2026 年 14 巻 4 号 p. 1-17
Cryopreservation is common in small ruminant breeding to conserve genetics and facilitate artificial insemination. On the other hand, oxidative stress occurring during the freezing and thawing process remains a major factor contributing to reduced sperm function and fertility. In this regard, the natural polyphenolic antioxidant agent resveratrol (RES) has been proposed as an additive to semen extenders. This study summarizes in vitro experimental studies assessing the impact of RES supplementation on sperm quality and oxidative status during semen cryopreservation (at −196 °C) and liquid storage at 5 °C in rams and bucks. According to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) 2020 checklist, ten peer-reviewed articles from 2010 to 2025 were selected for the analysis based on the experimental method, extender composition, RES dosage, preservation protocols, and measured parameters. The synthesis indicates that moderate RES concentrations (25–75 µM) are generally associated with improvements in total and progressive motility, plasma and acrosomal membrane integrity, and mitochondrial function, along with reductions in oxidative stress markers such as reactive oxygen species (ROS), malondialdehyde (MDA), and lipid peroxidation. The observed effects may be associated with enhanced cellular redox balance and mitochondrial function. Concentrations above 100 µM have been reported to exert potential adverse effects, suggesting a relatively narrow optimal dosage range. In general, the current data suggest that RES may improve sperm preservation efficiency. Nevertheless, additional standardized and in vivo studies are needed to verify its practical use in small ruminant production systems.
The combined success of artificial insemination (AI) and semen cryopreservation has made great breakthroughs in biotechnological reproduction [1]. These techniques are essential for genetic expansion and for the long-term storage and distribution of valuable livestock genetic materials [2, 3, 4]. In small ruminants, such as sheep and goats, artificial insemination and semen cryopreservation offer substantial economic and logistical advantages; however, these benefits are often constrained by technological challenges inherent to the cryopreservation process [5]. The “double-edged sword” of cryopreservation is the physical, osmotic, and biochemical injuries induced on the cells. These cryoinjuries are detrimental, affecting the sperm quality and fertilization potential due to poor motility, viability, and morphological integrity of the cryopreserved semen samples [6]. Sperm cells, especially ram and buck sperm, are the most vulnerable to the damaging effects of cryopreservation. In addition to poly unsaturated fatty acid (PUFA) components of membranes, the high cholesterol to phospholipid ratio is harmful [7]. The membranes’ cholesterol peroxidic ratio increases susceptibility to damaging lipid peroxidation (LPO) during cryopreservation. The freezing, thawing, and possible mitochondrial degeneration in hypoxic states induce the excess production of Reactive Oxygen Species (ROS). The oxidative state is detrimental, and it is a dominant state recognized [8].
Research that includes human beings as well as other animals such as bulls, boars, rabbits, and rams has highlighted positive outcomes by adding resveratrol while cryopreserving semen [9], e.g., enhanced total and progressive motility, preserved and improved plasma and acrosome membranes, decreased peroxidation and DNA fragmentation, and reduced acrosome loss [10]. In addition, it preserves and maintains mitochondrial ATP membrane potential, which is necessary for motility and fertilizing capacity [11]. However, despite these reported benefits, the existing literature remains inconsistent and heterogeneous, particularly with respect to optimal resveratrol concentrations, experimental conditions, and the magnitude of its protective effects. Some studies only report results at specific concentrations, which usually occur between 10 and 50 µM, while other research reported no results at all or at higher concentrations, which might be harmful due to possible pro-oxidant activity [12, 13].
For small ruminants, the existing literature also shows similar variability. Research on rams and bucks has showcased the differences species-wise due to breed, the composition of extenders (Tris, citrate, or egg-yolk based), cryopreservation processes, and individual semen quality [14]. Highly variable post-thaw parameters of evaluation in terms of motility, viability, acrosome status, and oxidative stress markers (malondialdehyde [MDA] and total antioxidant capacity [TAC], and antioxidant enzymes) have made direct comparisons across studies even more complicated [15]. Hence, in ruminant cryobiology, there remains an unclear understanding of extenders containing resveratrol [16].
There have been qualitative reviews on the potential functions of antioxidants against semen cryopreservation, but there exists no quantitative summary of evidence for resveratrol specifically in small ruminants [17, 18, 19]. A systematic review must be conducted to combine information across several independent studies on the magnitude of response associated with resveratrol exposure on major indicators of semen quality (motility, vitality) and to examine potential sources of heterogeneity among studies. In addition, the basic mechanism of resveratrol in mitigating the detrimental effects of oxidative stress during cryopreservation should be re-evaluated based on newer molecular research [20]. Resveratrol has been shown to modulate the nuclear factor erythroid 2–related factor 2–antioxidant response element (Nrf2–ARE) signaling between environmental stressors and organismal detoxification response, leading to increased expression of phase II detoxifying enzymes and enhanced mitochondrial antioxidant capacity [21]. In sperm, SIRT1 activation and downstream pathways may ultimately lead to increased expression of peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) and enhanced mitochondrial biogenesis and ATP production [22]. This and other similar biochemical pathways are critical for maintenance of motility and structural integrity through the freeze-thaw cycle [23]. Overall, exploration of such mechanisms in small ruminants would allow the development of novel strategies for reproductive biotechnology applied to preservation of genetic resources and artificial insemination programs targeted to areas of the tropics where oxidative stress presents a challenge [24, 25].
For studies evaluating the effects of resveratrol supplementation during semen cryopreservation in bucks or rams, the included studies were controlled in vitro experimental studies. As part of the intervention, resveratrol was supplemented into the semen extender at any concentration and formulation and had to be compared to a control group that did not receive a treatment or received a placebo or no antioxidant treatment. It also needed to provide quantitative measurements of at least one oxidative stress or sperm quality parameter (motility, viability, plasma membrane integrity, acrosome integrity, or biochemical markers, MDA, TAC, SOD, CAT, and GPx).
The inclusion criteria were defined a priori and applied consistently during the screening process, including: (i) studies conducted on ram or buck semen, (ii) evaluation of resveratrol as a single intervention, and (iii) reporting of extractable quantitative outcomes.
Reviews, conference abstracts, and dissertations without peer-reviewed primary data were not included. Studies involving cell lines, non-small ruminant species, or combined antioxidant treatments where the impact of resveratrol could not be separated were not included. In addition, studies with incomplete outcome reporting or no quantitative data that could be extracted were not included.
Exclusion criteria were also predefined, including studies lacking control groups, studies not involving cryopreservation (e.g., purely liquid storage without freezing context), and studies with insufficient methodological detail.
2.2 Methodology for literature searchesIn adherence to the PRISMA 2020, this study took the form of a systematic review [26]. Four scientific and scholarly databases (Web of Science, Scopus, PubMed, and Google Scholar) were searched and narrowed down to recent studies on utilizing antioxidants for the preservation of spermatozoa in small ruminants. The search covered studies from January 2010 up to March 2025.
A structured search strategy was applied using combinations of relevant keywords and Boolean operators, including (“resveratrol” AND “sperm” AND “cryopreservation” AND (“ram” OR “buck” OR “sheep” OR “goat”)). The search strategy was adapted for each database to maximize sensitivity and specificity.
In total, 86 records were located: 37 from the Web of Science, 34 from Scopus, and 15 from PubMed, while 2 were from Google Scholar but were not included as gray literature. After eliminating 22 duplicate records and 3 irrelevant records that surfaced through automated means, the preliminary screening process was conducted on the 61 records. Upon partial review of the remaining studies, it was determined that 14 studies were irrelevant or fell short of depth on cryopreservation. This decision was made based solely on the review titles and abstracts.
Title and abstract screening were performed systematically based on predefined eligibility criteria, followed by full-text assessment for final inclusion.
Of the 47 articles evaluated for full-text accessibility, there were 10 articles that could not be accessed due to access restrictions. Of the remaining 37 full-text articles eligible to be reviewed, 27 were excluded for the following reasons: 15 fell outside the scope of cryopreservation (studies on liquid storage or oxidative stress assays not relating to freezing) and 12 listed species were not related to ram and buck.
Ultimately, 10 studies were included in the qualitative synthesis and met all the inclusion criteria. Within these studies, sufficient data were extractable on at least one sperm functional parameter (motility, viability, or plasma membrane and acrosome integrity) or oxidative stress biomarker (MDA, SOD, CAT, GPx, TAC), allowing for a comparative analysis of resveratrol-supplemented semen samples compared with their respective control samples.
Due to the heterogeneity in study design, species, extender composition, and outcome measurements, a quantitative meta-analysis was not performed; instead, a structured qualitative synthesis was conducted by grouping findings into functional domains (motility, oxidative stress, mitochondrial function, and membrane integrity). PRISMA 2020 flow diagram of the literature search, screening, and selection of studies on the impact of resveratrol supplementation in semen cryopreservation in rams and bucks is shown in Figure 1.
The quality of the methodology and the risk of bias of the articles included in this systematic review were evaluated using a slightly modified version of the Systematic Review Center for Laboratory Animal Experimentation (SYRCLE) Risk of Bias (RoB) tool, which was specifically developed for experimental and in vitro animal research. Each study was assessed across standard domains including selection bias, performance bias, detection bias, and reporting bias, and categorized qualitatively (low, unclear, or high risk of bias). The assessment was based on the clarity and completeness of methodological reporting, including information related to sample allocation, semen handling procedures, outcome assessment, and data reporting. Because most included studies involved in vitro semen cryopreservation experiments, several SYRCLE domains were interpreted and adapted according to the experimental characteristics of reproductive biotechnology studies. The quality of the methodology of the 10 studies that eventually made it into the review was of a moderate to high quality. However, variability in reporting standards, lack of randomization, and limited replication in some studies were identified as potential sources of bias and are considered in the interpretation of results.
A qualitative synthesis was performed using 10 in vitro experimental studies that examined the impact of resveratrol (RES) administration during either cryopreservation or liquid storage of semen in small ruminants. The studies included ten using rams and six using bucks from different local and crossbred species such as Najdi, Santa Inês, Small-tailed Han, and Saanen goats. The ejaculates taken from healthy males with at least 70% motility before freezing were pooled for all studies to guarantee that the semen quality was similar across all treatments.
General characteristics of these studies, which cover the animal species, extender composition, cryopreservation protocols, RES treatment, and evaluation methods, are presented in Table 1.
| Study | Species / Breed | Sample (Ejaculates) | Extender | Cryopreservation / Storage Protocol | Resveratrol Concentration | Exposure Duration & Stage | Evaluation Methods | Main Parameters Evaluated |
|---|---|---|---|---|---|---|---|---|
| [5] | Buck | 50 ejaculates pooled from 4 bucks (n=50) | Optidyl® (Biovet, France) | Cooling to 5 °C (4 h) → vapor freezing | 0–250 µM | Added before freezing | CASA, flow cytometry | Motility, morphology, membrane & acrosome integrity, mitochondrial activity, ROS |
| [13] | Ram (Santa Inês) | 51 ejaculates pooled (n=51) | Tris–egg yolk + 5% glycerol | Cooling to 5 °C (2 h) → vapor freezing (10 min) | 0 or 50 µM | Post-thaw incubation (Talp-Hepes) | CASA, fluorescence microscopy, TBARS assay | Motility, capacitation status, mitochondrial activity, membrane integrity, TBARS, sperm–oocyte binding |
| [15] | Buck (Ankara) | 4 pooled samples (n=4) | Tris–citric–fructose + 20% EY + 5% glycerol | Cooling to 4 °C (3 h) → vapor freezing (15 min) | 500 µM | Added before freezing | Microscopy, fluorescence staining (SYBR-14/PI, JC-1) | Motility, membrane & acrosome integrity, mitochondrial activity |
| [27] | Buck (Saanen, Toggenburg, British Alpine) | 36 ejaculates pooled (n=36) | Skim milk-based extender + 7% glycerol | Automatic slow freezing → LN₂ | 0–100 µM | Added before freezing | CASA, microscopy | Kinematic parameters, membrane & acrosome integrity, morphology, oxidative stress |
| [28] | Ram (Small-tailed Han) | 3 adult rams (n=12) | OPTIXCELL® (IMV, France) | Cooling at 4 °C (4 h) → vapor freezing (8 min) | 15 µM | Before and after freezing | CASA, proteomics (LC-MS/MS), Western blot | Motility, kinematics, energy- and ROS-related proteins (DLD, NDUFB9) |
| [29] | Ram (Small-tailed Han) | 64 ejaculates pooled from 8 rams (n=64) | Tris–citric acid–fructose + 5% glycerol + 20% egg yolk | Cooling to 4 °C (3 h) → vapor freezing → LN₂ | 0–100 µM | Added before freezing | CASA, flow cytometry, biochemical assays | Motility, membrane & acrosome integrity, mitochondrial activity, ATP, antioxidant status (GSH, GPx, SOD, CAT), ROS, MDA |
| [30] | Buck (Kamori) | 32 ejaculates pooled from 4 bucks (n=32) | Tris–citric acid–egg yolk + 5% glycerol | Cooling (4 °C, 3 h) → vapor freezing (8 min) | 0–50 µM | Added before freezing | CASA, AO assay, biochemical assays | Motility, viability, acrosome & DNA integrity, antioxidants (SOD, CAT, TAC, TOS, MDA) |
| [31] | Ram | Pooled ejaculates (n=10) | Tris–egg yolk (15% EY) + 5% glycerol | Controlled cooling 0.2 °C/min → 5 °C; vapor freezing | 10, 20, 40 µg/mL | Pre-dilution before freezing | CASA, HOST, fluorescence microscopy, flow cytometry | Motility, capacitation status, membrane & acrosome integrity, mitochondrial and ROS levels |
| [32] | Ram (Najdi) | 30 ejaculates pooled (n=30) | Triladyl® + 20% clear egg yolk | Gradual cooling to 4 °C, stored up to 15 days | 0, 200, 400 µM | During liquid storage | CASA, microscopy, thermal incubation test | Total/progressive motility, viability, membrane & acrosome integrity, longevity |
| [33] | Ram (Najdi) | 30 ejaculates pooled from 3 rams (n=30) | Triladyl® (Tris, citric acid, sugar, glycerol) + 20% egg yolk | Liquid storage at 5 °C for 168 h | 0, 200, 400 µM | During cooling storage | CASA, SEM, biochemical assays | Motility, kinematics, morphology, oxidative biomarkers (MDA, SOD, GSH), IVF outcomes |
Most studies used Tris-based egg-yolk (TEY) or Triladyl® extenders with glycerol (5–7%) supplemented as a cryoprotectant. In addition to the above, a number of studies carried out the use of commercial formulations such as Optidyl® or OPTIXCELL® (Biovet, France; IMV, France), while only one research work opted for skim milk–based media [27]. The cryopreservation procedures of the studies were mostly the same and included cooling down to 4–5 °C, 2–4 hours of equilibration, followed by freezing in nitrogen vapor (−120 °C) before going to liquid nitrogen (−196 °C). Generally, thawing was performed at 37 °C for a period of 20–30 s.
The resveratrol concentrations differed greatly in the different studies, with one study reporting 10 µM and another 500 µM or 10–40 µg/mL in those using mass-based units. Most studies used resveratrol before freezing, during dilution or equilibration, whereas a few looked into post-thaw supplementation [13].
The evaluation approaches used for specimens included computer-assisted sperm analysis (CASA) mainly for motility and kinematic parameters, fluorescence microscopy and flow cytometry for assessing the integrity of the acrosome and plasma membrane, and biochemical assays for oxidative stress and antioxidant markers, including malondialdehyde (MDA), superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), total antioxidant capacity (TAC), and reduced glutathione (GSH). A few studies employed proteomic and mitochondrial analyses to evaluate the molecular mechanisms behind the antioxidant effect of resveratrol [28, 29].
3.2 Effects of resveratrol on sperm motility and kinematic parametersThe application of RES across all the studies performed on post-thaw sperm of both rams and bucks resulted in a uniform and observable enhancement of motility and motion kinematics. The magnitude of these effects, however, was significantly linked to the dose given, species, the composition of the semen extender, and the conditions of storage. The main findings about the motility and kinematic parameters in the different studies are shown in Table 2.
In the case of Small-tailed Han sheep, it was observed that 50 µM and 75 µM RES supplementation consistently led to significant increases in total motility (TM) and progressive motility (PM) relative to the untreated controls [29]. A similar trend of enhanced motility has been documented for Kamori bucks, whereby 40 µM and 50 µM RES resulted in the highest motility values [30]. On the other hand, when RES was applied alone at 20 µg/mL to Kivircik rams, motility was not significantly affected, hinting at the possibility that the reaction to RES may be influenced by breed-specific membrane characteristics or extender composition [31].
Evaluation of velocity attributes produced by CASA revealed that among Small-tailed Han rams, RES at 50 µM after thawing led to the highest curvilinear velocity (VCL), straight-line velocity (VSL), and average path velocity (VAP) [29]. Nevertheless, in the case of Santa Inês rams, kinematic parameters post-thawing did not improve with the use of 50 µM RES, thus indicating that the effect of RES exposure timing and different breeds may affect the degree of kinematic response [13].
When it comes to stability of motion pattern, both quercetin (100 µM) and very high levels of RES significantly diminished the oscillation index (wobble, WOB) in frozen–thawed sperm from bucks. This change has been linked to enhanced fertilization potential due to more linear and controlled flagellar movement [27]. On the other hand, RES at 50 µM and 75 µM led to a significant increase in WOB values in Small-tailed Han rams, indicating better kinematic adjustment under ideal antioxidant dosing [29].
Interestingly, post-thaw motility peaked at 61 ± 1.0% in Angora buck semen when RES was used in conjunction with catalase (CATR, 500 µM/mL), exceeding the results of either antioxidant alone [15]. This evidence is in line with the notion that combined antioxidant regimens have a better protective effect than single-compound supplementation in maintaining sperm motility during cryopreservation.
| Parameter | Key Findings (Focus on Resveratrol Effects) | Source |
|---|---|---|
| Total Motility (TM) & Progressive Motility (PM) | Resveratrol (RES) at 50 µM and 75 µM significantly improved total and progressive motility in Small-tailed Han rams. Similarly, concentrations of 40 µM and 50 µM yielded the highest motility in Kamori bucks. However, in Kivircik rams, RES at 20 µg/mL alone did not produce a significant effect. | [29, 30, 31] |
| Velocity Parameters (VCL, VSL, VAP) | RES at 50 µM produced the highest curvilinear (VCL), straight-line (VSL), and average path (VAP) velocities in Small-tailed Han rams after thawing. Conversely, post-thaw supplementation with 50 µM RES in Santa Inês rams showed no improvement in kinematic parameters. | [13, 29] |
| Wobble (WOB) | High concentrations of RES or quercetin (100 µM) reduced the oscillation index (WOB) in frozen–thawed buck sperm, which is considered beneficial for fertility. RES at 50 µM and 75 µM also significantly increased WOB in Small-tailed Han rams. | [27, 29] |
| Optimal Combination | The combination of Catalase + RES (CATR, 500 µM/mL) achieved the highest post-thaw motility (61 ± 1.0%) in Angora bucks, outperforming either antioxidant used alone. | [15] |
Altogether, Table 2 reveals that RES addition at a medium concentration range (roughly 40–75 µM) works best to enhance sperm motility and CASA-derived kinematic parameters in small ruminants, whereas outside of this concentration range, effects might be marginal or even non-existent. These results emphasize that dosing and experimental conditions determine the success of RES use in semen extenders for cryopreservation.
3.3 Effects of resveratrol on the membrane, acrosome, and DNA integrity of spermSupplementation with resveratrol (RES) markedly affected the integrity of the sperm structure, including the plasma membrane, acrosome, and nuclear DNA integrity. These parameters are considered key factors for sperm function and competence post‐cryopreservation, with membrane and acrosome integrity being important prerequisites to fertilize an oocyte and DNA integrity being related to the genetic quality of spermatozoa. A summary of the central results concerning the membrane and DNA integrity is shown in Table 3.
| Parameter | Key Findings (Focus on RES and CLC) | Sources |
|---|---|---|
| Plasma Membrane Integrity (PMI) | Either CLC alone or the combination of RES + CLC significantly improved post-thaw plasma membrane integrity in Kivircik rams compared to controls, while RES alone did not show a significant effect. | [31] |
| Acrosome Integrity (AI) | Similar to PMI, both CLC and RES + CLC markedly enhanced acrosome integrity. In addition, 50 µM RES improved AI in Small-tailed Han rams. | [29, 31] |
| DNA Integrity (TUNEL / 8-OHdG) | RES at 50 µM and 75 µM significantly reduced oxidative DNA damage (8-OHdG) in Small-tailed Han rams. In Sarda bucks, 50 µM RES also decreased early apoptotic cells as detected by the TUNEL assay. | [29, 30] |
| Premature Capacitation (F Pattern) | CLC supplementation effectively minimized membrane damage and reduced premature capacitation (F pattern) in ram spermatozoa, indicating improved membrane stability during freezing–thawing. | [31] |
Plasma membrane integrity was one of the most commonly reported parameters in the included studies. Post-thaw PMI in Kivircik rams was remarkably increased by treatment with CLC and/or RES vs untreated control [31]. The absence of this effect when RES was applied by itself points out that CLCs are key facilitators in keeping the lipid bilayer intact through freezing and thawing processes. The working of cholesterol and RES is like that of partners: cholesterol works through the membrane, making it less fluid and also preventing transitions from one phase to another at low temperatures, whereas RES is the champion that gets rid of the reactive oxygen species, thus stopping lipid peroxidation. This is consistent with the evidence that has been accumulating over the years that the use of cyclodextrins, which are loaded with cholesterol, can reduce cold-shock injury and enhance the lifespan of sperm. The combination of RES and CLC provides a double protection: the physicochemical stabilization by cholesterol and the biochemical antioxidant defense by RES, thus allowing the membrane to be optimal during the cryopreservation process.
The supplementation of CLC and RES + CLC also had large improvements in acrosomal membrane integrity, as it houses the enzymatic machinery required to penetrate the zona pellucida [31]. A significant increase in acrosome integrity in Small-tailed Han rams was observed following supplementation with 50 µM resveratrol compared to the control group [29]. Together, these results show that resveratrol, in particular, helps to maintain acrosome structure and enzymatic capacity during cryo-injury.
The acrosomal membrane, which has a high content of polyunsaturated fatty acids (PUFAs), is quite susceptible to oxidative damage. The ability of resveratrol to enhance total antioxidant capacity (TAC) and to eliminate reactive oxygen species, for instance, superoxide and hydroxyl radicals, safeguards this membrane. The noted improvement in acrosome integrity (AI) aligns with the increased post-thaw fertilization rates found in various research studies, which accentuates its importance regarding function. The slightly better stabilizing effect was noted when resveratrol was used together with cholesterol-loaded cyclodextrin (CLC), thus reinforcing the view that the combination of lipid conditioning with antioxidant supplementation gives the best safeguard for organelles with membranes. Oxidative stress caused by cryopreservation usually results in the fragmentation of nuclear DNA and the formation of oxidative base modifications, especially the production of 8-hydroxy-2’deoxyguanosine (8-OHdG). A few studies have provided evidence that the addition of resveratrol mitigates oxidative damage. The addition of 50 or 75 µM resveratrol to frozen– thawed Small-tailed Han ram sperm significantly reduced 8-hydroxy-2’-deoxyguanosine (8OHdG) levels [29]. In addition, the previously mentioned concentrations promoted optimal sperm motility and function in the fertilization mitochondria, thereby increasing overall sperm vitality.
In Sarda bucks, supplementation with 50 µM resveratrol significantly reduced the proportion of early apoptotic spermatozoa, as determined by the TUNEL assay [30]. This suggests that resveratrol can help maintain the integrity of sperm DNA, attributable not only to the overall reduction of oxidative base damage but also to the stabilization of a programmed death pathway, induced by faulty mitochondria. These positive effects are probably increased when resveratrol is combined with cholesterol-rich lipids.
The results reflect that the antioxidant and anti-apoptotic properties of resveratrol allowed the sperm DNA to remain stable under cryogenic stress, which, in turn, provided the opportunities for successful fertilization as well as normal embryo development. The combined supplementation of cholesterol-loaded cyclodextrin (CLC) and resveratrol significantly improved the cryosurvival of ram spermatozoa [31]. Moreover, the combination of resveratrol and cholesterol-loaded cyclodextrin (RES + CLC) prevents premature capacitation (F pattern) through the regulation of membrane cholesterol balance and the reduction of oxidative stress, thus enhancing sperm viability and facilitating the normal timing of fertilization. Both rams and bucks show comparable positive effects on DNA and acrosome integrity, although the choice of the extender and the species-specific differences in membrane lipid composition determine the most effective RES concentration. Buck sperm, characterized by a higher content of saturated lipids, is more resistant to oxidative stress but less fluid, which may account for their slightly different response to resveratrol.
3.4 Effects of resveratrol on oxidative stress, antioxidant enzymes, and molecular energy regulationCryopreservation leads to large amounts of reactive oxygen species (ROS) being produced, which in turn causes the breakdown of lipids, dysfunction of mitochondria, and damage to DNA thus, reducing the fertilization capacity. The present review reveals that resveratrol (RES), a strong natural polyphenol, has the ability to reduce oxidative stress effectively, boost the antioxidant enzyme defenses, and facilitate the mitochondrial energy balance in the semen of small ruminants (Table 4).
| Parameter | Key Findings (Focus on RES) | Sources |
|---|---|---|
| Oxidative Stress Markers (ROS, LPO, MDA) | 50 µM RES was the most effective concentration in reducing reactive oxygen species (ROS), lipid peroxidation (LPO), and malondialdehyde (MDA) levels in Small-tailed Han rams. In liquid-stored Najdi rams, 400 µM RES also decreased MDA levels. Conversely, in Kivircik rams, 20 µg/mL RES led to higher intracellular ROS compared with other groups, indicating dose-dependent cytotoxicity. | [29, 31, 23, 33] |
| Antioxidant Enzymes (SOD, GPx, CAT, GSH) | 50–75 µM RES enhanced the activities of scavenging enzymes, including superoxide dismutase (SOD), glutathione peroxidase (GPx), and catalase (CAT), while increasing intracellular glutathione (GSH) levels. In liquid-stored semen of Najdi rams, RES also elevated SOD and GSH activity, demonstrating antioxidant reinforcement across preservation systems. | [32, 33] |
| Total Antioxidant Capacity (TAC) | 40–50 µM RES significantly increased total antioxidant capacity (TAC) in Kamori bucks, confirming its role as an effective enhancer of overall redox balance. | [30] |
| Mitochondrial Activity (ΔΨm, ATP Content) | 50–75 µM RES improved mitochondrial membrane potential (hMMP) and adenosine triphosphate (ATP) content in Small-tailed Han rams post-thaw. A synergistic effect of RES + CLC was observed in Kivircik rams, further boosting mitochondrial function. | [29, 31] |
| Energy Regulation (NAD⁺/NADH Ratio) | 75 µM RES significantly elevated cellular NAD⁺ content and lowered the NADH/NAD⁺ ratio, reflecting improved oxidative phosphorylation efficiency and redox homeostasis. | [29] |
| Key Mitochondrial Proteins (DLD & NDUFB9) | Pre-freeze addition of RES upregulated DLD (dihydrolipoamide dehydrogenase) and NDUFB9 (NADH dehydrogenase 1 beta subcomplex 9) in ram spermatozoa. These proteins are involved in mitochondrial energy metabolism and the reduction of ROS generation from Complex I. | [28] |
| Signaling Pathways (SIRT1 / AMPK Axis) | RES at 25–75 µM enhanced SIRT1 expression and promoted AMPK phosphorylation (p-AMPK) in ram sperm, indicating activation of the SIRT1–AMPK pathway that mediates antioxidant defense and energy homeostasis under cryostress. | [28, 29] |
A clear result was found in most of the investigations that oxidative stress markers such as ROS, lipid peroxidation (LPO), and malondialdehyde (MDA) were significantly reduced when RES was used. Notably, supplementation with 50 µM resveratrol was sufficient to suppress reactive oxygen species generation and lipid peroxidation in frozen–thawed Small-tailed Han ram spermatozoa [29]. Thus, resveratrol, with its characteristics of free radical counteraction and lipid peroxide arresting, serves as a key element on the surfaces of both the plasma and the mitochondria to secure them from further damage.
However, the effects of higher RES concentrations appear to depend on the preservation system and experimental conditions. While concentrations of 200–400 µM were tolerated during liquid storage in Najdi ram semen, higher concentrations under cryopreservation conditions were associated with oxidative imbalance and possible cytotoxic effects [32, 33]. Their study demonstrated that upon exposure to 100 µg/mL RES, Kivircik rams’ intracellular ROS levels were lower than in the other groups; thus, the authors concluded that the decline in ROS occurred only in this group because of the whispers of potential pro-oxidant or cytotoxic effects.
Besides, resveratrol directly confronting ROS also triggers an enzyme-based antioxidant system existing within the sperm. The evidence suggests that there was an undoubtedly increased number of enzymes by 50–75 µM RES in the intracellular environment, an increase that came with the coordinated effects of the enzymes and glutathione [29, 32, 33]. The collaborative action upregulates the cell’s ability to nullify the generated products during cryopreservation. The most notable outcome of both SOD and CAT activity together in the liquid-stored Najdi rams spoke is the fact that resveratrol could serve as a ROS scavenger and also a stimulus of gene expression that eventually led to the triggering of antioxidant genes via Nrf2-dependent pathways. The genetic modification of some cells revitalizes their ability to protect, and therefore, oxidative stress is alleviated even when resveratrol is metabolized or diluted in the extender.
Furthermore, supplementation with 40–50 µM resveratrol significantly increased the total antioxidant capacity (TAC) in Kamori buck semen [30]. This thus confirms that it acts as a global redox regulator. The current findings thus imply that the proposed biological activity of resveratrol lies in the overall antioxidant scavenging capacity of the compound itself. In addition, it may act as an activator of the endogenous antioxidant system of the sperm.
The antioxidant properties of resveratrol also exist within the mitochondrial compartment, which acts as both a primary source and target of ROS in cryoinjury. Mitochondria play a central role in the motility and survival of sperm cells as the ultimate step of providing adenosine triphosphate (ATP) for flagellar movement. Supplementation with 50–75 µM resveratrol increased mitochondrial membrane potential (ΔΨm) and ATP content in post-thaw Small-tailed Han ram spermatozoa, indicating improved preservation of mitochondrial structure and function during the freeze–thaw process [29].
Synergistic effects were observed in Kivircik rams when resveratrol was combined with cholesterol-loaded cyclodextrin (CLC; a cholesterol-enriched cyclodextrin formulation, Cyclomen), enhancing sperm cryotolerance [31]. The total effect of both substances showed that there was a cumulative increase in mitochondrial activity above that of RES alone. This possibly indicated an increased stabilization of the mitochondrial membrane due to the cholesterol supplementation that could reduce ROS escape from the electron transport chain.
In addition, resveratrol can modify important molecular factors that regulate cell metabolism. At a concentration of 75 µM, RES showed a substantial increase in nicotinamide adenine dinucleotide (NAD⁺) levels and significantly reduced the ratio of nicotinamide adenine dinucleotide reduced form to nicotinamide adenine dinucleotide oxidized form (NADH/NAD⁺) [29]. The increase in the levels of the oxidized form of nicotinamide adenine dinucleotide may contribute to efficient oxidative phosphorylation and reduced leakage of electrons that generate ROS. The increased levels of the oxidized form of nicotinamide adenine dinucleotide also affect the activity of SIRT1.
Proteomic analysis suggested that pre-freeze supplementation with resveratrol was associated with increased expression of dihydrolipoamide dehydrogenase (DLD) and NADH dehydrogenase 1 beta subcomplex 9 (NDUFB9), both of which are essential components of the mitochondrial electron transport chain [28]. In terms of functionality, DLD assists in ATP synthesis and energy transport as part of the pyruvate dehydrogenase complex.
Based on currently available evidence, resveratrol has been suggested to modulate the SIRT1– AMPK signaling pathway. For example, supplementation with 25–75 µM resveratrol increased the expression of sirtuin 1 (SIRT1) and phosphorylated AMP-activated protein kinase (pAMPK) in ram spermatozoa [28, 29]. Modulation of this signaling pathway may contribute to mitochondrial biogenesis and reduced cellular damage through improved glucose and fatty acid metabolism.
The SIRT1/AMPK signaling route may be involved in the cellular response to resveratrol supplementation, potentially contributing to improved sperm resilience under cryogenic stress. The combined antioxidant and metabolic regulatory properties of resveratrol may contribute to reductions in ROS and MDA levels, as well as an increase in the activity of SOD, CAT, and GPx and the preservation of mitochondrial function. The maximum efficacy is at 50–75 µM, while concentrations above 100 µM lead to oxidative stress imbalance. The observed upregulation of DLD and NDUFB9 proteins following RES supplementation may be associated with improved ATP production and reduced ROS leakage, and this, in turn, keeps sperm quality in terms of motility, membrane integrity, and DNA stability.
The collected evidence presented in this review suggests that resveratrol (RES) can be effective in mitigating cryo-induced damage in spermatozoa, likely through its direct antioxidant activity and indirect metabolic regulation [27, 29, 34]. In small-ruminant research, RES has been associated with improvements in key post-thaw functional parameters, including total and progressive motility, membrane and acrosome integrity, mitochondrial activity, and biochemical markers of oxidative stress. However, the magnitude and consistency of these benefits are strongly dependent on resveratrol concentration, semen preservation conditions (liquid storage versus cryopreservation), and extender composition, indicating a clear dose- and context-specific response.
Our findings in rams and bucks are broadly consistent with observations reported in other mammalian species (boar, bull, and human), suggesting a potentially conserved biological role of RES in male gametes, although species-specific responses remain evident [15, 35, 36, 37]. Evidence synthesized from both species-specific and cross-species studies further indicates that moderate resveratrol concentrations are generally associated with improvements in sperm functional parameters and reductions in oxidative stress markers, thereby supporting the biological plausibility of these effects beyond the small-ruminant context [29].
The data converge mechanistically to two complementary modes of action. First, RES as a free-radical scavenger and enhancer of endogenous antioxidant defense systems: in numerous studies, it has been reported to reduce ROS and MDA levels while increasing antioxidant enzyme activities such as SOD, CAT, GPx, and TAC [38, 39, 40, 41]. Such biochemical modulation may contribute to reduced lipid peroxidation and improved membrane stability across different extenders and breeds. Second, RES appears to support mitochondrial bioenergetics, including mitochondrial membrane potential, ATP levels, and the expression of electron transport chain-related proteins (DLD, NDUFB9), as well as possible modulation of the SIRT1–AMPK signaling axis involved in redox balance and energy metabolism. These combined antioxidant and metabolic effects may partly explain the concurrent improvements in sperm motility and kinematic parameters observed in several studies [29, 31].
RES shows a biphasic dose-response pattern, in which low to moderate amounts are beneficial while high amounts become either inactive or harmful. Such a scenario is seen across different species like rams, bucks, boars, and even humans, highlighting the importance of dose optimization under specific experimental conditions. Sperm cells appear to tolerate higher RES concentrations (200–400 µM) under liquid storage conditions, whereas lower concentrations (25–75 µM) are more suitable during cryopreservation due to increased oxidative and osmotic stress during freezing–thawing [29, 34, 42, 43, 44]. Importantly, the potentially adverse effects of high RES concentrations were reported mainly under cryopreservation conditions, while higher concentrations appeared to be better tolerated during liquid storage at 5 °C. Moreover, the addition of RES during equilibration instead of post-thaw has been associated with more consistent protective effects, supporting the concept of pre-conditioning prior to cryo-stress [45, 46]. The use of RES in combination with cholesterol-loaded cyclodextrin (CLC) may further enhance membrane stability and reduce lipid peroxidation, although such combined approaches require further validation [47, 48, 49, 50]. The proposed mechanisms underlying the protective effects of resveratrol on sperm cryotolerance are summarized schematically in Figure 2.
The findings presented in this review contribute to the current understanding of sperm cryobiology, particularly regarding the interaction between oxidative stress regulation, mitochondrial function, and membrane stability. The protective effects of administering moderate doses of RES are consistent with the notion that cryo-injury is not merely the result of physical damage (e.g., ice formation and osmotic stress) but also of oxidative and metabolic disruptions.
At the molecular level, the observed changes in the SIRT1–AMPK signaling pathway and mitochondrial proteins (e.g., DLD, NDUFB9) suggest that resveratrol may exert both antioxidant and metabolic regulatory effects, thereby possibly increasing the cells’ ability to withstand the freezing-thawing process [28, 29]. Thus, the idea of using antioxidants can be broadened to encompass the combined regulation of redox balance, energy metabolism, and membrane integrity [51, 52, 53, 54, 55].
From an applied agricultural perspective, RES supplementation may represent a promising strategy for improving semen preservation in small ruminant breeding programs. The increased quality of sperm after freezing could, in theory, lead to higher efficiency in artificial insemination (AI), better fertility results, and easier spread of top genetics. Nevertheless, practical use of RES will depend on the right concentration, choice of extender, and timing of giving the supplement in animal production settings. Besides, the issue of cost and how easily it can fit into industrial livestock production should be considered before making it widespread.
4.3 Study limitationsThe positive impact of resveratrol (RES) has been frequently reported, yet there are a number of limitations that impede the interpretation and generalization of the results. One of the major differences among the studies is the animal breed, their age, diet, and even the extender composition (for instance, whether Tris-based or commercial formulations are used), which in turn may affect both the semen quality and RES absorption. Moreover, variations in cryopreservation procedures - for example, equilibration time, freezing rates, and post-thaw handling could also be a source of variability in oxidative stress levels and sperm responses. Another weakness of the methodology was most likely insufficient sample size justification, lack of randomization, and no replication in some studies, which could lower the strength of the reported results and also introduce bias. Besides, the combining of ejaculates for certain trials may hide the level of variation of each ejaculate, which is particularly important in male reproductive biology.
Besides, the majority of studies used in this review have mainly depended on in vitro parameters such as sperm motility, viability, and oxidative biomarkers; to the best of their knowledge, the evidence linking these parameters to in vivo fertility outcomes (e.g. conception or pregnancy rates) is limited [56]. Standard semen quality determinations, including concentration, morphology, and motility, have resulted in unreliable correlations with actual reproductive performance [57, 58]. Hence, future studies should include functional, molecular, and in vivo fertility data to more precisely determine the practical importance of RES supplementation in livestock reproduction systems [56, 59].
This systematic review suggests that resveratrol (RES) may provide protective effects on spermatozoa during cryopreservation and liquid storage in small ruminants, mainly through its antioxidant and metabolic regulatory properties. It can be concluded from the ten in vitro studies that were reviewed that RES at moderate concentrations (25–75 µM) was broadly found to be effective in enhancing post-thaw motility, plasma membrane and acrosome integrity, mitochondrial activity, and the activity of antioxidant enzymes, while at the same time reducing oxidative stress markers such as reactive oxygen species, malondialdehyde, and lipid peroxidation. At the mechanistic level, RES appears to exert its effects through possible modulation of the SIRT1–AMPK pathway and modulation of mitochondrial proteins such as NDUFB9, suggesting a role in maintaining cellular energy and redox balance rather than acting solely as a direct free radical scavenger. The study has shown that, depending on the dose, the response could be biphasic, such that good effects were seen at low to moderate concentrations, whereas negative or less pronounced effects might be found at higher concentrations (>100 µM). Different optimal concentrations for liquid storage and cryopreservation also highlight the need for RES supplementation in a context-specific manner.
From an applied agricultural perspective, the above-mentioned findings suggest that RES may represent a promising additive in semen extenders to improve the efficiency of semen preservation in small ruminant breeding programs, especially considering artificial insemination and conservation of genetic resources. However, given the limited number of studies and their methodological variability, further standardized and in vivo investigations are required before definitive recommendations can be made for routine application.
Ragil Angga Prastiya: Conceptualization, Methodology, Formal analysis, Data curation, Writing - original draft. Trilas Sardjito: Methodology, Validation, Supervision, Writing - review & editing. Amung Logam Saputro: Investigation, Data curation, Visualization. Rico Anggriawan: Investigation, Data curation, Visualization. Samira Musa Sasi: Formal analysis, Investigation, Writing - review & editing. Nagia Musa Alghoul: Formal analysis, Resources, Writing - review & editing, Validation.
This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.
The authors would like to express their sincere appreciation to their colleagues at the Faculty of Health, Medicine and Life Sciences, Universitas Airlangga, Indonesia, for their valuable insights and discussions during the preparation of this manuscript.
The authors declare no conflict of interest.
