Journal Pre-proof

Alpha-lipoic acid improves cryopreservation of rooster semen by

reducing oxidative stress

Xiaoxin Chen , Jianqiang Liu , Yi Liu , Xu Li , Dingjie An ,

Xiaohui Liu , Lichun Zhang

PII: S0032-5791(24)00211-6
DOI: https://doi.org/10.1016/j.psj.2024.103632
Reference: PSJ 103632

To appear in: Poultry Science

Received date: 12 January 2024

Accepted date: 4 March 2024

Please cite this article as: Xiaoxin Chen , Jianqiang Liu , Yi Liu , Xu Li , Dingjie An , Xiaohui Liu ,
Lichun Zhang , Alpha-lipoic acid improves cryopreservation of rooster semen by reducing oxidative
stress, Poultry Science (2024), doi: https://doi.org/10.1016/j.psj.2024.103632

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 Running Title: ALA IMPROVES ROOSTER SEMEN CRYOPRESERVATION

Alpha-lipoic acid improves cryopreservation of rooster semen by reducing

oxidative stress

Xiaoxin Chen, Jianqiang Liu, Yi Liu, Xu Li, Dingjie An, Xiaohui Liu, Lichun Zhang*

Institute of Animal Sciences, Jilin Academy of Agricultural Sciences, Changchun,

Jilin, 130033, China

*
Corresponding author: Lichun Zhang

Institute of Animal Sciences, Jilin Academy of Agricultural Sciences,

No. 1363 Sheng-Tai Street, Changchun, Jilin 130033, China

Tel:13214442365

Fax: NA

Email: zhang_lich@163.com

Scientific section: Physiology and Reproduction

1
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

2
 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 ~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 (DSBs) and

3
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 five 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. ALA has strong antioxidant

activity and scavenges free radicals directly by reducing form (Fasipe et al., 2022).

ALA 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.

ALA 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 (ALANs) 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%

4
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,

USA).

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 five 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).

5
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-1 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

6
powder, and equilibrated at 4 °C for 1 h. The tubes were lain flat on a pre-cooled

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 days 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 two frozen fine tubes in a 37-°C water bath to thaw for 30

s and analyzed five randomly selected fields in each sample (4 μL) at least five 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 four 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.

7
 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

8
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-1 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,

9
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 (Figure 1A–D). 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.

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

10
(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.

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.

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

11
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.

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

12
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 (Mervaala et al., 2003; Midaoui et al., 2003; Piotrowski et al.,

2001), and participates in mitochondrial dehydrogenase reactions (Arivazhagan et al.,

2001). Sperm mitochondria regulate ATP production and consumption but are also

important sources of ROS. ALA 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

13
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. ALA

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.

ACKNOWLEDGEMENTS

This work was supported by the Jilin Academy of Agricultural Sciences [grant

number E22224003]. 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.

COMPETING INTERESTS

The authors declare no competing interests.

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TABLES

Table 1. Kinematic parameters for CASA of sperm motility after ALA addition.

Alpha-lipoic acid (μg/mL) 0 2 4 8 16

72.16 ± 70.98 ± 72.86 ± 84.13 ± 82.48 ±

LIN (%)
3.49 2.22 6.90 1.26 2.73

30.64 ± 38.44 ± 34.66 ± 34.24 ± 35.42 ±

BCF (Hz)
4.12 1.51 3.44 3.31 1.42

10.38 ± 10.76 ± 10.65 ± 10.66 ± 10.65 ±

ALH (μm/s)
0.40 0.34 0.53 0.30 0.48

87.99 ± 86.55 ± 89.34 ± 96.27 ± 94.09 ±

STR (%)
2.14bc 0.74c 3.09bc 1.19a 2.10ab

78.66 ± 76.67 ± 82.82 ± 89.19 ± 85.57 ±

VCL (μm/s)
1.86bc 2.62c 0.73abc 2.56a 3.07ab

57.31 ± 59.25 ± 66.46 ± 75.19 ± 63.02 ±

VAP (μm/s)
1.81c 1.98bc 2.43b 4.08a 0.91bc

48.64 ± 50.75 ± 50.20 ± 58.36 ± 53.88 ±

VSL (μm/s)
1.44b 1.03b 2.57b 2.72a 0.85ab

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).

19
FIGURE CAPTIONS

Figure 1. 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.

20
Figure 2. 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.

Figure 3. 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.

Figure 4. Effect of ALA concentration on thawed rooster sperm motility, membrane

21
integrity, and acrosome integrity.

Figure 5. Effect of ALA concentration on MDA content and mitochondrial activity of

rooster semen post-thawing.

Declaration of Interests

☒ The authors declare that they have no known competing financial interests or personal
relationships that could have appeared to influence the work reported in this paper.

☐The authors declare the following financial interests/personal relationships which may be
considered as potential competing interests:

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