Research note: Effect of sample type, sperm concentration, and storage conditions on malondialdehyde levels in rooster semen

Abstract

Malondialdehyde (MDA) is a widely used biomarker of lipid peroxidation and oxidative stress in avian semen. However, inconsistencies in sample fraction, sperm concentration, and storage conditions complicate comparisons across studies. This study systematically evaluated how these factors influence MDA in rooster semen. In Experiment 1, pooled semen was diluted to 100, 150, 200, and 250 × 10⁶ spz/mL and fractionated into whole semen, seminal plasma, and sperm pellet before MDA quantification. In Experiment 2, the three fractions were stored at 4°C or -20°C and analyzed on Days 1, 3, and 7 (Day 0 baseline). In Experiment 1, both sperm concentration and sample fraction affected MDA (P < 0.05), with the highest values in sperm pellets, moderate in whole semen, and the lowest in seminal plasma. In Experiment 2, whole semen exhibited time-dependent increases at both temperatures, whereas seminal plasma and sperm pellets were comparatively stable. These findings indicate that sample origin and sperm density materially influence MDA readouts, and that storage particularly impacts whole semen. We recommend standardizing semen fraction and sperm concentration, and reporting storage conditions, to improve reproducibility of oxidative stress assessments in poultry reproduction.

Introduction

Accurate assessment of oxidative stress in rooster semen is crucial for evaluating sperm quality, predicting fertility outcomes, and refining assisted reproduction protocols. Lipid peroxidation (LPO), which damages sperm membranes and reduces fertilizing ability, is commonly quantified by measuring malondialdehyde (MDA), a stable by-product of polyunsaturated fatty acid degradation. The thiobarbituric acid reactive substances (TBARS) assay remains the most widely employed method for MDA quantification in animal reproduction research.

Despite its broad application, methodological inconsistencies limit the reliability and comparability of MDA data in avian semen studies. Different research groups have analyzed MDA in whole semen, seminal plasma, or sperm pellet fractions (Partyka et al., 2012; Authaida et al., 2024). yet systematic evaluation of how these sample origins influence oxidative stress measurements in poultry is lacking. This is critical because sperm pellets, rich in mitochondria and lipid membranes, are inherently more vulnerable to reactive oxygen species (ROS), whereas seminal plasma contains enzymatic antioxidants that may buffer oxidative damage. Without standardization, cross-study variation in sample fraction selection can lead to misleading conclusions about the oxidative status of avian semen.

Another overlooked factor is sperm concentration. In human studies, seminal MDA levels show variable associations with sperm density (Hsieh et al., 2006), but little is known about whether dilution levels in poultry semen influence oxidative stress profiles. Given that sperm density directly affects mitochondrial activity and ROS generation, ignoring this variable may obscure the true relationship between oxidative stress and semen quality.

Additionally, semen storage practices introduce further complexity. While some studies assess MDA immediately after collection, others measure it after refrigerated or frozen storage. In human plasma, MDA has been shown to accumulate during storage, even at subzero temperatures (Kumar et al., 2012). However, the stability of MDA in poultry semen across different semen fractions and storage protocols has not been systematically investigated. This gap raises concerns about whether observed oxidative stress reflects intrinsic sperm physiology or is influenced by storage conditions.

Accordingly, this study was designed to address these methodological gaps. Specifically, we aimed to (1) assess how sample fraction (whole semen, seminal plasma, and sperm pellet) and sperm concentration affect MDA levels, and (2) evaluate the effects of storage temperature (4°C and –20°C) and duration (1, 3, and 7 days) on MDA accumulation, using fresh samples as the reference control. By providing the first systematic comparison of these factors, this research offers critical insights for standardizing oxidative stress assessment in avian semen. Such standardization is essential to enhance reproducibility across laboratories and guide the design of antioxidant supplementation strategies in poultry reproduction research.

Materials and methods

Ethical approval

All experimental procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of Khon Kaen University (Record No. IACUC-KKU-64/68) and conducted in accordance with the National Research Council of Thailand’s guidelines for the ethical use of animals in research.

Experimental animals

Twenty-one 33-week-old native Thai roosters (Pradu Hang Dam) were housed individually in cages (60 × 45 × 45 cm) under an open-house system with natural daylight and temperature. They were fed a commercial diet at 100 g/day and had ad libitum access to clean water. Semen was collected twice weekly using the abdominal massage technique.

Semen collection and processing

Semen collection was conducted from individual roosters using the dorsal abdominal massage technique. Each ejaculate was immediately assessed for volume and transferred into a sterile 1.5 mL microtube containing 0.1 mL of IGGKPh diluent (Authaida et al., 2024) to prevent dehydration and preserve sperm viability during handling.

After collection, semen samples were maintained at 22-25°C and transported to the laboratory within 20 min. Upon arrival, each sample was evaluated for sperm concentration and motility. Sperm concentration was determined using a hemocytometer: 1 μL of semen was diluted in 999 μL of 4 % sodium chloride solution, and the diluted sample was counted under a compound microscope at 400 × magnification. Final values were expressed as sperm per milliliter (spz/mL).

Sperm motility was assessed using a mass movement scoring system on a scale of 0 to 5 (0 = no movement; 5 = vigorous swirling motion with >90 % motile sperm). A 5–10 μL drop of semen was placed directly on a microscope slide (without a coverslip) and examined at 100 × magnification (Olympus CH30, Tokyo, Japan).

Only ejaculates that met all of the following quality criteria were pooled to minimize individual variation: volume between 0.2 and 0.6 mL, sperm concentration ≥ 3 × 10⁹ spz/mL, and mass motility score > 3.5. The pooled semen was then aliquoted for experimental use.

Experimental design

A two-stage experimental design was implemented to investigate the effects of sperm concentration, sample type, and storage conditions on lipid peroxidation in rooster semen, as assessed by MDA levels (Fig. 1).

Fig. 1.

Experimental design. (A) Experiment 1: Semen was diluted to 4 concentrations (100, 150, 200, and 250 × 10⁶ spz/mL) and divided into whole semen, seminal plasma, and sperm pellet fractions by centrifugation. Lipid peroxidation was immediately assessed by TBARS assay. (B) Experiment 2: Semen diluted to 250 × 10⁶ spz/mL was fractionated as above and stored at 4°C or –20°C. Samples were analyzed by TBARS assay on Days 0, 1, 3, and 7.

Experiment 1: Effect of sperm concentration and sample type on lipid peroxidation

This experiment aimed to determine the effect of sperm concentration and sample type on MDA levels. Pooled rooster semen was diluted to four concentrations (100, 150, 200, and 250 × 10⁶ spz/mL) and separated into three sample types: whole semen, seminal plasma, and sperm pellet. MDA concentrations were quantified by the TBARS assay. The experiment was conducted in four independent replicates.

Experiment 2: Effect of storage conditions on MDA levels

This experiment evaluated the influence of storage temperature and duration on MDA levels. Semen was diluted to 250 × 10⁶ spz/mL to standardize sperm density across all samples since Experiment 1 demonstrated that sperm concentration affects MDA values. The diluted semen was then fractionated into whole semen, seminal plasma, and sperm pellet samples. Each sample was stored at 4°C (refrigeration) or −20°C (laboratory freezer), as these conditions reflect common short-term storage practices for semen fractions. Then MDA concentrations were analyzed on Days 1, 3, and 7, with Day 0 serving as the fresh control. The experiment was repeated four times.

Sample preparation

Whole semen samples were obtained as aliquots taken directly after dilution without centrifugation. To prepare seminal plasma, semen was centrifuged at 4,200 × g for 10 min at room temperature, and the supernatant was carefully collected. For sperm pellets, the fractions retained after centrifugation were washed three times with phosphate-buffered saline (PBS), with centrifugation after each wash, to minimize residual plasma contamination. Following the final centrifugation, each pellet was resuspended in deionized water. For consistency across all sample types, 250 µL of each fraction (whole semen, seminal plasma, and sperm pellet suspension) was used for subsequent lipid peroxidation analysis. The procedure was adapted from Partyka et al. (2012).

Lipid peroxidation assessment

MDA concentrations, as indicators of LPO, were measured using the TBARS assay. Briefly, 0.25 mL of 0.2 mM ferrous sulfate and 0.25 mL of 1 mM sodium ascorbate were added to each sample, followed by incubation at 37°C for 1 hour to promote peroxidation. Subsequently, 1 mL of 15 % (w/v) trichloroacetic acid and 1 mL of 0.375 % (w/v) thiobarbituric acid were added. The mixture was boiled at 100°C for 10 min, then rapidly cooled on ice (4°C), and centrifuged at 4,200 × g for 10 min. The MDA concentrations in the supernatants were quantified spectrophotometrically at 532 nm using a UV-1200 spectrophotometer (Shimadzu, Japan). Quality control was ensured in each assay by including calibration standards, blank controls, and replicate, with standard curves confirming the linearity of the TBARS assay (R² consistently >0.99).

Statistical analysis

Data were analyzed using SPSS version 28.0 (IBM Corp., Armonk, NY, USA). Experiment 1 was conducted as a 4 × 3 factorial design in a completely randomized design (CRD), with four replicates per treatment. The factors were sperm concentration (100, 150, 200, and 250 × 10⁶ spz/mL) and sample type (whole semen, seminal plasma, and sperm pellet). Data were subjected to analysis of variance (ANOVA), and treatment means were compared using Tukey’s post hoc test.

For Experiment 2, data were analyzed using a CRD with an augmented factorial arrangement to evaluate the effects of storage duration (Days 1, 3, and 7) and temperature (4°C and –20°C) on each sample type, using Day 0 as the baseline for comparison.

Results and discussion

Experiment 1: Effect of sperm concentration and sample type on lipid peroxidation

Analysis of variance revealed that both sperm concentration (P = 0.034; Fig. 2A1) and sample type (P < 0.001; Fig. 2A2) influenced MDA concentrations, whereas their interaction was not significant (P = 0.982; Fig. 2A3).

Fig. 2.

Effect of sperm concentration, sample type, and storage conditions on lipid peroxidation (MDA levels) in rooster semen. (A1) Effect of sperm concentration. (A2) Effect of sample type. (A3) Interaction between sperm concentration and sample type. (B1–B3) Effect of storage temperature (4°C and –20°C) and duration (Days 0, 1, 3, and 7) on MDA levels in whole semen (B1), seminal plasma (B2), and sperm pellet (B3). Values are means ± SEM. Different superscripts indicate significant differences (P < 0.05).

MDA levels increased progressively with sperm concentration, from 0.60 ± 0.11 µmol/mL at 100 × 10⁶ spz/mL to 0.95 ± 0.13 µmol/mL at 250 × 10⁶ spz/mL (Fig. 2A1). The highest concentration was significantly greater than the lowest (P < 0.05), confirming that denser semen suspensions exhibit greater oxidative stress, likely due to increased mitochondrial respiration and ROS generation.

These findings align with human studies reporting oxidative imbalance in high-density ejaculates (Patrício et al., 2016) and provide the first systematic evidence in poultry that sperm concentration must be considered when assessing oxidative biomarkers. From a practical standpoint, this suggests that antioxidant supplementation in semen extenders may need to be adjusted based on sperm density to maximize protective efficiency in artificial insemination (AI) programs.

Marked differences were also observed among sample fractions (Fig. 2A2). Sperm pellets showed the highest MDA concentrations (1.14 ± 0.12 µmol/mL), whole semen was intermediate (0.74 ± 0.06 µmol/mL), and seminal plasma exhibited the lowest values (0.41 ± 0.04 µmol/mL) (P < 0.05 for all comparisons). This confirms that spermatozoa are the primary site of lipid peroxidation, consistent with earlier findings in avian semen (Partyka et al., 2012). The elevated susceptibility of sperm pellets reflects their high content of polyunsaturated fatty acids and mitochondria, whereas seminal plasma appears to be partially protected by antioxidant enzymes (Partyka et al., 2010).

These results highlight that reliance on whole semen may obscure the true oxidative status, since cellular and plasma interactions continue during handling. Fractionated samples, particularly sperm pellets, therefore provide a more accurate and reproducible substrate for oxidative stress assessment in poultry semen research.

Experiment 2: Effect of storage conditions on MDA levels

The interaction between storage temperature and duration was significant only in whole semen (P < 0.05; Fig. 2B1). In contrast, neither the interaction nor the main factors of temperature and storage duration were significant in seminal plasma or sperm pellet (P > 0.05; Fig. 2B2-B3).

In whole semen (Fig. 2B1), MDA concentrations increased significantly over time at both storage temperatures (P < 0.001). This rise was observed as early as Day 1 under frozen conditions (–20°C), whereas at 4°C, lipid peroxidation levels remained stable until Day 3 before increasing. This pattern suggests that freezing triggers acute oxidative injury, likely due to the formation of ice crystals and associated membrane disruption during the critical cooling phase (0 to –10°C) (Check et al., 1994). In contrast, chilling induces a more gradual but progressive accumulation of lipid peroxidation. The heightened vulnerability of whole semen reflects the ongoing metabolic activity of spermatozoa and their enzymatic interactions with seminal plasma, which together generate a dynamic environment for continuous ROS production and oxidative damage.

MDA concentrations in seminal plasma (Fig. 2B2) and sperm pellets (Fig. 2B3) remained relatively stable throughout the 7-day storage period at both 4°C and –20°C (P > 0.05). This stability suggests that lipid peroxidation was substantially minimized or arrested in these fractionated components. This stability is likely due to two key factors. First, centrifugation used to separate the fractions may have caused immediate sperm cell death or metabolic arrest, particularly in the sperm pellet, thereby halting ROS generation at the mitochondrial level (Rijsselaere et al., 2002). Second, the removal of spermatozoa from seminal plasma eliminated the primary cellular source of reactive species and enzymatic pro-oxidants, leading to a relatively inert biochemical environment.

Although sperm pellets stored at –20°C showed a slight, non-significant rise in MDA (∼16 % by Day 7), this likely reflected residual peroxidation in membrane-compromised cells. Interestingly, seminal plasma exhibited remarkable oxidative stability at –20°C but showed a delayed increase at 4°C (∼41 % by Day 7). This unexpected rise may result from non-enzymatic oxidative degradation processes that proceed more readily at chilling temperatures, particularly affecting lipids and extracellular nucleic acids lacking antioxidant protection (Chari and Colagar, 2011; Zagoskin et al., 2017).

Taken together, these findings confirm that whole semen is far more susceptible to oxidative deterioration during storage than fractionated samples. Although the overall trend of increasing lipid peroxidation with prolonged storage and the mitigating effect of lower temperature were expected, the present study adds novel insight by demonstrating that these effects are strongly fraction-dependent. Whole semen showed pronounced vulnerability, whereas seminal plasma and sperm pellets remained comparatively stable. This fraction-specific response provides practical guidance, highlighting the need for greater caution when handling whole semen in oxidative stress assessments.

Collectively, this study demonstrates that both sperm concentration and sample origin are critical determinants of MDA levels in rooster semen. Sperm pellets exhibited the highest lipid peroxidation, while seminal plasma showed the lowest, confirming that spermatozoa are the primary physiological source of oxidative stress. Furthermore, storage at 4°C and –20°C induced progressive MDA accumulation in whole semen, whereas fractionated samples remained relatively stable, indicating that storage conditions further contribute to oxidative changes. In summary, both methodological factors (sample type and sperm concentration) and storage conditions (temperature and duration) strongly influence oxidative stress assessment. Standardizing these parameters is therefore essential to achieve reliable oxidative stress assessment in avian semen.

CRediT authorship contribution statement

Melada Chaithongsri: Writing – original draft, Methodology, Data curation. Kitiya Pachamon: Writing – original draft, Methodology. Thirawat Koedkanmark: Methodology, Data curation. Wuttigrai Boonkum: Writing – original draft, Formal analysis, Data curation. Monchai Duangjinda: Formal analysis. Vibuntita Chankitisakul: Writing – review & editing, Writing – original draft, Validation, Project administration, Funding acquisition, Conceptualization.

Disclosures

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.