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. 2026 Aug 5;12(5):e71139. doi: 10.1002/vms3.71139

Mito‐TEMPO Improves Post‐Thaw Quality and Fertilizing Capacity of Rooster Semen Cryopreserved in Beltsville Extender

Mahdi Mokhtari 1, Mahdi Khodaei‐Motlagh 1,✉, Reza Masoudi 2,✉, Mohsen Sharafi 3
PMCID: PMC13440482  PMID: 42555276

ABSTRACT

Background

Rooster semen cryopreservation is a reproductive technology carried out to boost genetic gain and productivity in chicken's commercial flocks. However, semen cryopreservation significantly decreases the quality and fertilizing potential of sperm cells.

Objectives

This study evaluated cryoprotective effects of the mitochondria‐targeted antioxidant Mito‐TEMPO added to the freezing medium by assessing post‐thaw characteristics of rooster sperm.

Methods

Semen samples were diluted in the Beltsville extender supplemented with 0, 1, 10, 100 or 1000 µM of Mito‐TEMPO. Following the thawing of frozen semen doses, we examined the following sperm parameters: motility, membrane integrity, morphology, mitochondrial activity, acrosome integrity, viability, apoptosis status, lipid peroxidation, DNA fragmentation, reactive oxygen species (ROS) accumulation, and fertilizing ability.

Results

Semen diluted in extenders containing 10 or 100 µM of Mito‐TEMPO significantly exceeded all other groups of frozen‐thawed semen samples in sperm motility, average path velocity as well as membrane/acrosome integrity and mitochondrial activity. Moreover, the addition of 10 and 100 µM of Mito‐TEMPO significantly decreased lipid peroxidation, apoptosis rate, DNA fragmentation, and ROS concentrations compared with all cryopreserved groups and was associated with a higher (p ≤ 0.05) fertilization rate compared with a non‐supplemented control group, albeit it was still significantly lower compared with that obtained using fresh semen.

Conclusions

It could be concluded that Mito‐TEMPO improved an array of quality parameters and fertilizing potential of rooster semen and hence can be recommended for use as a diluent additive in semen cryopreservation procedures employed in commercial poultry operations.

Keywords: Beltsville, freezing, Mito‐TEMPO, rooster, sperm

This study reported the effect of Mito‐TEMPO on rooster semen quality cryopreserved in Beltsville extender. Semen diluted in extenders containing 10 or 100 µM of Mito‐TEMPO presented higher motility, average path velocity as well as membrane/acrosome integrity, mitochondrial activity, and fertility rate; and lower lipid peroxidation, apoptosis rate, DNA fragmentation, and reactive oxygen species (ROS) concentrations compared with all cryopreserved groups.

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1. Introduction

Sperm cryopreservation represents an essential approach for the long‐term preservation of genetic resources in avian species. However, the process is challenged by the high susceptibility of avian spermatozoa to cryoinjury, primarily due to thermal shock during freezing. The plasma membrane of rooster sperm is enriched with polyunsaturated fatty acids, which are prone to lipid peroxidation triggered by reactive oxygen species (ROS) generated during the freeze–thaw process (Aitken et al. 2014). Consequently, rooster spermatozoa exhibit marked sensitivity to oxidative stress, resulting in impaired motility, DNA fragmentation, and diminished fertility following exposure to subzero temperatures (Cerolini et al. 2005).

Although spermatozoa possess endogenous antioxidant defences, such as glutathione peroxidase, superoxide dismutase, and catalase, these intrinsic systems are insufficient to counteract the excessive ROS generated during cryopreservation (Cerolini et al. 2005). Accordingly, the supplementation of semen extenders with exogenous antioxidants has been proposed as an effective strategy to mitigate cryodamage and improve post‐thaw sperm quality (Shahverdi et al. 2015). Nevertheless, accumulating evidence suggests that the efficacy of many antioxidants is limited because the efficient neutralization of ROS requires sufficient concentrations within mitochondria. The impermeability of the inner mitochondrial membrane to most ions and small molecules restricts the mitochondrial accumulation of many conventional antioxidants, thereby limiting their effectiveness (Esmaeilkhanian et al. 2023).

Mito‐TEMPO, a mitochondria‐targeted antioxidant, has been developed to overcome this limitation. It consists of the superoxide dismutase mimetic piperidine nitroxide (Tempo) conjugated to the lipophilic cation triphenylphosphonium (TPP+). Tempo mimics superoxide dismutase activity in the catalytic dismutation of superoxide radicals, while TPP+ facilitates selective mitochondrial uptake due to its membrane permeability and accumulation driven by the organelle's negative membrane potential (Trnka et al. 2008). This unique structure allows Mito‐TEMPO to act as a potent scavenger of mitochondrial ROS. Supplementation of human semen freezing extenders with Mito‐TEMPO has been reported to enhance post‐thaw sperm quality (Lu et al. 2018).

Semen extenders maintain the physicochemical stability of spermatozoa by providing isosmotic conditions and near‐neutral pH. Among these, the Beltsville extender is one of the most widely used media for rooster sperm cryopreservation (Shahverdi et al. 2015). The physicochemical properties of Beltsville are unique to poultry and differ substantially from extenders used in mammals. The interaction of Mito‐TEMPO with this specific extender composition has never been investigated. Thus, incorporating mitochondria‐targeted antioxidants such as Mito‐TEMPO into the Beltsville extender may represent a promising approach to improve rooster semen cryopreservation outcomes. The present study was designed to evaluate the effects of Mito‐TEMPO supplementation in the Beltsville extender on functional parameters of rooster sperm during cryopreservation and subsequent fertility following insemination with frozen‐thawed semen.

2. Materials and Methods

2.1. Chemicals

All chemicals used in this study were purchased from Sigma Aldrich (St. Louis, MO, USA) or Merck (Darmstadt, Germany).

2.2. Rooster Management and Semen Sample Collections

Ten Ross 308 roosters aged 32 weeks were used in this experiment. The roosters were kept individually in cages (70 × 60 × 75 cm) at a temperature of 20–23°C, under a photoperiod of 15 h of light:9 h of darkness. Roosters were fed the same diet according to Ross 308 catalogue, and water was freely available. Semen was collected from each rooster biweekly using the abdominal massage method (Safari Asl et al. 2018). Immediately after collection, semen samples were placed in a water bath at 37°C and sent to the laboratory for initial sperm quality assessment. The following parameters were recorded: individual sample volumes, sperm concentration, total and progressive sperm motility, and morphology. The samples were selected for further study if their volume was ≥0.5 mL, sperm concentration exceeded 3 × 109 sperm/mL, with a minimal total and progressive motility of 75% and 60%, respectively, and sperm had at least 85% of normal morphology. The qualified samples were pooled to omit male individual differences in each sampling day, and each replicate refers to collection days.

2.3. Cryopreservation

The Beltsville extender contains potassium hydrogen phosphate trihydrate (K2HPO4•3H2O, 12.7 g/L), sodium glutamate (C5H8NNaO4, 8.61 g/L), fructose (C6H12O6, 5 g/L), sodium acetate trihydrate (C2H9NaO5, 4.3 g/L), n‐Tris (hydroxymethyl) methyl‐2‐aminoethanesulfonic acid (C6H15NO6S, 1.9 g/L), potassium citrate (K3C6H5O7, 0.64 g/L), monopotassium phosphate (KH2PO4, 0.6 g/L), magnesium chloride hexahydrate (MgCl2•6H2O, 0.34 g/L), and glycerol (2 % v/v) (Shahverdi et al. 2015). The extender's pH and osmolarity were 7.1 and 310 mOsm/kg, respectively. Semen samples were diluted (1:10) with the Beltsville extender containing 0, 1, 10, 100, or 1000 µM of Mito‐TEMPO. French straws (0.25 mL, IMV, L'Aigle, France) were loaded with diluted semen (100×106 cells/straw), sealed with polyvinyl alcohol powder, and equilibrated at 5°C for 3 h. After equilibration, the straws were placed in a cryo‐box approximately 5 cm above the surface of liquid nitrogen for 12 min and were then plunged into liquid nitrogen and stored. For evaluation of sperm quality, one month after freezing, the frozen semen samples were thawed for 30 s in a water bath (37°C). This experiment was conducted in six replicates. After thawing and before any evaluation, glycerol was removed using a discontinuous Accudenz, which was equipped with a 12% (5.0 mL) layer and a 30% (0.5 mL) layer. Centrifugation of semen (1200 g; 20 min) maintained the glycerol and extender above the 12 % layer, whereas the purified sperm was deposited between the 12% and 30% layers. After Accudenz, up to 500 µL, extender was added to the semen layer to readjust the semen's original volume and resuspend before recovering the sperm.

2.4. Gross Assessment of Post‐Thawed Semen Samples

2.4.1. Motion Characteristics

Sperm class analysis software (version 5.1; Microptic, Barcelona, Spain) was used to evaluate the sperm motion characteristics (Masoudi et al. 2022). In this evaluation, semen samples were initially diluted with phosphate‐buffered saline (PBS) buffer (1:7). Subsequently, 5 µL of diluted sample was placed on the pre‐warmed chamber slide (38°C, Leja 4, 20 µm height, Leja Products, Luzernestraat B.V., Holland). Sperm in at least six fields that contained a minimum of 400 spermatozoa were evaluated for each sample, and the values for the following variables were recorded: total motility (TM, %), progressive motility (PM, %), average path velocity (VAP, m/s), straight‐line velocity (VSL, m/s), curvilinear velocity (VCL, m/s), and linearity (LIN, %).

2.4.2. Malondialdehyde Concentration

Values for malondialdehyde (MDA) concentration were considered to be an indicator of the extent of lipid peroxidation and were recorded after there was an induced reaction with thiobarbituric acid (Esterbauer and Cheeseman 1990). The MDA concentration was determined by making absorption assessments and a standard curve of MDA equivalent that was generated using the findings when there was acid catalyzed hydrolysis of 1,1,3,3‐tetramethoxypropane. Briefly, 1 mL of the diluted semen sample (400 ×106 sperm cells/mL) was mixed with 1 mL of cold 20 % (w/v) tricholoro acetic acid to precipitate protein. The precipitate was pelleted by centrifugation (960 g for 15 min), and 1 mL of the supernatant was incubated with 1 mL of 0.67% (w/v) thiobarbituric acid in a boiling water bath at 100°C for 10 min. After cooling, the absorbance was determined using a spectrophotometer (UV‐1200, Shimadzu, Japan) at 532 nm. All MDA concentrations were expressed as nmol/mL.

2.4.3. Membrane Functionality

The hypo‐osmotic swelling test (HOST) was used to determine the percentage of sperm with functional membranes (Revell and Mrode 1994). The HOST is based on the resistance of the sperm membrane to stressful conditions in a hypoosmotic medium. With this assessment, 5 µL of semen sample was mixed with 50 µL hypo‐osmotic solution (57.6 mM fructose and 19.2 mM sodium citrate, 100 mOsm/L) and incubated for 20 min. There was subsequent analysis of 300 spermatozoa using a phase‐contrast microscope (×400 magnification, CKX41, Olympus, Tokyo, Japan), and numbers of sperm in samples with swollen and non‐swollen tails were recorded as sperm cells with functional and non‐functional membranes, respectively.

2.4.4. Normal Morphology

The Hancock solution was used to determine percentages of post‐thawed sperm with normal morphology. A drop of thawed sample was placed in an Eppendorf tube containing 1 mL of Hancock solution (150 mL sodium saline solution, 500 mL double‐distilled water, 62.5 mL formalin [37%], and 150 mL buffer solution) (Schaäfer and Holzmann 2000). Subsequently, there were 300 spermatozoa evaluated using a microscope, and the percentages of sperm cells with abnormal heads and/or tails were recorded.

2.5. Flow Cytometric Study of Post‐Thawed Semen Samples

A FACSCalibur (Becton Dickinson, San Jose, CA, USA) flow cytometer was used to evaluate mitochondrial activity, viability, DNA fragmentation, and hydrogen peroxide concentration (Lee et al. 2008). A minimum of 10,000 spermatozoa were examined per assay at a flow rate of 100 cells/s. The sperm population was gated using 90° and forward‐angle light scatter to exclude debris and aggregates. The excitation wavelength was 488 nm supplied by an argon laser. Green fluorescence (Rh123 and Annexin V) was detected on FL1 detector with a 530/30 nm band‐pass filter, and red fluorescence (propidium iodide [PI]) was detected on FL2 detector with a 585/42 nm band‐pass filter. Single‐stranded DNA as sign of fragmentation emits a red fluorescence detected at a 670‐band pass filter (FL3). The FlowJo software was used to analyze the data obtained with the use of these procedures (Treestar, Inc., San Carlos, CA, USA).

2.5.1. Mitochondrial Activity

To assess the quantity of mitochondrial activity, 10 µL of Rhodamin‐123 solution (Rh123, 0.01 µg/mL; Invitrogen TM, Eugene, OR, USA) was added to 300 µL of diluted semen and incubated in a dark room for 20 min. The sperm suspension was subsequently centrifuged at 500 × g for 3 min and again resuspended in 500 µL Tris buffer. Subsequently, 10 µL of PI (1 µg/mL) was added to the sperm suspension. Numbers of sperm cells with positive signal for Rh123 and negative signal for PI (Rh123+/PI−) were recorded as spermatozoa with active mitochondria (Figure 1) (Barfourooshi et al. 2023).

FIGURE 1.

FIGURE 1

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Evaluation of mitochondrial activity via Rhodamin‐123 (Rh123) and PI. Sperm cells with positive signal for Rh123 and negative signal for PI (Rh123+/PI−) were recorded as spermatozoa with active mitochondria.

2.5.2. Viability and Apoptotic‐Like Changes

Annexin V‐FITC kit (IQP, Groningen, and the Netherlands) and PI were used to assess phosphatidyl serine externalization as an index of apoptotic‐like changes in spermatozoa (Zarei et al. 2022). The samples were washed in calcium buffer and adjusted to the concentration of 1 × 106 spermatozoa/mL followed by the addition of 10 µL Annexin V–FITC (0.01 µg/mL) to 100 µL of the sperm suspension, which were then incubated for 20 min in room temperature (22°C). There were subsequently 10 µL of PI added to the sperm suspension, and there was incubation for at least 10 min at 22°C. This suspension was subsequently evaluated using a flow cytometer, and sperm subpopulations were classified into four groups: (1) viable cells negative for both Annexin V and PI (A−/PI−), (2) primary apoptotic cells positive for Annexin V but negative for PI (A+/PI−), (3) secondary apoptotic cells positive for both Annexin V and PI (A+/P+), and (4) necrotic cells negative for Annexin V but positive for PI (A−/P+) (Figure 2).

FIGURE 2.

FIGURE 2

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Annexin V‐FITC kit and PI were used to assess phosphatidyl serine externalization as an index of apoptotic‐like changes in spermatozoa. Sperm subpopulations were classified into four groups: (Q1) live cells negative for both Annexin V and PI (A−/PI−), (Q2) cells with primary apoptotic‐like changes positive for Annexin V but negative for PI (A+/PI−), (Q3) cells with secondary apoptotic‐like changes positive for both Annexin V and PI (A+/PI+), and (Q4) necrotic cells negative for Annexin V but positive for PI (A−/PI+).

2.5.3. Assessment of DNA Fragmentation

The extent of DNA fragmentation was assessed using the sperm chromatin structure assay (SCSA) (Evenson and Wixon 2005). The samples were washed and diluted in PBS buffer. Subsequently, there was treatment of the sperm suspension containing 3 × 106 spermatozoa/mL with an acid detergent solution (0.15 mol/L NaCl, 0.1 % Triton X‐100 and 0.08 N HCl) for 40 s, and sperm cells were stained with 6 mg/L purified acridine orange (AO) in a phosphate‐citrate buffer. Percentages of spermatozoa with fragmented DNA were recorded using the DFI frequency dot plot obtained from the ratio between the red and total (red plus green) fluorescence intensity (Figure 3) (Hosseinifar et al. 2015).

FIGURE 3.

FIGURE 3

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Evaluation of sperm DNA fragmentation via sperm chromatin structure assay (SCSA) based on green (FL1) and red (FL3) fluorescence. Main population includes sperm without DNA fragmentation. The percentage of abnormal spermatozoa was calculated from the DFI frequency dot plot obtained from the ratio between the red and total (red plus green) fluorescence intensity.

2.5.4. Hydrogen Peroxide Content

Dichlorofluorescin diacetate (DCFH‐DA) was used to evaluate the intracellular hydrogen peroxide content (Sharma et al. 2017). Semen samples were rinsed with PBS to a concentration of 3–5 × 106 spermatozoa/mL. There was addition of DCFH‐DA (25 µM) to the sperm suspension and incubation for 40 min at 25°C. After washing, 2 µL of PI was added to the semen samples, and samples were analyzed using flow cytometry procedures (Figure 4).

FIGURE 4.

FIGURE 4

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Dichlorofluorescin diacetate (DCFH‐DA) assessed the intracellular H2O2 content that introduced as ROS concentration based on fluorescence of H2O2 − and PI.

2.6. Assessment of Acrosome Integrity Using Fluorescent Analysis Procedures

Pisum sativum agglutinin (PSA) was used to determine sperm acrosome integrity (AI) (Thys et al. 2009). Note that 5 µL of semen sample was added to 100 µL ethanol, and after 20 min, 10 µL of the suspended sample was mixed with 30 µL of PSA on a glass slide. There was a subsequent evaluation of 200 spermatozoa on a slide using a fluorescent microscope (BX51, Olympus) that had fluorescence illumination and a FITC filter capacity at 400× magnification. Spermatozoa with and without green heads were recorded as cells with intact acrosomes and disrupted/damaged acrosomes, respectively.

2.7. Artificial Insemination

Fertilization capacity was determined using the semen samples for artificial insemination of hens (Long and Kulkarni 2004). Ross broiler breeder hens were assigned to one of the four following groups randomly (20 hens in each group): (1) inseminated with fresh semen; or (2–4) inseminated with a semen sample containing 0, 10, and 100 µM Mito‐TEMPO, respectively. Each straw contained 100 × 106 spermatozoa, and artificial insemination was performed twice a week for 1 month by an expert technician. The eggs were collected for 5 days after the last artificial insemination. For each group, four weekly batches (96 eggs in each batch) were selected for incubation. After 7 days of incubation, there was candling of eggs to determine numbers of eggs fertilized so that there could be calculation of the percentage of eggs fertilized. The percentage of eggs from which there was hatching was determined from data collected 21 days after the initiation of the incubation period based on numbers of fertilized eggs.

2.8. Statistical Analysis

The Levene's and Kolmogorov–Smirnov and Shapiro–Wilk tests were used to examine the data for homogeneity of variances and normality of data distributions, respectively. The data were analyzed using Proc GLM procedures of the SAS 9.1 program (SAS Institute, version 9.1, 2002, Cary, NC, USA). The Tukey's test was used to determine statistical differences among the various groups. The results are presented as mean ± SE. Fertility and hatching rates were analyzed using the GENMOD procedure utilizing chi‐Square analyses. Mean differences were considered statistically significant at p ≤ 0.05.

3. Results

The effects of different concentrations of Mito‐TEMPO on the quality parameters and fertility potential of rooster sperm are summarized in Tables 1, 2, 3, 4, 5. In almost all quality parameters evaluations, the best results were related to the fresh semen group. In cryopreserved samples, total and progressive sperm motility, and the average path velocity was greater (p < 0.05) in semen samples supplemented with 10 or 100 µM of Mito‐TEMPO compared with all other groups (Table 1). For the values of the VSL, VCL, and LIN variables, there were no differences when Mito‐TEMPO was added at differing concentrations.

TABLE 1.

The effects of different Mito‐TEMPO concentrations on motility parameters (motion kinetics) in rooster semen.

Mito‐TEMPO concentration (µM)
Parameter Fresh 0 1 10 100 1000 SEM
TM (%) 91.0a 50.5c 52.1c 58.4b 60.3b 50.0c 1.2
PM (%) 72.5a 22.3c 24.0c 33.6b 35.0b 22.7c 1.0
VAP (µm/s) 61.2a 39.4c 40.8c 48.5b 50.3b 38.6c 1.4
VSL (µm/s) 48.6a 30.0b 30.5b 32.0b 32.4b 30.4b 1.3
VCL (µm/s) 69.5a 54.1b 54.5b 55.0b 56.1b 54.5b 1.5
LIN (%) 69.9a 55.4b 55.9b 58.1b 57.7b 55.7b 1.8
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Note: Different letters within the same row denote significant differences among the groups (P ≤ 0.05). Mean ± SEM of total motility (TM), progressive motility (PM), average path velocity (VAP), straight‐line velocity (VSL), curvilinear velocity (VCL), and linearity (LIN).

TABLE 2.

The effects of different Mito‐TEMPO concentrations on the percentage of membrane integrity, abnormal morphology, mitochondrial activity, and acrosome integrity of rooster sperm.

Mito‐TEMPO concentration (µM)
Parameter Fresh 0 1 10 100 1000 SEM
MI (%) 90.5a 52.4c 53.5c 60.3b 62.7b 52.6c 1.5
AM (%) 14.2a 17.6b 18.4b 17.0b 16.5b 18.2b 1.0
MA (%) 87.6a 50.0c 51.5c 68.4b 71.6b 52.2c 2.1
AI (%) 89.9a 55.6c 57.2c 66.4b 70.5b 55.3c 2.3
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Note: Different letters within the same row denote significant differences among the groups (p ≤ 0.05). Mean ± SEM of membrane integrity (MI), abnormal morphology (AM), mitochondrial activity (MA), and acrosome integrity (AI).

TABLE 3.

The effects of different Mito‐TEMPO concentrations on MDA concentrations, DNA fragmentation, and ROS concentrations of rooster sperm.

Mito‐TEMPO concentration (µM)
Parameter Fresh 0 1 10 100 1000 SEM
MDA (nmol/mL) 1.65a 5.12c 4.97c 3.51b 3.23b 5.02c 0.32
DNA F. (%) 6.4a 16.8c 15.5c 10.4b 9.1b 16.0c 1.1
ROS C. (%) 10.0a 28.5c 26.2c 20.7b 18.5b 28.9c 2.1
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Note: Different letters within the same row denote significant differences among the groups (p ≤ 0.05). Mean ± SEM of MDA concentrations (MDA), DNA fragmentation (DNA F.), and ROS concentrations (ROS C.).

TABLE 4.

The effects of different Mito‐TEMPO concentrations on the viability and apoptosis status of rooster sperm.

Mito‐TEMPO concentration (µM)
Parameter Fresh 0 1 10 100 1000 SEM
VC (%) 75.5a 45.5c 46.0c 61.5b 64.0b 45.4c 1.8
PA (%) 5.5 4.5 4.2 5.0 5.3 5.6 1.0
SA (%) 14.5a 45.5c 45.0c 28.0b 25.6b 42.8c 2.3
NC (%) 4.5 4.5 4.8 5.5 5.1 6.2 1.1
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Note: Different letters within the same row denote significant differences among the groups (p ≤ 0.05). Mean ± SEM of viable cells (VC), primary apoptosis (PA), secondary apoptosis (SA), and necrotic cells (NC).

TABLE 5.

Fertility and egg hatchability after artificial insemination of hens with fresh or cryopreserved rooster semen.

Mito‐TEMPO concentration (µM)
Type of semen/ Variable Fresh 0 10 100
Fertility (%) 87.2a (335/384) 46.8c (180/384) 67.7b (260/384) 70.5b (271/384)
Hatchability (%) 92.5a (310/335) 75.0b (135/180) 77.3b (201/260) 77.4b (210/271)
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Note: Mean values with different letters in the same row are significantly different (p ≤ 0.05).

In addition, semen samples containing 10 or 100 µM of Mito‐TEMPO had higher (p ≤ 0.05) percentages of spermatozoa with swollen tails/intact cell membranes (hypoosmotic test) and normal acrosomes compared with the other groups (Table 2). Moreover, mitochondrial activity was also the highest (p < 0.05) for semen cryopreserved in extender supplemented with 10 or 100 µM of Mito‐TEMPO (Table 2). There were no effects on sperm morphology post‐thawing when there was supplementation of cryopreservation medium with different concentrations of Mito‐TEMPO.

Further, rooster semen frozen in extenders containing 10 or 100 µM of Mito‐TEMPO exhibited the lowest (p < 0.05) incidence of MDA concentration, DNA fragmentation, accumulation of ROS (Table 3), and frequency of secondary apoptosis as well as the highest proportion of viable spermatozoa (Table 4). There were no differences (p > 0.05) among control and Mito‐TEMPO‐supplemented semen samples in the rates of primary apoptosis or necrosis (Table 4).

Finally, the mean fertility rates after inseminating the hens with frozen‐thawed rooster semen cryopreserved in the Beltsville extender containing 10 or 100 µM of Mito‐TEMPO were greater (p ≤ 0.05) compared with those after using non‐supplemented semen, but they were still lower (p < 0.05) compared with fertility after AI using fresh semen (Table 5). The hatching rate calculated as a proportion of fertilized eggs did not vary (p > 0.05) among frozen‐thawed Mito‐TEMPO‐supplemented and control inseminates rooster semen.

4. Discussion

The incorporation of antioxidants into cryopreservation media is acknowledged as a viable strategy to alleviate the adverse effects associated with freezing‐thawing on sperm quality and fertilization potential in both ruminant species (Barfourooshi et al. 2023) and avian species (Alipour‐Jenaghard et al. 2023). The sperm plasma membrane, characterized by a high content of polyunsaturated fatty acids, exhibits significant vulnerability to oxidative damage during the cryopreservation process, which subsequently compromises sperm functionality and diminishes fertilization capacity following thawing (Cerolini et al. 2006). The introduction of antioxidants into freezing extenders has the potential to shield spermatozoa from oxidative and mechanical stresses induced by the freezing process.

The current investigation assessed the impact of Mito‐TEMPO, a novel antioxidant specifically targeted to mitochondria, on the post‐thaw quality and fertility of rooster semen. Mito‐TEMPO is characterized by its positive charge, which facilitates selective accumulation within the mitochondrial matrix, thereby enhancing its capacity to inhibit lipid peroxidation and diminish the production of mitochondrial ROS more efficiently than traditional antioxidants (Yang et al. 2018). The results of our study indicated that the supplementation of 10 and 100 µM Mito‐TEMPO significantly enhanced sperm motility parameters (including total and progressive motility, as well as average path velocity), membrane functionality, mitochondrial activity, acrosome integrity, viability, and fertility rates when compared to untreated control samples. Concurrently, there was a notable reduction in lipid peroxidation, secondary apoptotic changes, DNA fragmentation, and hydrogen peroxide levels in samples treated with Mito‐TEMPO, aligning with previous studies that substantiated the ROS scavenging properties of TEMPO derivatives (Lu et al. 2018; Azadi et al. 2017).

Mitochondria play a crucial role in sperm energy metabolism through the processes of oxidative phosphorylation and ATP synthesis (Ruiz‐Pesini et al. 2007); however, thermal shock and excessive generation of ROS during cryopreservation can severely hinder mitochondrial function, resulting in disruptions in ATP transport (Fang et al. 2014). The presence of oxidative stress induces mitochondrial fission, aggregation, and dysfunction (P.‐I. Chen et al. 2017), which leads to a decrease in ATP production (H. Chen et al. 2005) and triggers the activation of mitochondrial apoptosis pathways. This apoptotic process involves the nuclear translocation of apoptosis effectors and an increase in mitochondrial membrane permeability, which is facilitated by pore formation and membrane rupture (Bajt et al. 2008).

The capacity of Mito‐TEMPO to inhibit the translocation of mitochondrial Bax underscores its protective function in maintaining mitochondrial integrity and sperm viability throughout the cryopreservation process (Hu and Li 2016). By reducing mitochondrial superoxide levels, it is probable that Mito‐TEMPO enhances mitochondrial function and mitigates apoptosis and necrosis triggered by cryogenic stress (Park et al. 2015). Considering the robust correlation between sperm mitochondrial activity, DNA integrity, and post‐thaw sperm quality as well as fertilization potential (Gómez‐Torres et al. 2017), Mito‐TEMPO supplementation could be able to improve these parameters by stabilizing the electron transport chain and mitochondrial membranes (Yang et al. 2018; Trnka et al. 2009).

Furthermore, the advantageous effects of Mito‐TEMPO may be facilitated through the modulation of critical metabolic enzymes, such as glucose‐6‐phosphate isomerase (GPI), whose enzymatic activity is diminished by cryo‐injury yet is maintained through antioxidant supplementation (Jiang et al. 2015).

Importantly, the morphology of spermatozoa, which is predominantly established during the process of spermatogenesis, exhibited no alterations due to either cryopreservation or Mito‐TEMPO treatment, corroborating previous findings (Zarei et al. 2022).

In the avian reproductive system, spermatozoa are retained within specialized sperm storage tubules (SST), with only those sperm exhibiting sustained fertilization potential proceeding to the sites of fertilization (Feyzi et al. 2018). The decline in sperm quality induced by the freeze‐thaw cycle adversely affects this transport mechanism and, consequently, fertility. Our findings suggest that supplementation with Mito‐TEMPO enhances sperm viability, potentially promoting the transit of spermatozoa and improving fertility outcomes post‐thaw. The fertility rates observed in hens inseminated with semen treated with Mito‐TEMPO (10 and 100 µM) were markedly elevated compared to control groups and demonstrated a strong correlation with enhanced semen quality parameters. Notably, hatching rates remained unaffected by Mito‐TEMPO treatment, highlighting that embryonic development may be governed by variables that are independent of sperm quality (Askarianzadeh et al. 2018).

5. Conclusion

Supplementation of the Beltsville semen extender with 10 and 100 µM of Mito‐TEMPO contributed to the effective cryopreservation of rooster semen, preserving post‐thaw sperm quality. Moreover, the highest fertility rates were obtained with cryopreserved sperm diluted in the freezing extender supplemented with the same two concentrations of Mito‐TEMPO. Rooster semen fertility is strongly dependent on sperm motility and viability, which in turn is dependent on mitochondrial function. Hence, the application of mitochondria‐targeted antioxidants such as Mito‐TEMPO appears to be an optimal strategy for maintaining the activity and fertilizing potential of rooster semen during the cryopreservation process.

Author Contributions

Mahdi Mokhtari: investigation, visualization, conceptualization, supervision, validation, and editing. Mahdi Khodaei‐Motlagh, Reza Masoudi, and Mohsen Sharafi: Writing – reviewing, methodology, data curation, conceptualization. All authors reviewed the results and approved the final version of the manuscript.

Funding

The authors have nothing to report.

Ethics Statement

The current study was approved by the Research Ethics Committee of the Animal Science Research Institute of Iran (IR. ASRI. REC. 2023. 008769).

Conflicts of Interest

The authors declare no conflicts of interest.

Acknowledgements

The authors would like to acknowledge Animal Science Research Institute of Iran (ASRI) for supporting this study.

Contributor Information

Mahdi Khodaei‐Motlagh, Email: mmotlagh2002@gmail.com.

Reza Masoudi, Email: reza.masoodi@gmail.com.

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

References

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

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