density and affinity. It catalyzes various mitochondrial multienzyme complexes, such as branched chain ketone dehydrogenase and pyruvate dehydrogenase, and plays a crucial role in stabilizing and regulating these multienzyme complexes. Alpha-lipoic acid has strong antioxidant activity and scavenges free radicals directly by reducing form (Fasipe et al., 2023). Alpha-lipoic acid exerts various beneficial clinical effects (Carpenter and Hovda, 2022; Laganà et al., 2022). In summary, ALA is a key factor in ensuring cell growth and mitochondrial function integrity.

Alpha-lipoic acid further exhibits lipophilic and water-soluble properties that have contributed greatly to its applications in terms of semen preservation. Onder et al. (2022) reported that ALA improved ram sperm motility and DNA integrity after cryothawing via oxidative stress inhibition. Furthermore, the addition of various concentrations of ALA nanoliposomes (ALAN) enhanced buffalo spermatozoa freezability and acrosome membrane integrity, decreased lipid peroxidation contents during cryopreservation, and maintained sperm motility (Hassan et al., 2022). One such ALAN, ALAN150, exhibited remarkable sperm motility and membrane integrity preservation following either 30-s thawing at 37°C or 2-h incubation at 37°C/5% CO2. A large number of studies have shown that ALA has strong antioxidant ability, but it has not been used in the cryopreservation of rooster semen.

Based on these previous findings, we investigated the possible role of ALA in the oxidative stability of semen during cryopreservation, the effects of ALA on semen quality, and the potential of ALA in the cryopreservation of rooster semen.

MATERIALS AND METHODS

Chemicals

All reagents and materials were obtained from Sigma-Aldrich (St. Louis, MO).

Experimental Design

We used egg yolk extender with varying levels of ALA (0, 2, 4, 8, and 16 μg/mL). Each experiment was repeated at least 5 times.

Animals

We collected semen from Jilin Luhua rooster (n = 28) who were provided with feed ad libitum with no added antioxidants. The study was conducted at the Animal Husbandry Branch of Jilin Academy of Agricultural Sciences in the People's Republic of China. It was approved by the Animal Ethics Committee of the Institute of Animal Husbandry and Veterinary Medicine, Jilin Academy of Agricultural Sciences (approval no. JNK20211108).

Rooster Semen Collection

We used a back–abdominal massage method to collect sperm from each rooster every other day. The sperm had a fresh essence vitality of ≥ 80%, density of ≥ 28 × 108/mL, and deformity rate of ≤ 5%.

Basic Extender

We used Lake's as the base fluid (1.920 g sodium glutamate, 1.000 g fructose, 0.815 g sodium acetate, 0.128 g potassium citrate, 0.068 g magnesium chloride and added 100,000 IU penicillin–streptomycin. The chemicals were added to a volumetric flask, followed by the addition of 100 mL of double-distilled water to achieve a stable volume. We maintained the osmotic pressure at 333 mOsm/kg under continuous magnetic stirring for 30 min, added 15% egg yolk and 10% glycerol, stirred for another 30 min to completely disperse the solution, and finally added different concentrations of ALA. We first prepared a 1,000-fold concentrated storage solution by dissolving 2 mg of ALA in 1 mL of DMSO and then added an appropriate amount of this solution to achieve different final concentrations. For example, for a final concentration of 2 μg/mL, we added 25 μL of the concentrated ALA storage solution to 25 mL of freezer solution.

Semen Processing

Rooster semen samples were divided into 0.25-mL fine tubes, sealed with sealing powder, and equilibrated at 4°C for 1 h. The tubes were lain flat on a precooled fumigation rack 3 cm above the surface of liquid nitrogen vapor for 10 min and quickly treated with liquid ammonia. The tubes were stored in liquid nitrogen for at least 30 d before thawing for evaluation. To thaw semen, it was placed in a 37°C water bath and remove after 30 s.

Post-Thawed Sperm Analysis

Computer Assisted Semen Analysis. We analyzed sperm motility parameters using CASA. We immersed 2 frozen fine tubes in a 37°C water bath to thaw for 30 s and analyzed 5 randomly selected fields in each sample (4 μL) at least 5 times. The amplitude of lateral head displacement (ALH), beat-cross frequency (BCF), linearity (LIN), average path velocity (VAP), straight-line velocity (VSL), curvilinear velocity (VCL), and straightness (STR) were recorded for each group.

Acrosome Integrity. Acrosome integrity rates were assessed by Giemsa staining. After thawing the semen, we air dried a semen smear, fixed it in formaldehyde for 20 min, and stained it with Giemsa for 10 h. The smear was rinsed and air dried before examining 4 randomly selected fields under an optical microscope (Nikon ECLIPSE Tis, Japan); no less than 500 sperm were counted in each field. We finally calculated the ratio of the number of intact acrosome spermatozoa to the total number of spermatozoa to represent the acrosome integrity rate.

Plasma Membrane Integrity. Sperm plasma membrane integrity was measured using the hypoosmotic swelling test (HOST). The structure of the hypoosmotic solution was as follows: 7.85 g of sodium citrate, 1.4 g of fructose, and 100 mL of ultra-pure water, all combined into a 37°C incubator for preheating. First, the HOST solution was preheated ahead of time at 37°C, 50 µL of semen was treated with 500 µL of HOST solution, and the mixture was left at 37°C for 10 min. Four randomly selected fields were examined under an optical microscope and no less than 500 sperms were counted. Finally, the ratio of the sperm count to the total sperm count was calculated to represent sperm plasma membrane integrity.

Determination of Intracellular Antioxidant Enzyme Content in Thawed Sperm. We used a Beijing Solarbio Technology enzyme-linked immunosorbent assay (ELISA) kit (Regen Biology, Anhui, China) to determine the contents of the endogenous antioxidant enzymes T-AOC, SOD, CAT, and GSH-Px. We collected semen samples into centrifuge tubes, added 1 mL of extraction solution for every 5 million cells, and performed ultrasonic vibration. Subsequently, 8,000 g were centrifuged at 4°C for 10 min, and the supernatant was taken and placed on ice until testing. When testing, we mixed the supernatant with the working diluent; then, after incubation, the enzyme-labeling reagent and stop solution were added in sequence. The resulting mixture was placed into a microplate reader for analysis.

MDA Content Determination. The MDA concentration was determined using an ELISA kit (Beijing Solarbio Technology, Beijing, China). Under acidic and high-temperature conditions, MDA reacts with thiobarbituric acid to produce a brownish-red product. We performed colorimetry at a wavelength of 532 nm to evaluate the content of lipid peroxides in sperm cells. We collected semen samples into centrifuge tubes, added 1 mL of extraction solution for every 5 million cells, and performed ultrasonic vibration. Subsequently, 8,000 g was centrifuged at 4°C for 10 min, and the supernatant was taken and placed on ice until testing. In the experiment, the supernatant was mixed with the working diluent, kept warm in a 100°C water bath for 60 min (the cover was tightly wrapped to prevent it from bursting), and then cooled in an ice bath. Finally, 10,000 g was centrifuged for 10 min at room temperature, and 200 µL of supernatant was analyzed using the microplate reader.

Mitochondrial Activity Assay. Mitochondrial activity was analyzed using Rhodamine 123 (Rh123) staining. We added dye at a final concentration of 100 nmol/L to 20 µL of semen sample, wrapped the sample in tin foil, and let it sit at 37°C for 30 min. Under non-pathological conditions, the sperm tail exhibits green fluorescence, representing mitochondrial activity. Using a fluorescence phase contrast microscope, mitochondrial activity was calculated as the ratio of sperm with green fluorescence to the total number of sperms.

Preparation of TEM Samples. After fixing the thawed semen in 1% osmic acid•0.1 M phosphate buffer and dehydration with different concentrations of alcohol, rinsing, embedding, slicing, and staining, the semen samples were observed using TEM and imaged for analysis.

Statistical Analysis

One-way analysis of variance (ANOVA) was performed to assess differences between groups using GraphPad Prism 9 (GraphPad Software, La Jolla, CA) and SPSS Statistics 26 (IBM SPSS, Armonk, NY). Data are expressed as the mean ± standard error of the mean (SEM), with statistical significance set at p < 0.05.

RESULTS

Ultrastructural Changes in Rooster Sperm After Freezing

At different stages of freeze–thaw, nuclei varied slightly in particle size and density (Figures 1A–1D). The outer membrane of the sperm acrosome was clearly vesicular after freezing, with some sperm acrosomes lost (Figure 2). The plasma membrane outside the sperm head appeared relatively blurry and damaged. The mitochondrial sheaths outside the "9+2″ microtubule structure (Figure 2B) appeared fuzzy and elliptical, unevenly distributed, and some mitochondria were swollen.

Figure 1.

Figure 1

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Longitudinal profiles of late-stage sperm. (A), (B), (C), (D). N, nucleus; C and C', proximal and distal centrioles, respectively; V, vesicles; T, tail; and Mt, mitochondrial sheath. An outline of the proximal centriole (C) can be seen in the neck region as a dense ring with a clear center.

Figure 2.

Figure 2

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Cross section of sperm. (A) Longitudinally cut sperm head. N, nucleus; Mt, mitochondria; Ac, acrosome; T, tail. (B) Transversely cut sperm tail showing the mitochondrial sheath and “9+2″ microtubules.

Effects of ALA on Antioxidant Parameters of Rooster Semen Post Thawing

Table 1 shows that the addition of 2, 4, 8, and 16 μg/mL ALA improved the kinematic parameters of frozen semen. The beneficial effect of 8 μg/mL ALA on the STR, VCL, VAP, and VSL kinematic parameters exceeded that at other concentrations (p < 0.05).

Table 1.

Kinematic parameters for CASA of sperm motility after ALA addition.

Alpha-lipoic acid (μg/mL) 0 2 4 8 16
LIN (%) 72.16 ± 3.49 70.98 ± 2.22 72.86 ± 6.90 84.13 ± 1.26 82.48 ± 2.73
BCF (Hz) 30.64 ± 4.12 38.44 ± 1.51 34.66 ± 3.44 34.24 ± 3.31 35.42 ± 1.42
ALH (μm/s) 10.38 ± 0.40 10.76 ± 0.34 10.65 ± 0.53 10.66 ± 0.30 10.65 ± 0.48
STR (%) 87.99 ± 2.14bc 86.55 ± 0.74c 89.34 ± 3.09bc 96.27 ± 1.19a 94.09 ± 2.10ab
VCL (μm/s) 78.66 ± 1.86bc 76.67 ± 2.62c 82.82 ± 0.73abc 89.19 ± 2.56a 85.57 ± 3.07ab
VAP (μm/s) 57.31 ± 1.81c 59.25 ± 1.98bc 66.46 ± 2.43b 75.19 ± 4.08a 63.02 ± 0.91bc
VSL (μm/s) 48.64 ± 1.44b 50.75 ± 1.03b 50.20 ± 2.57b 58.36 ± 2.72a 53.88 ± 0.85ab
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a,b,c

in the same column and row indicate a significant difference (p < 0.05); identical lowercase letters indicate no difference (p > 0.05).

Effects of ALA on Antioxidant Capacity of Rooster Sperm

T-AOC, SOD, GSH-Px, and CAT estimates after semen cryopreservation in 0, 2, 4, 8, and 16 μg/mL ALA are shown in Figure 3. As the concentration of ALA increased, the antioxidant enzyme contents gradually increased. The T-AOC, GSH-PX, and SOD contents were highest in the ALA 8 μg/mL group (p < 0.05). The activities of GSH-Px and CAT decreased in the ALA 16 μg/mL group.

Figure 3.

Figure 3

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Effect of ALA concentration on the antioxidant capacity of rooster sperm. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 compared with the control group.

Effects of ALA on Sperm Plasma Membrane and Acrosome Integrity and Vitality Post Thawing

The outcomes of semen cryopreservation with varying concentrations of ALA (0, 2, 4, 8, and 16 μg/mL) on the plasma membrane and acrosome integrity, and sperm vitality are shown in Figure 4. The results showed that the sperm motility of the 2, 4, 8, and 16 μg/mL groups increased slowly. The membrane and acrosome integrity of the 2, 4, and 8 μg/mL groups were higher than that of the control, and the effect of 8 μg/mL was the best; the 16 μg/mL group showed significantly lower values than the other groups.

Figure 4.

Figure 4

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Effect of ALA concentration on thawed rooster sperm motility, membrane integrity, and acrosome integrity.

Impact of ALA on MDA Content and Mitochondrial Activity Post Thawing

As the concentration of ALA increased, the intracellular MDA content decreased (Figure 5). The MDA contents were lowest with 8 μg/mL ALA (p < 0.05), at which mitochondrial activity was also slightly higher than that in the other groups. The MDA content increased and mitochondrial activity decreased in the 16 μg/mL group, indicating that excessive addition of ALA not only failed to protect sperm, but dysregulated the antioxidant environment and sperm structure.

Figure 5.

Figure 5

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Effect of ALA concentration on MDA content and mitochondrial activity of rooster semen post-thawing.

DISCUSSION

The results of this study show that adding ALA to semen cryopreservation solution can improve sperm quality after freeze–thaw by enhancing antioxidant capacity and reducing lipid peroxidation.

With the popularization of artificial insemination and increasing emphasis on germplasm resource conservation, the development of frozen semen technologies has gradually progressed. However, the large amount of easily oxidizable polyunsaturated fatty acids in the plasma membrane of rooster sperm cells poses a challenge to effective cryopreservation (Saleh and Agarwal, 2002). The proportion of straight-line moving sperm in total sperm counts directly affects the conception rate of female animals, and is an important index for routine detection of semen quality. We found that adding ALA improved the motility of freeze–thawed sperm along with the integrity rate of the sperm plasma membrane and acrosome, which may improve hatching rates in breeding eggs. This indicates that ALA acted as an effective antioxidant, reducing the rate of sperm deformity and potentially enhancing fertilization rates through improved motility.

After cryopreservation, the fluidity, permeability, and internal environment of the sperm membrane changes. The antioxidant level inside the sperm becomes dysregulated, and the content of toxic substances increases. The occurrence of oxidative stress reactions damages the sperm membrane and inevitably decreases mitochondrial membrane potential. ALA has strong detoxifying and antioxidant effects owing to a high oxygen scavenging capacity in tissues including the heart, brain, and kidneys (Piotrowski et al., 2001; Mervaala et al., 2003; Midaoui et al., 2003), and participates in mitochondrial dehydrogenase reactions (Arivazhagan et al., 2001). Sperm mitochondria regulate ATP production and consumption but are also important sources of ROS. Alpha-lipoic acid is an important coenzyme in the mitochondria of cells. It is involved in the posttranslational modification of key mitochondrial proteins, and is also a natural antioxidant molecule. Previous studies have demonstrated that ALA protects sperm motility and mitochondrial functioning (Koenig and Meyerhoff, 2003; Selvakumar et al., 2006). Its antioxidant activity reduces ROS production in sperm and protects against DNA damage (Aly et al., 2009; Taherian et al., 2019). Similarly, our experiments showed that increasing concentrations of ALA promoted the total antioxidant capacity (based on CAT, GSH-Px, and SOD enzyme activity) of freeze–thawed sperm. Total antioxidant capacity increased significantly with treatments of 4, 8, and 16 μg/mL ALA compared with the control treatment. The SOD enzyme activity of the 4- and 8-μg/mL groups was significantly higher than that in the control group, and GSH-Px activity was higher in the 8-μg/mL group than that in the control group.

Collectively, our findings suggest that ALA-treated cryodiluent improves the motility of freeze–thawed sperm; integrity of membranes, acrosomes, and organelles; and ability to resist oxidative stress. In particular, the addition of 8 μg/mL ALA mitigated the detrimental impact of lipid peroxidation on rooster semen quality. Alpha-lipoic acid effectively enhanced the antioxidant capacity of rooster sperm and thus represents a promising antioxidative agent for cryoprotectants in the field of reproduction. To maximize the benefits in production, the addition of ALA should be considered when preparing cryoprotectants to improve the freezing efficiency of rooster semen.

ACKNOWLEDGMENTS

This work was supported by the Key R&D projects in Jilin Province [grant number 20210202028NC]. The funder did not play any role in the study design; in the collection, analysis, and interpretation of data; in the writing of the report; or in the decision to submit the article for publication.

DISCLOSURES

The authors declare no competing interests.

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