Skip to main content
NCBI home page
Veterinary Medicine and Science logoLink to Veterinary Medicine and Science
. 2026 Sep 28;12(5):e71186. doi: 10.1002/vms3.71186

Effects of Supplementing Semen Extenders With Spirulina platensis Extract on Post‐Thaw Ram Sperm Quality

Kimia Jaberi 1, Seyyed Mojtaba Mousavi 1,✉, Ali Forouharmehr 1
PMCID: PMC13618925  PMID: 42804407

ABSTRACT

Background

Supplementation of semen extender with natural antioxidants represents a promising strategy to mitigate cryopreservation‐induced damage in ram spermatozoa.

Objectives

This study evaluated the dose‐dependent effects of Spirulina platensis extract (0‐70 µg/mL) supplemented to Tris‐based extenders on post‐thaw sperm quality in Lori rams.

Methods

Semen was collected twice weekly over a four‐week period (eight replicates) from four mature rams using an artificial vagina. On each collection day, ejaculates meeting the acceptable criteria were pooled and extended in a Tris‐based diluent containing 0, 10, 20, 30, 40, 50, 60, or 70 µg/mL of Spirulina extract, designated as Spir‐0 through Spir‐70, respectively. All samples were equilibrated at 5°C for 3 h, then cryopreserved in liquid nitrogen and subsequently thawed in a 37°C water bath for 30 s. Post‐thaw sperm quality was comprehensively assessed for motility (using CASA), viability, plasma membrane integrity, acrosome morphology, mitochondrial membrane potential (ΔΨm), DNA denaturation (via AO), and oxidative biomarkers, including malondialdehyde (MDA), hydrogen peroxide (H2O2), and antioxidant enzyme activities (superoxide dismutase [SOD], catalase [CAT], and glutathione peroxidase [GPx]). Data were analysed using a completely randomised design with one‐way ANOVA, followed by Tukey's HSD post‐hoc test, with statistical significance set at P < 0.05.

Results

Supplementation at the concentration of 50 µg/mL Spirulina extract (Spir‐50) yielded the highest post‐thaw total (TM; 50.23%) and progressive motility (PM; 33.0%), as compared to the control (TM; 32.30%; PM; 18.8%). Moreover, this concentration significantly enhanced viability (59.16%), plasma membrane functionality (56.95%), and the proportion of spermatozoa exhibiting high mitochondrial membrane potential (hMMP; 54.5%). In addition, Spir‐50 markedly increased glutathione peroxidase (GPx) activity (35.49 vs. 23.73 mU/mL in control) and superoxide dismutase (SOD) activity (213.07 vs. 190.74 U/mL in control), along with total antioxidant capacity (TAC; 4.53 µmol/L vs. 3.34 µmol/L in control). Conversely, this treatment substantially reduced malondialdehyde (MDA; 2.60 nmol/mL vs. 7.69 nmol/mL in control), DNA denaturation (2.87% vs. 6.67% in control), and hydrogen peroxide levels (H2O2; 16.4% vs. 27.7% in control).

Conclusion

Supplementation with the concentration of 50 µg/mL Spirulina extract exhibited the most effective cryoprotective effects among the tested concentrations by improving sperm motility, viability, membrane and DNA integrity, mitochondrial function, and endogenous antioxidant defences, while attenuating lipid peroxidation and oxidative stress. These findings support the incorporation of Spirulina extract in Tris‐based extenders to improve post‐thaw ram sperm quality in ram semen cryopreservation protocols.

Keywords: cryopreservation, mitochondrial membrane potential, lipid peroxidation, ram semen, Spirulina

Supplementation of Tris‐based semen extender with 50 µg/mL Spirulina platensis methanolic extract significantly improved post‐thaw ram sperm motility, viability, membrane integrity, mitochondrial function, and DNA stability while enhancing antioxidant enzyme activities and markedly reducing lipid peroxidation and oxidative stress. These findings demonstrate Spirulina extract as an effective natural cryoprotectant for ram semen cryopreservation.

graphic file with name VMS3-12-e71186-g002.webp

1. Introduction

Sperm cryopreservation combined with artificial insemination is a key technology in modern assisted reproductive technologies in livestock. These techniques allow for efficient delivery of superior genetic material from elite sires, reduce the cost of maintaining large breeding herds, and contribute to preserving genetic diversity within domestic breeds (Lecewicz et al. 2019). However, their effectiveness is critically dependent upon the post‐thaw recovery of high‐quality, viable and functionally competent spermatozoa with preserved fertilising capacity (Aisen et al. 2002). Cryopreservation exerts severe physicochemical stresses on spermatozoa, resulting in marked reductions in viability, motility, and fertilising capacity, largely via oxidative stress, lipid peroxidation, mitochondrial dysfunction, and DNA fragmentation (Berean et al. 2024; Kameni et al. 2022; Sanocka and Kurpisz 2004).

Ram spermatozoa are notably vulnerable to cryopreservation‐induced damage owing to their exceptionally high membrane content of polyunsaturated fatty acids (PUFAs) and intrinsically lower endogenous antioxidant capacity compared with other domestic species (Muiño‐Blanco et al. 2008). Consequently, supplementation of freezing extenders with exogenous natural antioxidants has gained considerable scientific attention (Berean et al. 2024).

Spirulina platensis, a filamentous cyanobacterium, represents a valuable source of potent antioxidants, including phycocyanin, β‐carotene, vitamins C and E, selenium, and polysaccharides (Calella et al. 2022).

Recent studies have underscored the cryoprotective properties and reproductive benefits of Spirulina platensis in rams. Ben Moula et al. (2024) demonstrated that acetonic and hexanoic extracts of Spirulina platensis (1.25 and 3.75 µg/mL) notably improved post‐thaw motility, viability, and membrane integrity, while reducing lipid peroxidation in ram semen cryopreserved and stored at −80°C (Ben Moula et al. 2024). Similarly, purified C‐phycocyanin (2.4 µg/mL) substantially enhanced total and progressive motility, velocity parameters, viability, and antioxidant status during chilled storage (5°C) in skim‐milk extender, accompanied by a numerical increase in in vivo fertility rate (76% vs. 63% in controls) (Rahim et al. 2024). Dietary supplementation with Spirulina platensis (5 g/kg diet) in ram lambs improved semen quality (motility, viability and spermconcentration), increased antioxidant enzyme activities (SOD, GPx, CAT and glutathione), and decreased MDA concentrations (Assar et al. 2023). Certain Spirulina extracts also preserved sperm quality during liquid storage at 4°C and maintained fertility following artificial insemination (Ben Moula et al. 2023). These benefits are not confined to rams: Spirulina extract supplementation in bovine and buffalo semen extenders likewise enhanced post‐thaw motility and viability while substantially reducing lipid peroxidation (Badr et al. 2021; Mizera et al. 2019).

Despite these promising findings, several critical gaps remain unaddressed. First, the methanolic extract of Spirulina platensis, which may exhibit a distinct phytochemical profile and differing antioxidant potential relative to other solvent extracts, has not yet been evaluated in ram semen cryopreservation; previous studies have exclusively utilised acetonic/hexanoic extracts, purified C‐phycocyanin, or dietary supplementation. Second, the combination of Spirulina with the globally standardised Tris‐egg yolk extender and liquid nitrogen storage (−196°C) has likewise remained unexplored, as prior research has been restricted to skim‐milk extenders and −80°C freezing. Third, a comprehensive dose‐response assessment (0–70 µg/mL) is lacking, with previous investigations limited to narrow concentration ranges (≤3.75 µg/mL). To address these gaps, this study is the first to evaluate graded concentrations (0–70 µg/mL) of methanolic Spirulina platensis extract—chosen through a preliminary dose‐finding study—in Tris‐egg yolk extender on post‐thaw ram sperm quality following liquid nitrogen cryopreservation. We hypothesised that the extract's rich antioxidant components (phycocyanin, β‐carotene, polyphenols and tocopherols) would alleviate cryo‐induced oxidative stress and enhance overall sperm cryosurvival in a dose‐dependent manner.

2. Material and Methods

Unless otherwise stated, all chemicals and reagents were purchased from Sigma‐Aldrich (St. Louis, MO, USA). This study was approved by the institutional Animal Ethics Committee.

2.1. Animals and Semen Collection

This experiment was conducted during the non‐breeding season (winter) at the experimental livestock facility of Khorramabad, western Iran (47°17′ E, 33°26′ N; 1171 m above sea level). Over the study period, ambient temperature ranged from −4 to 17°C, with a mean relative humidity of 65%. Four mature Lori rams (aged 2–4 years; mean body weight: 68.2  ± 5.1 kg) were housed in a single shaded pen (4 × 5 m) and provided with ad libitum access to fresh water and a total mixed ration formulated to meet the nutritional requirements of adult rams (NRC, 2007).

Semen was collected from four mature rams of proven fertility (n = 4) using an artificial vagina, twice weekly over 4 consecutive weeks (eight replicates), yielding a total of 32 ejaculates (i.e., 8 ejaculates per ram). Immediately after collection, ejaculates were transferred to graduated tubes and transported to the laboratory in a thermos flask maintained at 35°C. Only ejaculates meeting the following quality criteria were retained and pooled to minimise inter‐individual variability: volume 0.5–2.0 mL, sperm concentration > 3 × 109 spermatozoa/mL, progressive motility > 80%, and morphologically abnormal spermatozoa < 10%. Pooled semen (representing the experimental unit) was diluted 1:20 (v/v) with a Tris‐based extender (292 mM Tris, 55.5 mM fructose, 81.4 mM citric acid, 15% [v/v] egg yolk in 100 mL of redistilled water; pH 7.2; osmolality 320 mOsm/kg) (Emamverdi et al. 2013). The diluted semen was then divided into eight equal aliquots and supplemented with Spirulina platensis extract at final concentrations of 0 (control), 10, 20, 30, 40, 50, 60, or 70 µg/mL, representing the experimental treatment groups (Spir 0 to Spir 70). Samples were gradually cooled to 5°C over a 3 h equilibration period, loaded into 0.25‐mL French straws (IMV, L'Aigle France) and sealed with haematocrit sealing powder (Lab Tron Co., Tehran, Iran). The straws were exposed to liquid nitrogen vapour at 5 cm above the liquid surface for 12 min, then plunged into liquid nitrogen for cryopreservation. Thawing was performed by immersion in a 37°C water bath for 30 s (Najafi et al. 2013). To prepare the required Spirulina dilutions, a stock solution was first made by dissolving 0.01 g of Spirulina extract in 1 mL of ethanol. Thereafter, 0, 1, 2, 3, 4, 5, 6, and 7 µL of the stock solution were added to the base diluent to produce 1 mL of each treatment: Spir‐0 (control, without ethanol), Spir‐10, Spir‐20, Spir‐30, Spir‐40, Spir‐50, Spir‐60 and Spir‐70, respectively.

2.2. Preparation of Spirulina Extract

Spirulina platensis extract was prepared by mixing 10 g of finely ground Spirulina powder (Giyah Kala Co., Tehran, Iran) with 100 mL of 80% (v/v) methanol in an Erlenmeyer flask. The mixture was subjected to agitation on an orbital shaker for 72 h at room temperature (≈22–25°C), then filtered through Whatman No. 1 filter paper. The filtrate was concentrated to dryness under reduced pressure using a rotary evaporator to remove the solvent and concentrate the bioactive compounds (Van Tuan et al. 2011). The resulting dry extract was stored in airtight containers, kept in a cool, dry place away from direct light, and maintained at a constant temperature of 25°C until further analysis. The methanolic fraction samples were subsequently freeze‐dried to yield Spirulina platensis extract powder. Total phenolic content was quantified using the Folin‐Ciocalteu method and expressed as milligrams of gallic acid equivalents (GAE) per gram of extract (mg GAE/g extract) (Singleton et al. 1999). Total flavonoid content was measured using the aluminium chloride colorimetric method and expressed as milligrams of quercetin equivalents (QE) per gram of extract (mg QE/g extract) (Zhishen et al. 1999). The FRAP assay was performed according to Benzie and Strain (1996), and results were expressed as µmol Fe2 + per gram of extract (µmol Fe2 +/g extract) (Benzie and Strain 1996). DPPH radical scavenging activity was calculated as the percentage inhibition of the DPPH radical. The total phenolic content, total flavonoid content, in vitro antioxidant activity, and DPPH radical scavenging activity of the prepared Spirulina extract are summarised in Table 1.

TABLE 1.

Total phenolic, flavonoid, antioxidant activity and DPPH assay of Spirulina extract.

Parameter Amount
Total phenols (mg GAE /g extract) 20.15
Total flavonoid (mg QE /g extract) 5.56
FRAP (µmol Fe2 +/g extract) 78.65
DPPH˙ radical scavenging activity (%) 46.34
Open in a new tab

Abbreviations: DPPH, 1,1‐diphenyl‐ 2‐picrylhydrazyl; FRAP, ferric reducing antioxidant power; GAE, gallic acid equivalent; QE, quercetin equivalent.

2.3. Post‐Thaw Sperm Quality Assessments

2.3.1. Sperm Motility and Kinematic Parameters

Post‐thaw sperm motility was assessed immediately post‐thaw using a computer‐assisted sperm analyser (CASA; HFT system, Houshmand Fanavar, Tehran, Iran) (Mogheiseh et al. 2022). A 10‐µL aliquot of each thawed sample was diluted to a final concentration of 20 × 106 sperm/mL with PBS and loaded into a pre‐warmed (37°C) Leja standard count 4‐chamber slide (20‐µm depth; ISOLAB, Germany). Slides were examined using a phase‐contrast microscope (Olympus BX60) equipped with a heated stage (37°C) and integrated with the CASA system. The analysis was performed at a frame rate of 60 Hz, acquiring 30 frames per field, with a minimum track length of 25 frames. The following kinematic parameters were recorded: total motility (TM, %), progressive motility (PM, %; defined as having STR ≥ 80% and VAP ≥ 25 µm/s), average path velocity (VAP, µm/s), straight‐line velocity (VSL, µm/s), curvilinear velocity (VCL, µm/s), amplitude of lateral head displacement (ALH, µm), beat‐cross frequency (BCF, Hz), straightness (STR, %), and linearity (LIN, %). At least five randomly selected fields were analysed per sample, with a minimum of 500 spermatozoa counted. All analyses were completed within 1–2 min of chamber loading to minimise time‑dependent changes in sperm kinematics.

2.3.2. Sperm Viability

Sperm viability was assessed using eosin‐nigrosin differential staining according to standard protocols (Benmoula et al. 2018). The staining solution was prepared by dissolving 1.67 g eosin Y and 2.9 g sodium citrate in 100 mL distilled water under gentle heating, followed by the addition of 10 g nigrosin and brief boiling of the mixture. After cooling to room temperature (∼20°C), the solution was filtered through a 0.2‐µm syringe filter. A 10‐µL aliquot of thawed semen was mixed with an equal volume of the stain on a pre‐warmed slide, smeared thinly, and air‐dried. Smears were examined under bright‐field microscopy at 400× magnification. At least 200 spermatozoa per sample were evaluated and classified as live (unstained, white‐headed) or dead (pink/purple‐headed). Results were expressed as the percentage of membrane‐intact (live) spermatozoa.

2.3.3. Sperm Plasma Membrane Functionality

Plasma membrane functionality was assessed using the hypo‐osmotic swelling (HOS) test (Revell and Mrode 1994). A 30 µL aliquot of thawed semen was mixed with 300 µL of hypo‐osmotic solution (100 mOsm/kg, containing 9 g/L fructose and 4.9 g/L sodium citrate in distilled water) and then incubated at 37°C for 30 min. A drop of the mixture was transferred to a slide, coverslipped, and evaluated under phase‐contrast microscopy at 400× magnification. At least 200 spermatozoa per sample were assessed, and the percentage exhibiting coiled or swollen tails—indicative of functional, osmotically responsive membranes—was recorded.

2.3.4. Sperm Morphology

Sperm morphology was evaluated using Hancock's differential staining method (Schäfer and Holzmann 2000). The fixative solution was prepared by combining 62.5 mL 37% formalin, 150 mL 0.9% saline, 150 mL phosphate‐buffered saline (PBS), and 500 mL double‐distilled water. Thawed semen (20 µL) was added to 2 mL pre‐warmed (37°C) Hancock's solution. A 10‐µL drop of the mixture was placed on a pre‐warmed slide, covered with a coverslip, and immediately examined under phase‐contrast microscopy at 1000× magnification. A minimum of 200 spermatozoa per sample were assessed, and the total abnormality rate—defined as the percentage of spermatozoa exhibiting any morphological defect in the head, midpiece, or tail—was calculated.

2.3.5. Lipid Peroxidation

Lipid peroxidation was quantified by measuring malondialdehyde (MDA) levels using the thiobarbituric acid reactive substances (TBARS) method (Esterbauer and Cheeseman 1990). Thawed semen samples were first diluted with PBS to a standardised concentration of 25 × 106 sperm/mL, as determined by hemocytometer counting. Thereafter, 1 mL of diluted semen was mixed with 1 mL EDTA, 1 mL butylated hydroxytoluene (BHT), and 2 mL trichloroacetic acid (TCA) to precipitate proteins and inhibit further oxidation. The mixture was vortexed and centrifuged at 1200 × g for 15 min at 4°C, and the supernatant was collected. Aliquots (1 mL) of supernatant were then incubated with 1 mL thiobarbituric acid (TBA) and heated in a boiling water bath (95°C) for 20 min. After cooling to room temperature for 30 min, absorbance was measured at 532 nm using a spectrophotometer (Biotek Epoch, USA). MDA concentrations were calculated from a standard curve using the following formula and expressed as nmol/mL:

MDA (nmol/mL)=(Absorbance532(sample)−Absorbance532(blank))−ba×Dilution factor

Where: Absorbance532 ( s a m p l e ) = absorbance of the sample at 532 nm; Absorbance532 (bl a n k ) = absorbance of the blank (all reagents without sample); b = y‐intercept of the standard curve; a = slope of the standard curve and Dilution factor = volume of supernatant (1 mL) / volume of original semen sample (1 mL) = 1.0

The correction using the blank accounts for any background absorbance from the reagents, and the dilution factor was applied to express the final MDA concentration per mL of the original semen sample. All samples were analysed in duplicate.

2.3.6. Antioxidant Enzyme Activities and Total Antioxidant Capacity

Activities of superoxide dismutase (SOD) and glutathione peroxidase (GPx), along with total antioxidant capacity (TAC), were quantified using commercially available assay kits (Navand Salamat Co., Iran) in accordance with the manufacturer's instructions (Izanloo et al. 2021).

2.3.6.1. Sample Preparation for Intracellular Enzymes (SOD and GPx)

For SOD and GPx measurements, spermatozoa were separated from seminal plasma by centrifugation at 800 × g for 10 min at 4°C. The supernatant (seminal plasma) was collected and frozen at −80°C for subsequent TAC analysis. The sperm pellet was washed three times with phosphate‐buffered saline (PBS) to remove residual seminal plasma and subsequently resuspended in ice‐cold lysis buffer (50 mM Tris‐HCl, 5 mM EDTA, 1 mM dithiothreitol [DTT], pH 7.5). Sperm were lysed by three cycles of freeze‐thawing (−80°C to 37°C) followed by sonication on ice (three 10‐s pulses at 40% amplitude). The lysate was centrifuged at 12,000 × g for 15 min at 4°C, and the supernatant was recovered for enzyme activity measurements. SOD and GPx activities were expressed as U/mL of the lysate (Keles et al. 2001).

2.3.6.2. Total Antioxidant Capacity (TAC)

TAC was determined in the seminal plasma using the commercial kit (Navand Salamat Co., Iran), consistent with previous studies in ram semen (Izanloo et al. 2021 ). Seminal plasma samples were analysed directly without further dilution.

2.3.7. Intracellular Hydrogen Peroxide

Intracellular hydrogen peroxide (H2O2) levels were quantified by flow cytometry using 2′,7′‐dichlorodihydrofluorescein diacetate (DCFH‐DA; D6883, Sigma‐Aldrich, USA) following the protocol described by Mahfouz et al. (2009) with minor modifications. Spermatozoa were diluted to a concentration of 1 × 106 sperm/mL in PBS. An aliquot of 500 µL of the sperm suspension was incubated with 10 µM DCFH‐DA and 5 µM propidium iodide (PI) at 37°C for 30 min in the dark. After incubation, samples were washed twice with PBS by centrifugation at 400 × g for 5 min to remove excess probe. The sperm pellet was resuspended in 500 µL PBS for flow cytometric analysis. Flow cytometric analysis was performed on a BD FACSCalibur flow cytometer (Becton Dickinson, San Jose, CA, USA) equipped with a 488‑nm argon laser. Green fluorescence from DCFH‑DA (excitation: 488 nm; emission: 530 nm) was detected in the FL‑1 channel, and PI fluorescence (excitation: 488 nm; emission: 670 nm) was detected in the FL‑3 channel. For each sample, 10,000 events were acquired. Spermatozoa were identified based on forward scatter (FSC) and side scatter (SSC) properties to distinguish them from debris and non‑sperm particles. Viable spermatozoa were then selected as the PI‑negative (PI−) population (Mahfouz et al. 2009). An unlabelled sperm sample was used as the negative control to set the autofluorescence threshold. The percentage of H2O2‑positive viable spermatozoa (DCFH‑DA+/PI−) was determined by setting the threshold based on the fluorescence intensity of the negative control. Results were expressed as the percentage of viable spermatozoa positive for H2O2 (Naderi et al. 2020).

2.3.8. Mitochondrial Membrane Potential

Mitochondrial membrane potential (ΔΨm) was evaluated using the MitoProbe JC‐1 Assay Kit (Catalogue number: M34152, Thermo Fisher Scientific, Eugene, OR, USA) according to the manufacturer's instructions with minor modifications for ram spermatozoa (Mohammadi et al. 2024). Spermatozoa were diluted to a concentration of 2 × 106 sperm/mL in PBS. An aliquot of 500 µL of the sperm suspension was incubated with 8 µM JC‐1 at 37°C for 30 min in the dark. A parallel sample was treated with 50 µM carbonyl cyanide p‑trifluoromethoxyphenylhydrazone (FCCP) as a positive control for depolarisation. After incubation, samples were washed twice with PBS by centrifugation at 400 × g for 5 min to remove excess dye. The pellet was resuspended in 500 µL PBS and analysed immediately. Flow cytometric analysis was performed on a BD FACSCalibur flow cytometer (Becton Dickinson, San Jose, CA, USA) equipped with a 488‑nm argon laser. Flow cytometric analysis was performed on a BD FACSCalibur flow cytometer (Becton Dickinson, San Jose, CA, USA) equipped with a 488‑nm argon laser. JC‑1 emits green fluorescence (FL‑1 channel, 529 nm) in its monomeric form (depolarised mitochondria) and red fluorescence (FL‑2 channel, 590 nm) in its aggregate form (polarised mitochondria, high ΔΨm). For each sample, 10,000 events were acquired. Spermatozoa were first gated based on forward scatter (FSC) and side scatter (SSC) properties to exclude debris and non‑sperm particles. Results were expressed as the percentage of viable spermatozoa exhibiting high mitochondrial membrane potential (hMMP), calculated as the ratio of red (aggregate) to green (monomeric) fluorescence (FL‑2/FL‑1) (Mohammadi et al. 2024).

2.3.9. Sperm Chromatin Stability

Sperm DNA denaturation was assessed using the acridine orange (AO) assay (Piri et al. 2024). Air‐dried smears were fixed in Carnoy's solution (methanol:glacial acetic acid, 3:1 v/v) at 4°C for 2 h, subsequently air‐dried, stained with freshly prepared AO solution (1 mg/mL) for 10 min at room temperature. After rinsing in distilled water, smears were examined under an Olympus BX51 epifluorescence microscope at 1000× magnification. Acridine orange intercalates into double‐stranded DNA, emitting green fluorescence, while binding to single‐stranded or denatured DNA emits red fluorescence. A total of 200 spermatozoa were scored per replicate, yielding a total of 600 spermatozoa evaluated per treatment group. All slides were scored by a single experienced observer who was blinded to the treatment groups to avoid observer bias. The percentage of spermatozoa exhibiting red fluorescence was recorded as an indicator of chromatin instability and susceptibility to DNA denaturation (Evenson and Jost 1994). Results were expressed as the percentage of spermatozoa presenting DNA denaturation.

2.4. Statistical Analysis

The experiment was conducted as a completely randomised design with eight treatments corresponding to Spirulina extract concentrations of 0, 10, 20, 30, 40, 50, 60 and 70 µg/mL, with eight replicates per treatment. The experimental unit was the pooled semen sample per collection day (replicate), to which the treatments were applied. Collection day (replicate) was included as a random effect to account for potential non‐independence among samples derived from the same pooled ejaculate. Data normality was verified by the Shapiro–Wilk test, and homogeneity of variance was evaluated using Levene's test. Since both assumptions were met, data were analysed using one‐way ANOVA via the General Linear Model (GLM) procedure in SAS (version 9.1; SAS Institute Inc., Cary, NC, USA), with treatment as a fixed effect and replicate as a random effect. Treatment means were compared using Tukey's honestly significant difference (HSD) test, with statistical significance defined as p < 0.05.

3. Results

Post‐thaw sperm kinematic parameters, as assessed by computer‐assisted sperm analysis (CASA), revealed significant improvements in specific kinematic parameters in the Spir‐50 group (Table 2). Total motility (TM) and progressive motility (PM) reached values of 50.23% and 33.0%, respectively—which were significantly (p < 0.05) higher than the control values (TM: 32.3%; PM: 18.82%). The highest linearity (LIN) was recorded in the Spir‐50 group (89.64%), which was significantly higher than those observed in Spir‐0, Spir‐10, and Spir‐70 (P < 0.05), but not significantly different from Spir‐30 and Spir‐40. Intermediate improvements were recorded in Spir‐40 and Spir‐30 groups for these parameters. Other kinematic parameters, including VSL, VCL, VAP, STR, ALH, and BCF, did not reveal significant differences among treatments (p > 0.05).

TABLE 2.

Comparison of ram sperm kinematic parameters after the freeze‐thawing process.

Treatment TM (%) PM (%) LIN (%) VSL (µm/s) STR (%) VCL (µm/s) VAP (µm/s) ALH (µm) BCF (Hz)
Spir‐0 32.303 bc 18.823 b 42.01 b 73.35 87.46 111.61 83.67 6.207 28.07
Spir‐10 30.492 c 18.415 b 30.42 b 47.95 82.02 80.01 58.72 4.391 22.51
Spir‐20 40.871 abc 27.297 ab 71.47 ab 77.89 92.60 103.54 84.61 5.701 27.03
Spir‐30 40.612 abc 24.809 ab 83.14 a 62.14 85.12 111.27 72.33 5.402 26.57
Spir‐40 46.364 ab 31.807 a 84.24 a 87.09 89.10 142.99 97.54 6.621 36.08
Spir‐50 50.231 a 33.007 a 89.64 a 106.89 80.68 144.90 101.97 7.632 40.14
Spir‐60 37.734 abc 24.093 ab 44.93 ab 74.25 82.35 105.87 84.70 5.885 27.03
Spir‐70 29.536 c 16.090 b 34.12 b 70.44 83.58 113.31 83.16 6.184 32.60
SEM 4.486 3.737 9.30 18.88 9.44 19.35 17.10 1.42 8.70
Open in a new tab

Note: a,b,cColumns with different superscripts differ (p < 0.05).

Spir‐0, Spir‐10, Spir‐20, Spir‐30, Spir‐40, Spir‐50, Spir‐60 and Spir‐70 represent 0, 10, 20, 30, 40, 50, 60 and 70 µg/mL Spirulina extract in the diluent, respectively.

Abbreviations: ALH, amplitude of lateral head displacement, BCF, beat cross frequency; LIN, linearity percentage, PM, progressive motility; STR, straightness, TM, total motility; VAP, average path velocity, VCL, curvilinear velocity; VSL, straight‐line velocity

The Spir‐50 group demonstrated the highest post‐thaw sperm viability (59.16% live spermatozoa), as compared to 40.52% in the control group (p < 0.05; Table 3). Plasma membrane functionality showed a similar trend: the Spir‐50 group scored the highest value (56.95%), significantly surpassing Spir‐40 (48.75%), with both values being substantially higher than all other treatments (p < 0.05).

TABLE 3.

Comparative effects of Spirulina‐supplemented diluents on ram sperm viability, plasma membrane functionality, and morphology.

Parameters
Treatment Normalities (%) HOS (%) Viability (%)
Spir‐0 77.15 38.832 cd 40.52 b
Spir‐10 75.82 40.528 bcd 44.16 b
Spir‐20 85.02 43.153 bcd 46.33 b
Spir‐30 83.18 44.432 bc 49.34 ab
Spir‐40 82.100 48.751 ab 53.16 ab
Spir‐50 84.70 56.955 a 59.16 a
Spir‐60 82.44 47.43 bc 50.16 ab
Spir‐70 78.50 35.014 d 41.83 b
Treatment Normalities (%) HOS (%) Viability (%)
Open in a new tab

Note: a,b,c,d Columns with different superscripts differ (p < 0.05). Spir‐0, Spir‐10, Spir‐20, Spir‐30, Spir‐40, Spir‐50, Spir‐60 and Spir‐70 represent 0, 10, 20, 30, 40, 50, 60 and 70 µg/mL Spirulina extract in the diluent, respectively.

Malondialdehyde (MDA) concentration was significantly lower in the Spir‐50 group (2.60 nmol/mL) compared with the control (7.69 nmol/mL) and Spir‑10 (6.06 nmol/mL) and Spir‑30 (5.29 nmol/mL) groups (p < 0.05; Table 4). Superoxide dismutase (SOD) activity was highest in the Spir‐50 group (213.07 U/mL), significantly higher than in the control (190.74 U/mL; p < 0.05). Glutathione peroxidase (GPx) activity was highest in Spir‐60 (40.98 mU/mL) and Spir‐70 (39.89 mU/mL), although these values were not significantly different from Spir‑50 (35.49 mU/mL). Total antioxidant capacity (TAC) was significantly higher in all Spir‑supplemented groups compared with the control (p < 0.05), with the numerically highest value observed in Spir‑70 (4.75 µmol/L), but without significant differences among the Spir‑containing treatments.

TABLE 4.

Comparison of antioxidant enzymes activity and MDA levels across treatments.

 Treatment MDA (nmol/ml) GPX (mU/mL) TAC (µmol/L) SOD (U/ mL)
Spir‐70 3.62cd 39.89 ab 4.75 a 199.51abc
Spir‐60 3.09 cd 40.98 a 4.56 a 199.38abc
Spir‐50 2.60d 35.49 ab 4.54 a 213.07 a
Spir‐40 3.27 cd 32.03 abc 4.48 a 202.50abc
Spir‐30 5.29bc 24.10 c 4.53 a 206.18 ab
Spir‐20 3.54 cd 29.79 bc 4.28 a 193.90 bc
Spir‐10 6.06ab 29.66 bc 4.31 a 197.98abc
Spir‐0 7.69 a 23.73 c 3.34 b 190.74 c
SEM 0.72 3.44 0.26 4.531
Open in a new tab

Note: a,b,c,d Columns with different superscripts differ (p < 0.05).

Spir‐0, Spir‐10, Spir‐20, Spir‐30, Spir‐40, Spir‐50, Spir‐60 and Spir‐70 represent 0, 10, 20, 30, 40, 50, 60 and 70 µg/mL Spirulina extract in the diluent, respectively.

As presented in Table 5, DNA denaturation and H2O2 levels were significantly lower in the Spir‐50 group (2.87% and 16.43%, respectively) compared with the control (6.67% and 27.66%, respectively; p < 0.05). The percentage of spermatozoa with high mitochondrial membrane potential (hMMP) was highest in the Spir‐50 group (54.46%), which was significantly higher than in the control (39.1%; p < 0.05; Table 5). However, hMMP values in Spir‑50 were statistically similar to those in Spir‑40, Spir‑60, and Spir‑70 groups.

TABLE 5.

Comparison of DNA denaturation rate, mitochondrial activity, and H2O2 levels across treatments following the freeze‐thawing process.

Treatment Damaged DNA (%) H2O2 (%) hMMP (%)
Spir‐0 6.67 a 27.66 a 39.10 b
Spir‐10 6.03 ab 23.33 ab 42.33 ab
Spir‐20 3.91 bcd 18.35 ab 46.05 ab
Spir‐30 3.40 cd 18.67 ab 41.66 ab
Spir‐40 3.47 cd 20.66 ab 50.01 ab
Spir‐50 2.87 d 16.43 b 54.46 a
Spir‐60 4.30 bcd 21.34 ab 51.34 ab
Spir‐70 5.40 abc 23.01 ab 47.03 ab
SEM 0.70 3.23 4.27
Open in a new tab

Note: a,b,c,d Columns with different superscripts differ (p < 0.05).

Spir‐0, Spir‐10, Spir‐20, Spir‐30, Spir‐40, Spir‐50, Spir‐60 and Spir‐70 represent 0, 10, 20, 30, 40, 50, 60 and 70 µg/mL Spirulina extract in the diluent, respectively. hMMP: Sperm with high mitochondrial membrane potential.

Figure 1 provides a representative micrograph of acridine orange staining, illustrating the fluorescence patterns used to assess sperm chromatin stability (intact vs. denatured DNA) in the control group.

FIGURE 1.

FIGURE 1

Open in a new tab

Assessment of sperm chromatin stability using acridine orange (AO) fluorescent staining. Representative fluorescence micrograph of spermatozoa from the control group (Spir‑0). Spermatozoa with green fluorescence over the head region indicate intact, double‐stranded DNA, while spermatozoa with yellow‑to‑orange fluorescence indicate denatured, single‑stranded DNA. Samples were examined under an epifluorescence microscope at 400× magnification.

Figure 2 presents representative flow cytometric dot plots illustrating JC‐1 staining patterns for mitochondrial membrane potential across treatments.

FIGURE 2.

FIGURE 2

Open in a new tab

Representative flow cytometric dot plots illustrating JC‑1 staining patterns for mitochondrial membrane potential (ΔΨm) in post‐thaw ram spermatozoa. The upper right quadrant (high FL2‐H/red fluorescence) indicates spermatozoa with high mitochondrial membrane potential (hMMP; J‑aggregates), while the lower right quadrant indicates spermatozoa with low mitochondrial membrane potential (J‑monomers). These images are descriptive examples of the fluorescence patterns observed across treatments. All quantitative hMMP data are presented in Table 4. Spir‐0, Spir‐10, Spir‐20, Spir‐30, Spir‐40, Spir‐50, Spir‐60, and Spir‐70 correspond to 0, 10, 20, 30, 40, 50, 60, and 70 µg/mL Spirulina extract in the Tris‐based extender, respectively.

4. Discussion

The freeze–thaw process triggers reactive oxygen species (ROS) production, inflicting molecular damage to sperm cells and impairing critical functional parameters such as motility, membrane integrity, and DNA stability (Berean et al. 2024; Riesco et al. 2021).

In the present study, supplementation of the Tris‐based semen extender with 50 µg/mL Spirulina extract (Spir‐50) significantly enhanced post‐thaw total motility, progressive motility, viability, and plasma membrane functionality in ram spermatozoa. These findings are consistent with previous studies highlighting the cryoprotective potential of Spirulina and its bioactive components. Ethanolic Spirulina extract enhanced motility during chilled storage (Kameni et al. 2022), while acetonic and hexanoic extracts of Spirulina platensis improved post‐thaw sperm quality and reduced lipid peroxidation. (Ben Moula et al. 2024). Likewise, purified C‐phycocyanin markedly increased motility, kinematic parameters, viability, and antioxidant status during cooled storage (Rahim et al. 2024), and Spirulina extracts maintained sperm quality in liquid storage at 4°C without compromising fertility (Ben Moula et al. 2023). Furthermore, dietary supplementation with Spirulina platensis improved both systemic and seminal antioxidant capacity and overall semen quality in ram lambs (Assar et al. 2023).

These protective effects are largely attributed to the Spirulina’s potent antioxidant profile, encompassing carotenoids (e.g., β‐carotene), phycocyanin and tocopherols (vitamin E), vitamin C, and selenium (Karkos et al. 2011; Wu et al. 2016). These compounds effectively neutralise ROS, inhibit lipid peroxidation, and restore redox homeostasis, thereby mitigating cryopreservation‐induced oxidative damage (Fouda and Ismail 2017; Naeini et al. 2021). Beyond these direct antioxidants, Spirulina also provides polyunsaturated fatty acids (PUFAs), such as γ‐linolenic acid, which support antioxidant defence indirectly by serving as substrates for the activation of antioxidant enzymes (Yang et al. 2020).

Sperm plasma membrane integrity is vital for fertilising competence and the ability to undergo the acrosome reaction (Berlinguer et al. 2017). Lipid peroxidation of polyunsaturated fatty acids (PUFAs) disrupts membrane structure and function (Pintus and Ros‐Santaella 2021). The marked post‐thaw improvements in sperm viability and plasma membrane functionality observed in the Spir‐50 group align with the membrane‐protective and anti‐apoptotic effects of Spirulina supplementation (Badr et al. 2021; Ben Moula et al. 2024; Rahim et al. 2024).

Post‐thaw sperm morphology remained largely unaffected by Spirulina supplementation, in line with evidence that extender antioxidants generally do not influence morphological abnormalities, which predominantly originate during spermatogenesis rather than during cryopreservation (Amini et al. 2019; Zarei et al. 2021). Although some studies reported reduced morphological defects with Spirulina enrichment (Kameni Leugoué et al. 2022; Hassan et al. 2023), these discrepancies likely arise from differences in extract composition, antioxidant potency, preparation method, or morphological assessment protocols.

Enzymatic antioxidants—including SOD, catalase (CAT), GPx, and glutathione reductase—represent the primary cellular defence mechanism against ROS (Bansal and Bilaspuri 2011). In the present study, supplementation with 50 µg/mL Spirulina platensis extract resulted in the lowest MDA level (2.60 nmol/mL), reflecting minimal lipid peroxidation and oxidative damage. This was accompanied by markedly elevated SOD activity and TAC, consistent with prior evidence that Spirulina supplementation reduces lipid peroxidation and potentiates antioxidant defences in cryopreserved spermatozoa, thereby supporting improved fertility outcomes (Badr et al. 2021). Although acrosomal integrity was not assessed in the present study, previous research has demonstrated that Spirulina supplementation preserves acrosomal and plasma membrane integrity in cryopreserved spermatozoa (Badr et al. 2021). Comparable protective effects have also been reported for Spirulina platensis and Salvia verbenaca extracts in ram spermatozoa cryopreserved at −80°C (Ben Moula et al. 2024).

GPx plays a critical role in neutralising peroxides and protecting sperm DNA from oxidative damage (Tabatabaei Vakili et al. 2025), while SOD maintains motility by scavenging superoxide radicals (Qamar et al. 2022). Although some studies in rams have found no significant correlation between motility and GPx activity (Tabatabaei Vakili et al. 2025), positive associations have been documented in human infertility cases (Crisol et al. 2012). Associations between seminal SOD activity and motility are similarly inconsistent across species and experimental contexts, with positive correlations reported in infertile men (Murawski et al. 2007) but not observed in other populations (Patricio et al. 2016). In rams, ethanolic Spirulina extract at concentrations of 40 and 80 µg/mL has been shown to decrease MDA while increasing SOD and CAT activities during chilled storage (Kameni et al. 2022). It should be noted that excessive antioxidant supplementation may paradoxically induce pro‐oxidant effects by disrupting physiological ROS homeostasis, which is essential for normal sperm function (Suwimonteerabutr et al. 2024).

The optimal cryoprotective response at 50 µg/mL is likely attributable to a concentration‐dependent effect, wherein an intermediate concentration provides optimal antioxidant protection without compromising essential redox signalling pathways.

The Spir‐50 group demonstrated the lowest hydrogen peroxide (H2O2) levels, minimal DNA denaturation (2.87%), and the highest proportion of spermatozoa with high mitochondrial membrane potential (hMMP = 54.46%). These protective effects are likely attributed to Spirulina’s diverse antioxidant constituents, which counteract ROS‐induced DNA denaturation and mitochondrial dysfunction—processes that trigger caspase activation, disrupt mitochondrial membrane permeability, and impair ATP synthesis (Martin et al. 2004).

The marked motility improvement in the Spir‐50 group is likely associated with reduced DNA denaturation and preserved mitochondrial function. Sperm motility is critically dependent on mitochondrial ATP production and particularly susceptible to thermal and oxidative stresses during cooling and cryopreservation (Zarei et al. 2021). These findings align with prior reports demonstrating that Spirulina supplementation enhances viability, motility, and overall sperm quality (Putri and Budi 2020). Taken together, these findings underscore Spirulina extract as a promising natural additive for mitigating cryo‐induced oxidative damage in ram spermatozoa. Future research should delineate the precise molecular pathways involved and evaluate in vivo fertility outcomes following artificial insemination with Spirulina‐supplemented cryopreserved semen.

Pooling ejaculates is a widely adopted strategy to reduce individual variation and increase experimental efficiency in ram semen cryopreservation studies (Üstüner et al. 2014). However, this approach obscures inter‐ and intra‐individual variability, restricting the generalisability of results (Dayanıklı et al. 2025). Therefore, findings from pooled samples should be applied with appropriate caution, and confirmatory studies using individual rams are recommended when considering practical applications such as breeding programmes (Rickard et al. 2016).

5. Conclusion

Under the experimental conditions employed herein, supplementation of Tris‐based extenders with 50 µg/mL Spirulina extract notably improved several post‑thaw sperm quality parameters in ram spermatozoa. This improvement was associated with reduced oxidative stress—evidenced by significantly lower MDA and H2O2 levels—while bolstering endogenous antioxidant defences through enhanced GPx and SOD activities along with TAC. These biochemical improvements were accompanied by enhanced plasma membrane integrity, mitochondrial membrane potential, and nuclear DNA stability. Consequently, post‐thaw functional competence was markedly improved, as reflected in significant increases in total motility, progressive motility, sperm viability, and membrane functionality. Together, these results suggest that Spirulina extract may serve as a promising natural antioxidant supplement for mitigating cryopreservation‐induced oxidative damage in ram spermatozoa, although further in vivo studies are warranted to fully confirm its efficacy.

Author Contributions

Kimia Jaberi: methodology, investigation, data curation and writing – original draft. Seyyed Mojtaba Mousavi: conceptualisation, investigation, methodology, data curation, supervision, formal analysis, project administration, visualisation, resources, writing – review and editing. Ali Forouharmehr: writing – review and editing, visualisation, resources, supervision, data curation, conceptualisation and investigation.

Ethics Statement

This study was approved by the Animal Ethics Committee of Lorestan University, Iran.

Conflicts of Interest

The authors declare no conflicts of interest.

Acknowledgements

We would like to thank the vice‐chancellor for research at Lorestan University for providing the facilities and the financial support to carry out this study [grant number 40211314009.3870].

Data Availability Statement

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

References

  1. Aisen, E. , Medina V., and Venturino A.. 2002. “Cryopreservation and Post‐Thawed Fertility of Ram Semen Frozen in Different Trehalose Concentrations.” Theriogenology 57, no. 7: 1801–1808. 10.1016/S0093-691X(02)00653-2. [DOI] [PubMed] [Google Scholar]
  2. Amini, S. , Masoumi R., Rostami B., Shahir M. H., Taghilou P., and Arslan H. O.. 2019. “Effects of Supplementation of Tris‐egg Yolk Extender With Royal Jelly on Chilled and Frozen‐Thawed Ram Semen Characteristics.” Cryobiology 88: 75–80. 10.1016/j.cryobiol.2019.03.008. [DOI] [PubMed] [Google Scholar]
  3. Assar, D. , Al‐Wakeel R., Elbialy Z., et al. 2023. “Spirulina Platensis Algae Enhances Endogenous Antioxidant Status, Modulates Hemato‐Biochemical Parameters, and Improves Semen Quality of Growing Ram Lambs.” Advances in Animal and Veterinary Sciences 11, no. 4: 595–605. 10.17582/journal.aavs/2023/11.4.595.605. [DOI] [Google Scholar]
  4. Badr, M. , Rawash Z., Azab A., Dohreg R., Ghattas T., and Fathi M.. 2021. “ Spirulina Platensis Extract Addition to Semen Extender Enhances Cryotolerance and Fertilizing Potentials of Buffalo Bull Spermatozoa.” Animal Reproduction 18, no. 2: e20200520. 10.1590/1984-3143-AR2020-0520. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bansal, A. K. , and Bilaspuri G.. 2011. “Impacts of Oxidative Stress and Antioxidants on Semen Functions.” Veterinary Medicine International 2011, no. 1: 686137. 10.4061/2011/686137. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Benmoula, A. , Badi A., Hilali A., et al. 2018. “Evaluation of Camel Milk as an Extender for Short‐Term Storage of Ram Semen at 15 C.” Journal of Livestock Science and Technologies 6, no. 1: 57–64. 10.22103/jlst.2017.10987.1212. [DOI] [Google Scholar]
  7. Ben Moula, A. , Rahim A., Chentouf M., Raes M., Kirschvink N., and El Amiri B.. 2023. “Inclusion of Spirulina platensis and Salvia verbenaca Extracts to Boost Semen Quality and Fertilization Ability in Sheep.” Reproduction in Domestic Animals 58, no. 5: 637–645. 10.1111/rda.14332. [DOI] [PubMed] [Google Scholar]
  8. Ben Moula, A. , Salhi S., Rahim A., et al. 2024. “Enhancing Cryopreserved Ram Sperm Quality at −80°C With Spirulina platensis and Salvia verbenaca Extracts.” Acta Veterinaria Hungarica 72, no. 2: 125–132. 10.1556/004.2024.01042. [DOI] [PubMed] [Google Scholar]
  9. Benzie, I. F. , and Strain J. J.. 1996. “The Ferric Reducing Ability of Plasma (FRAP) as a Measure of ‘Antioxidant Power’: The FRAP Assay.” Analytical Biochemistry 239, no. 1: 70–76. 10.1006/abio.1996.0292. [DOI] [PubMed] [Google Scholar]
  10. Berean, D. I. , Bogdan L. M., and Cimpean R.. 2024. “Advancements in Understanding and Enhancing Antioxidant‐Mediated Sperm Cryopreservation in Small Ruminants: Challenges and Perspectives.” Antioxidants 13, no. 6: 624. 10.3390/antiox13060624. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Berlinguer, F. , Pasciu V., Succu S., et al. 2017. “REAC Technology as Optimizer of Stallion Spermatozoa Liquid Storage.” Reproductive Biology and Endocrinology 15, no. 1: 11. 10.1186/s12958-017-0229-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Calella, P. , Cerullo G., Di Dio M., et al. 2022. “Antioxidant, Anti‐inflammatory and Immunomodulatory Effects of Spirulina in Exercise and Sport: A Systematic Review.” Frontiers in Nutrition 9: 1048258. 10.3389/fnut.2022.1048258. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Crisol, L. , Matorras R., Aspichueta F., et al. 2012. “Glutathione Peroxidase Activity in Seminal Plasma and Its Relationship to Classical Sperm Parameters and in Vitro Fertilization‐Intracytoplasmic Sperm Injection Outcome.” Fertility and Sterility 97, no. 4: 852–857. e851. 10.1016/j.fertnstert.2012.01.097. [DOI] [PubMed] [Google Scholar]
  14. Dayanıklı, C. , Bülbül B., Doğan Ş., et al. 2025. “Improving Ram Semen Low Cryotolerance by Replacing the Seminal Plasma with That of High‐Cryotolerant Rams or Extender.” Veterinary Medicine and Science 11: e70381. 10.1002/vms3.70381. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Emamverdi, M. , Zhandi M., Zare Shahneh A., Sharafi M., and Akbari‐Sharif A.. 2013. “Optimization of Ram Semen Cryopreservation Using a Chemically Defined Soybean Lecithin‐Based Extender.” Reproduction in Domestic Animals 48, no. 6: 899–904. 10.1111/rda.12183. [DOI] [PubMed] [Google Scholar]
  16. Esterbauer, H. , and Cheeseman K. H.. 1990. “[42]Determination of Aldehydic Lipid Peroxidation Products: Malonaldehyde and 4‐hydroxynonenal.” In Methods in Enzymology edited by Packer L. and Glazer A. N., 186: 407–421. Elsevier. 10.1016/0076-6879(90)86134. [DOI] [PubMed] [Google Scholar]
  17. Evenson, D. , and Jost L.. 1994. “Sperm Chromatin Structure Assay: DNA Denaturability.” In Methods in Cell Biology edited by Darzynkiewicz Z., Robinson J. P., and Crissman H. A., 42: 159–176. 10.1016/s0091-679x(08)61073-0. [DOI] [PubMed] [Google Scholar]
  18. Fouda, S. F. , and Ismail R. F.. 2017. “Effect of Spirulina Platensis on Reproductive Performance of Rabbit Bucks.” Egyptian Journal of Nutrition and Feeds 20, no. 1: 55–66. 10.21608/ejnf.2017.75118. [DOI] [Google Scholar]
  19. Hassan, M. A. E. , Shehabeldin A. M., Omar M. E. A., et al. 2023. “Effect of Spirulina Nanoparticles or Selenium Coated Spirulina Nanoparticles Supplemented to Freezing Extender on Bull Sperm Freezability.” Pakistan Veterinary Journal 43, no. 4: 739‑747. 10.29261/pakvetj/2023.111. [DOI] [Google Scholar]
  20. Izanloo, H. , Soleimanzadeh A., Bucak M. N., Imani M., and Zhandi M.. 2021. “The Effects of Varying Concentrations of Glutathione and Trehalose in Improving Microscopic and Oxidative Stress Parameters in Turkey Semen During Liquid Storage at 5 C.” Cryobiology 101: 12–19. 10.1016/j.cryobiol.2021.07.002. [DOI] [PubMed] [Google Scholar]
  21. Kameni, S. L. , Dongmo A. B. N., Tebug T. T., Bomba F. D. T., Meutchieye F., and Ngoula F.. 2022. “Spirulina (Arthrospira platensis) Extract Promotes Motility, Microscopic, and Antioxidative Parameters of Ram Semen During Refrigerated Storage.” Black Sea Journal of Agriculture 5, no. 4: 481–490. 10.47115/bsagriculture.1162523. [DOI] [Google Scholar]
  22. Karkos, P. D. , Leong S. C., Karkos C. D., Sivaji N., and Assimakopoulos D. A.. 2011. “Spirulina in Clinical Practice: Evidence‑Based Human Applications.” Evidence‑Based Complementary and Alternative Medicine 2011: 531053. 10.1093/ecam/nen058. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Keles, M. S. , Taysi S., Sen N., Aksoy H., and Akcay F.. 2001. “Effect of Corticosteroid Therapy on Serum and CSF Malondialdehyde and Antioxidant Proteins in Multiple Sclerosis.” The Canadian Journal of Neurological Sciences 28, no. 2: 141–143. 10.1017/S0317167100052823. [DOI] [PubMed] [Google Scholar]
  24. Lecewicz, M. , Strzeżek R., Majewska A. M., Purpurowicz P. S., and Kordan W.. 2019. “The Effect of Different Concentrations of Caffeine, Pentoxifylline and 2'‐deoxyadenosine on the Biological Properties of Frozen‐Thawed Canine Semen.” Annals of Animal Science 19, no. 3: 733–746. 10.2478/aoas-2019-0025. [DOI] [Google Scholar]
  25. Mahfouz, R. , Sharma R., Lackner J., Aziz N., and Agarwal A.. 2009. “Evaluation of Chemiluminescence and Flow Cytometry as Tools in Assessing Production of Hydrogen Peroxide and Superoxide Anion in human Spermatozoa.” Fertility and Sterility 92, no. 2: 819–827. 10.1016/j.fertnstert.2008.05.087. [DOI] [PubMed] [Google Scholar]
  26. Martin, G. , Sabido O., Durand P., and Levy R.. 2004. “Cryopreservation Induces an Apoptosis‐Like Mechanism in Bull Sperm.” Biology of Reproduction 71, no. 1: 28–37. 10.1095/biolreprod.103.024281. [DOI] [PubMed] [Google Scholar]
  27. Mizera, A. , Kuczaj M., and Szul A.. 2019. “Impact of the Spirulina Maxima Extract Addition to Semen Extender on Bovine Sperm Quality.” Italian Journal of Animal Science 18, no. 1: 601–607. 10.1080/1828051X.2018.1548914. [DOI] [Google Scholar]
  28. Mogheiseh, A. , Divar M. R., Vesal N., and Mahdivand Moradloo F.. 2022. “Effects of Epididymal Sperm Recovery Methods on Fresh and Frozen–Thawed Sperm Characteristics in Dogs.” Reproduction in Domestic Animals 57, no. 9: 1038–1045. 10.1111/rda.14171. [DOI] [PubMed] [Google Scholar]
  29. Mohammadi, A. , Jafarpour F., Vash N. T., Hajian M., and Nasr‐Esfahani M. H.. 2024. “Supplementation of Sperm Cryopreservation Media With H2S Donors Enhances Sperm Quality, Reduces Oxidative Stress, and Improves in Vitro Fertilization Outcomes.” Scientific Reports 14, no. 1: 12400. 10.1038/s41598-024-62485-2. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Muiño‐Blanco, T. , Pérez‐Pé R., and Cebrián‐Pérez J.. 2008. “Seminal Plasma Proteins and Sperm Resistance to Stress.” Reproduction in Domestic Animals 43: 18–31. 10.1111/j.1439-0531.2008.01228. [DOI] [PubMed] [Google Scholar]
  31. Murawski, M. , Saczko J., Marcinkowska A., Chwiłkowska A., Gryboś M., and Banaś T.. 2007. “Evaluation of Superoxide Dismutase Activity and Its Impact on Semen Quality Parameters of Infertile Men.” Folia histochemica et cytobiologica 45, no. I: 123–126. [PubMed] [Google Scholar]
  32. Naderi, N. , Souri M., Nasr Esfahani M. H., Hajian M., and Tanhaei Vash N.. 2020. “Ferulago Angulata Extract Ameliorates Epididymal Sperm Toxicity in Mice Induced by Lead and Diazinon.” Andrology 8, no. 3: 706–718. 10.1111/andr.12730. [DOI] [PubMed] [Google Scholar]
  33. Naeini, F. , Zarezadeh M., Mohiti S., Tutunchi H., Ebrahimi Mamaghani M., and Ostadrahimi A.. 2021. “Spirulina Supplementation as an Adjuvant Therapy in Enhancement of Antioxidant Capacity: A Systematic Review and Meta‐Analysis of Controlled Clinical Trials.” International Journal of Clinical Practice 75, no. 10: e14618. 10.1111/ijcp.14618. [DOI] [PubMed] [Google Scholar]
  34. Najafi, A. , Zhandi M., Towhidi A., et al. 2013. “Trehalose and Glycerol Have a Dose‐Dependent Synergistic Effect on the Post‐thawing Quality of ram Semen Cryopreserved in a Soybean Lecithin‐based Extender.” Cryobiology 66, no. 3: 275–282. 10.1016/j.cryobiol.2013.03.002. [DOI] [PubMed] [Google Scholar]
  35. Patricio, A. , Cruz D. F., Silva J. V., et al. 2016. “Relation Between Seminal Quality and Oxidative Balance in Sperm Cells.” Acta Urológica Portuguesa 33, no. 1: 6–15. 10.1016/j.acup.2015.10.001. [DOI] [Google Scholar]
  36. Pintus, E. , and Ros‐Santaella J. L.. 2021. “Impact of Oxidative Stress on Male Reproduction in Domestic and Wild Animals.” Antioxidants 10, no. 7: 1154. 10.3390/antiox10071154. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Piri, M. , Mahdavi A. H., Hajian M., Nasr‐Esfahani M. H., Soltani L., and Vash N. T.. 2024. “Effects of Nano‐Berberine and Berberine Loaded on Green Synthesized Selenium Nanoparticles on Cryopreservation and in Vitro Fertilization of Goat Sperm.” Scientific Reports 14, no. 1: 24171. 10.1038/s41598-024-75792-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Putri, M. , and Budi D.. 2020. “Effect of Spirulina Platensis Supplementation in the Diet to Sperm Performance of Silver Rasbora (Rasbora argyrotaenia).” In Proceedings of the IOP Conference Series: Earth and Environmental Science , IOP Publishing. 10.1088/1755-1315/441/1/012041. [DOI] [Google Scholar]
  39. Qamar, A. Y. , Naveed M. I., Raza S., et al. 2022. “Role of Antioxidants in Fertility Preservation of Sperm—A Narrative Review.” Animal Bioscience 36, no. 3: 385. 10.5713/ab.22.0325. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Rahim, A. , Salhi S., El Khelfaoui N., Badaoui B., Essamadi A., and El Amiri B.. 2024. “Effect of C‐phycocyanin Purified From Spirulina platensis on Cooled Ram Semen Quality and in Vivo Fertility.” Theriogenology 215: 234–240. 10.1016/j.theriogenology.2023.12.007. [DOI] [PubMed] [Google Scholar]
  41. Revell, S. , and Mrode R.. 1994. “An Osmotic Resistance Test for Bovine Semen.” Animal Reproduction Science 36, no. 1–2: 77–86. 10.1016/0378-4320(94)90055-8. [DOI] [Google Scholar]
  42. Rickard, J. P. , Schmidt R. E., Maddison J. W., et al. 2016. “Variation in Seminal Plasma Alters the Ability of Ram Spermatozoa to Survive Cryopreservation.” Reproduction, Fertility and Development 28, no. 4: 516–523. 10.1071/RD14123. [DOI] [PubMed] [Google Scholar]
  43. Riesco, M. F. , Alvarez M., Anel‐Lopez L., et al. 2021. “Multiparametric Study of Antioxidant Effect on Ram Sperm Cryopreservation—From Field Trials to Research Bench.” Animals 11, no. 2: 283. 10.3390/ani11020283. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Sanocka, D. , and Kurpisz M.. 2004. “Reactive Oxygen Species and Sperm Cells.” Reproductive Biology and Endocrinology 2, no. 1: 12. 10.1186/1477-7827-2-12. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Schäfer, S. , and Holzmann A.. 2000. “The Use of Transmigration and Spermac™ Stain to Evaluate Epididymal Cat Spermatozoa.” Animal Reproduction Science 59, no. 3–4: 201–211. 10.1016/s0378-4320(00)00073-7. [DOI] [PubMed] [Google Scholar]
  46. Singleton, V. L. , Orthofer R., and Lamuela‑Raventós R. M.. 1999. “Analysis of Total Phenols and Other Oxidation Substrates and Antioxidants by Means of Folin‐Ciocalteu Reagent.” Methods in Enzymology 299: 152–178. 10.1016/S0076-6879(99)99017-1. [DOI] [Google Scholar]
  47. Suwimonteerabutr, J. , Yamsrikaew U., Damthongsen K., et al. 2024. “Improving the Quality of Chilled Semen From Thai Native Chicken Using Phosphorus and Vitamin B12 Supplementation in Semen Extender.” Poultry Science 103, no. 1: 103262. 10.1016/j.psj.2023.103262. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Tabatabaei Vakili, S. , Zeidi R., and Nabhani R.. 2025. “Protective Effect of B1 and B12 Vitamins on Post‐Thaw Sperm Quality Parameters and Seminal Plasma Antioxidant Status in Rams.” Veterinary Medicine and Science 11, no. 2: e70241. 10.1002/vms3.70241. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Üstüner, B. , Nur Z., Alçay S., Toker M. B., and Sağırkaya H.. 2014. “Effect of Egg Yolk and Soybean Lecithin on Tris‐based Extender in Post‐Thaw Ram Semen Quality and In Vitro Fertility.” Kafkas Üniversitesi Veteriner Fakültesi Dergisi 20, no. 3: 393–398. 10.9775/kvfd.2013.10248. [DOI] [Google Scholar]
  50. Van Tuan, H. , Son P. H., and Thuy P. T.. 2011. “A Study on Extraction and Determination of Some Properties of Spirulina Extracts.” Academia Journal of Biology 33, no. 4: 60–65. 10.15625/0866-7160/v33n4.783. [DOI] [Google Scholar]
  51. Wu, Q. , Liu L., Miron A., Klímová B., Wan D., and Kuča K.. 2016. “The Antioxidant, Immunomodulatory, and Anti‑Inflammatory Activities of Spirulina: An Overview.” Archives of Toxicology 90, no. 8: 1817‑1840. 10.1007/s00204-016-1744-5. [DOI] [PubMed] [Google Scholar]
  52. Yang, Y. , Du L., Hosokawa M., and Miyashita K.. 2020. “Spirulina Lipids Alleviate Oxidative Stress and Inflammation in Mice Fed a High‑Fat and High‑Sucrose Diet.” Marine Drugs 18, no. 3: 148. 10.3390/md18030148. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Zarei, F. , Kia H. D., Masoudi R., Moghaddam G., and Ebrahimi M.. 2021. “Supplementation of Ram's Semen Extender With Mito‐TEMPO I: Improvement in Quality Parameters and Reproductive Performance of Cooled‐Stored Semen.” Cryobiology 98: 215–218. 10.1016/j.cryobiol.2020.10.018. [DOI] [PubMed] [Google Scholar]
  54. Zhishen, J. , Mengcheng T., and Jianming W.. 1999. “The Determination of Flavonoid Contents in Mulberry and Their Scavenging Effects on Superoxide Radicals.” Food Chemistry 64, no. 4: 555–559. 10.1016/S0308-8146(98)00102-2. [DOI] [Google Scholar]

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.

Articles from Veterinary Medicine and Science are provided here courtesy of Wiley

RESOURCES