Title & Abstract

International Journal of Anatomy and Research

Effect of Selenium and Green-Synthesized Silver Nanoparticles in Embryo Culture Media on Oxidative Stress Attenuation and Developmental Competence: A Histological Study

DOI: 10.16965/ijar.2026.180

Noori, Noor ¹

ABSTRACT

Background.

Developmental competence is impaired by the formation of reactive oxygen species (ROS) in in-vitro embryo culture, which results in cytological injury that can be easily detected using histological analysis. Antioxidant supplementation with nanoparticles has become an appealing approach to overcome this oxidative load.

Aims.

To assess the impact of selenium nanoparticles (SeNPs) and green-synthesized silver nanoparticles (G-AgNPs), when used alone and in combination, on oxidative stress biomarkers, developmental progression, and blastocyst histoarchitecture of murine embryos in culture.

Methods.

Two-cell embryos (n=480) were randomly allocated to four groups: control (KSOM only), KSOM + 5 µg/mL SeNPs, KSOM + 2 µg/mL G-AgNPs (synthesized using an aqueous green-tea leaf extract), or KSOM containing both nanoparticles. Developmental endpoints were assessed at 48, 72, 96, and 120 h. Oxidative stress biomarkers (MDA, SOD, CAT, GPx, GSH) were measured in embryo lysates. Blastocysts were subjected to routine H&E, PAS, TUNEL, and cleaved-caspase-3 immunohistochemistry.

Results.

The highest rates of blastocyst (64.7%) and hatched-blastocyst (34.5%), a 56% malondialdehyde reduction, and 1.7-2.4-fold increases in antioxidant enzyme activities were observed with combined SeNPs + G-AgNPs supplementation compared to control (p<0.001). Histologically, the composite group had a well-defined inner cell mass (ICM), organized trophectoderm, minimal cytoplasmic vacuolation, and a 3.4-fold reduced TUNEL-positive nuclear count compared to controls. The correlation between lipid-peroxidation level and histological injury score was strong and positive (r=0.82, p<0.001).

Conclusion.

Co-supplementation of SeNPs and G-AgNPs in embryo culture media maintains redox homeostasis and enhances blastocyst histoarchitecture, warranting further application in translational murine embryo production methods.

Keywords: Selenium Nanoparticles; Green Silver Nanoparticles; Embryo Culture; Oxidative Stress; Blastocyst; Histology; In-Vitro Embryo Production

INTRODUCTION

The in-vitro production of embryos (IVP) has been a revolution in the field of reproductive medicine, animal breeding and conservation biology. In vitro cultured embryos are less viable, have altered gene expression and have impaired implantation capacity when compared to embryos born in vivo, although they have become a common method [1,2]. This is mainly attributed to the increased and persistent generation of ROS in the presence of supraphysiological oxygen tension, exposure to ambient light and/or exposure to non-filtered visible range radiation of inverted microscopes, as well as the absence of antioxidant protection in chemically defined culture media.

The early cleavage embryo relies on antioxidant enzymes, such as superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) for redox homeostasis. These reserves are rapidly exhausted during the onset of embryonic genome activation, and the morula and blastocyst are particularly vulnerable to oxidative damage [2]. Excessive ROS leads to lipid peroxidation in membranes (MDA), base oxidation (8-OHdG), mitochondrial damage and ultimately to mitochondria-dependent apoptosis through the activation of caspase [3]. Microscopically, the changes are characterized by cytoplasmic vacuolation, blebbing of the trophectoderm, disorganisation of the inner cell mass (ICM), shrinkage of the blastocoel and pyknotic nuclei.

The classical antioxidant supplementation with ascorbate, alpha-tocopherol, N-acetyl-cysteine or beta-mercaptoethanol has shown inconsistent results, with poor half-life in culture media, and with variable cellular uptake and poor therapeutic indices [1,3]. The answer is nanotechnology: Nanoscale antioxidants have a high surface to volume ratio, tunable release characteristics, better cellular uptake and enzyme mimicking (nanozyme) properties that are not seen with classical molecular antioxidants.

Selenium nanoparticles (SeNPs) are especially attractive due to the fact that selenium is a structural component of GPx and thioredoxin reductase [4]. SeNPs can also liberate bioavailable Se which triggers de-novo synthesis and activity of these selenoenzymes, and the particulate form has been shown to be a direct scavenger of peroxyl and superoxide radicals, with higher antioxidant activity and therapeutic-toxic ratio than inorganic selenite and organic selenomethionine [5,6]. Despite their potential to be cytotoxic at high concentrations [7], when synthesized by green methods using plant polyphenols, flavonoids and terpenoids, silver nanoparticles (AgNPs) are known to have ROS-scavenging and Nrf2-activating properties at low concentrations [8,9]. These phytochemicals not only cap the emerging nanoparticles, but also possess intrinsic antioxidant activity. G-AgNPs are therefore not only colloidally stable, but also have a surface chemistry that is biologically compatible.

Despite independent study of each nanoparticle in reproductive biology, head-to-head comparisons—particularly with detailed histological analyses of cultured embryos—are sparse [10]. We postulated that co-supplementation of SeNPs and G-AgNPs in embryo culture media would be more effective in reducing oxidative stress than either nanoparticle individually, thereby increasing blastocyst formation and maintaining normal histoarchitecture. Specific objectives were to: (i) synthesize and characterize SeNPs and G-AgNPs; (ii) establish non-cytotoxic supplementation levels; (iii) measure developmental endpoints in four experimental groups; (iv) quantify oxidative-stress biomarkers in embryo lysates; and (v) quantify blastocyst histology, apoptotic indices, and immunohistochemical markers.

METHODS

Nanoparticle Synthesis and Characterization

A modified ascorbic-acid reduction protocol was used to synthesize SeNPs by vigorously stirring chilled 50 mM ascorbic acid solution (0.5% w/v bovine serum albumin) with sodium selenite (Na2SeO3, 25 mM). The red-orange colloid was centrifuged (10,000 x g, 20 min), rinsed, re-suspended in sterile PBS (pH 7.2), and 0.22-µm filtered. For green-synthesized silver nanoparticles (G-AgNPs), dried leaves of Camellia sinensis were extracted in Milli-Q water (10% w/v, 80°C, 20 min), filtered, and reacted with silver nitrate solution (1 mM, 1:9 v/v at 60°C, ~45 min) until a constant yellow-brown color appeared, then purified by centrifugation (12,000 x g, 25 min).

Dynamic light scattering (DLS; Malvern Zetasizer Nano ZS) measured hydrodynamic diameter and polydispersity index. Transmission electron microscopy (TEM; JEOL JEM-1400) evaluated morphology and core size. UV-Vis spectrophotometry (200-800 nm) verified surface plasmon resonance, zeta potential confirmed colloidal stability, and FTIR spectroscopy (4000-500 cm-1) confirmed organic functional capping.

Animals, Oocyte Retrieval, and Embryo Culture

Adult Swiss albino female mice (6-8 weeks old, 25-30 g) and stud males were housed under a 12 h light/dark cycle with ad libitum food and water (IACUC-Ref: 232/2024; ARRIVE 2.0 guidelines) [20]. Superovulation was induced with 7.5 IU PMSG followed 48 h later by 7.5 IU hCG. Cumulus-oocyte complexes were retrieved from oviducts and fertilized in vitro with capacitated epididymal spermatozoa in TYH medium. At 18 h post-insemination, presumptive 2-cell embryos (n=480) were washed and allocated to pre-equilibrated KSOM droplets under mineral oil (37°C, 5% CO2, 5% O2, 90% N2) across four experimental groups (n = 120 embryos per group; Table 1).

Table 1

Experimental groups and nanoparticle supplementation regimens.

Group | Designation | Supplementation | No. of Embryos
I | Control | KSOM medium only | 120
II | SeNPs | KSOM + 5 µg/mL SeNPs | 120
III | G-AgNPs | KSOM + 2 µg/mL G-AgNPs | 120
IV | SeNPs + G-AgNPs | KSOM + 5 µg/mL SeNPs + 2 µg/mL G-AgNPs | 120

Developmental Scoring, Oxidative Biomarkers, and Histopathology

Embryos were graded at 24, 48, 72, 96, and 120 h for cleavage, morula, blastocyst, and hatched blastocyst rates (Gardner scheme) [11]. Blastocyst pools (30 embryos x 10 replicates) at 96 h were lysed in ice-cold RIPA buffer for protein-normalized assays: MDA (TBARS), SOD (xanthine-oxidase), CAT (H2O2 decomposition), GPx (NADPH-coupled), and GSH (DTNB). Representative blastocysts (n = 25 per group) fixed in 4% PFA were agarose-paraffin embedded, sectioned at 4 µm, and stained with H&E and PAS. Apoptosis was visualized by fluorometric TUNEL (Roche) and cleaved-caspase-3 IHC (HRP-DAB polymer). A blinded histologist scored each blastocyst on a 12-point composite injury scale (ICM compaction [0-3], trophectoderm cohesion [0-3], cytoplasmic vacuoles [0-3], pyknotic nuclei [0-3]).

Statistical Analysis

Data are presented as mean ± SD. Normality and homogeneity of variance were assessed by Shapiro-Wilk and Levene tests. Group differences were evaluated by one-way ANOVA with Tukey HSD post-hoc test, Kruskal-Wallis with Dunn's test for ordinal scores, and Chi-square for proportions. Pearson correlation was used for bivariate relationships. Statistical significance was set at p < 0.05 using SPSS v26.

RESULTS

SeNPs exhibited a hydrodynamic diameter of 48 ± 8 nm, PDI of 0.21, zeta potential of -28.4 ± 2.1 mV, quasi-spherical TEM core size of 40-60 nm, and UV-Vis absorption maximum at 395 nm. G-AgNPs showed a hydrodynamic size of 22 ± 4 nm, zeta potential of -32.6 ± 2.4 mV, UV-Vis peak at 430 nm, and FTIR plant-capping bands at 3400 cm-1 (hydroxyl), 1640 cm-1 (carbonyl), and 1050 cm-1 (C-O-C) (Figure 2).

Figure 2

Developmental progression improved significantly with nanoparticle supplementation (Table 2, Figure 3). Blastocyst formation rates at 96 h were 38.5 ± 3.8% in Control, 55.8 ± 3.3% in SeNPs, 51.2 ± 3.5% in G-AgNPs, and 64.7 ± 2.9% in the combined SeNPs + G-AgNPs group (p < 0.001 vs Control). Hatched blastocyst rates doubled in the combined group (34.5 ± 3.1% vs. 16.9 ± 2.7%, p < 0.001), with 45.9 ± 3.2% achieving good-quality grade (Gardner >= 3BB).

Figure 3

Embryo lysate oxidative stress profiling (Table 3, Figure 4) demonstrated a 56% reduction in MDA in the combined group (2.14 ± 0.18 nmol/mg protein vs. 4.82 ± 0.31 in Control, p < 0.001). Concurrently, SOD (11.30 ± 0.61 U/mg), CAT (20.9 ± 1.3 U/mg), GPx (7.62 ± 0.38 nmol/min/mg), GSH (2.64 ± 0.16 µmol/mg), and GSH/GSSG ratio (8.4 ± 0.6) were maximally elevated in the combined group (p < 0.001).

Figure 4

Histological H&E analysis (Figure 5) revealed poorly compacted ICM, irregular blastocoel, cytoplasmic vacuolation, and pyknotic nuclei in controls, which normalized to a tight ICM and cohesive single-layered trophectoderm in the combined group. Composite injury score (Table 4) declined from 8.6 ± 1.1 in controls to 2.3 ± 0.5 in the combined group, correlating strongly and positively with MDA levels (r = 0.82 / Pearson r = 0.98, p < 0.001; Figure 6C). Apoptotic assessment (Table 5, Figure 6A-B) showed marked suppression of TUNEL-positive nuclei (3.9 ± 1.0 vs. 13.2 ± 1.8 in Control, p < 0.001) and cleaved caspase-3 IHC score (0.7 ± 0.25 vs. 3.2 ± 0.4, p < 0.001), alongside higher total blastocyst cell counts (86 ± 7 vs. 52 ± 6) and ICM/TE ratio (0.34 ± 0.03 vs. 0.27 ± 0.03).

Figure 5

Figure 6

Table 2

Developmental outcomes (% of cultured embryos reaching each stage).

Stage (time) | Control | SeNPs | G-AgNPs | SeNPs + G-AgNPs
Cleavage (48 h) | 78.4 ± 3.1 | 86.1 ± 2.6* | 83.7 ± 2.9* | 90.5 ± 2.3***
Morula (72 h) | 58.9 ± 3.6 | 71.4 ± 3.0** | 68.2 ± 3.2** | 78.9 ± 2.8***
Blastocyst (96 h) | 38.5 ± 3.8 | 55.8 ± 3.3*** | 51.2 ± 3.5*** | 64.7 ± 2.9***
Hatched blastocyst (120 h) | 16.9 ± 2.7 | 27.6 ± 3.0*** | 24.1 ± 2.8*** | 34.5 ± 3.1***
Good-quality blastocyst (Gardner >=3BB) | 21.7 ± 3.0 | 36.4 ± 3.1*** | 32.8 ± 3.4*** | 45.9 ± 3.2***

*p < 0.05, **p < 0.01, ***p < 0.001 versus Control (ANOVA, Tukey HSD). Values are mean ± SD, n = 120 embryos per group.

Table 3

Oxidative stress biomarkers in embryo lysates after 96 h of culture (mean ± SD, n = 10 pooled replicates).

Biomarker | Control | SeNPs | G-AgNPs | SeNPs + G-AgNPs
MDA (nmol/mg protein) | 4.82 ± 0.31 | 2.81 ± 0.22*** | 3.26 ± 0.27*** | 2.14 ± 0.18***
SOD (U/mg protein) | 6.10 ± 0.48 | 9.45 ± 0.55*** | 8.72 ± 0.52*** | 11.30 ± 0.61***
CAT (U/mg protein) | 11.2 ± 1.0 | 17.6 ± 1.1*** | 16.1 ± 1.2*** | 20.9 ± 1.3***
GPx (nmol NADPH/min/mg) | 3.15 ± 0.25 | 6.48 ± 0.33*** | 5.12 ± 0.29*** | 7.62 ± 0.38***
GSH (µmol/mg protein) | 1.78 ± 0.17 | 2.10 ± 0.14*** | 1.97 ± 0.13*** | 2.64 ± 0.16***
GSH/GSSG ratio | 3.1 ± 0.4 | 6.2 ± 0.5*** | 5.5 ± 0.5*** | 8.4 ± 0.6***

***p < 0.001 versus Control (ANOVA, Tukey HSD). MDA = malondialdehyde; SOD = superoxide dismutase; CAT = catalase; GPx = glutathione peroxidase; GSH = reduced glutathione; GSSG = oxidized glutathione.

Table 4

Histological injury subscores and composite score (mean ± SD; higher = more injury).

Parameter (0-3) | Control | SeNPs | G-AgNPs | SeNPs + G-AgNPs
ICM compaction (loss) | 2.4 ± 0.4 | 1.1 ± 0.3*** | 1.3 ± 0.3*** | 0.6 ± 0.2***
Trophectoderm cohesion (loss) | 2.1 ± 0.4 | 0.9 ± 0.3*** | 0.8 ± 0.3*** | 0.5 ± 0.2***
Cytoplasmic vacuolation | 2.0 ± 0.4 | 0.9 ± 0.3*** | 1.2 ± 0.3*** | 0.6 ± 0.2***
Pyknotic nuclei density | 2.1 ± 0.3 | 0.9 ± 0.3*** | 1.1 ± 0.3*** | 0.6 ± 0.2***
Composite injury score (0-12) | 8.6 ± 1.1 | 3.8 ± 0.7*** | 4.4 ± 0.8*** | 2.3 ± 0.5***

***p < 0.001 versus Control (Kruskal-Wallis followed by Dunn's test).

Table 5

Apoptotic indices across groups (mean ± SD, n = 25 blastocysts per group).

Parameter | Control | SeNPs | G-AgNPs | SeNPs + G-AgNPs
TUNEL-positive nuclei / blastocyst | 13.2 ± 1.8 | 6.5 ± 1.3*** | 7.8 ± 1.4*** | 3.9 ± 1.0***
Caspase-3 IHC score (0-4) | 3.2 ± 0.4 | 1.4 ± 0.3*** | 1.8 ± 0.35*** | 0.7 ± 0.25***
Total cell count / blastocyst | 52 ± 6 | 74 ± 7*** | 69 ± 6*** | 86 ± 7***
ICM cell number | 11 ± 2 | 18 ± 2*** | 17 ± 2*** | 22 ± 3***
TE cell number | 41 ± 5 | 56 ± 6*** | 52 ± 5*** | 64 ± 6***
ICM/TE ratio | 0.27 ± 0.03 | 0.32 ± 0.03* | 0.33 ± 0.03* | 0.34 ± 0.03**

*p < 0.05, **p < 0.01, ***p < 0.001 versus Control.

DISCUSSION

Co-supplementation of SeNPs and G-AgNPs in embryo culture media restores redox homeostasis, enhances developmental competence, and preserves blastocyst histoarchitecture through distinct, synergistic antioxidant mechanisms (Figure 7) [1-3].

Figure 7

SeNPs act primarily via an enzymatic axis: intracellular release of bioavailable selenium augments GPx and thioredoxin reductase cofactor pools and elevates the GSH/GSSG ratio [4-6,17]. Phyto-capped G-AgNPs act at the cell-surface interface, transferring electrons directly to incoming ROS while priming cytoprotection via low-level Nrf2 activation (upregulating HO-1, NQO1, and SOD2) [7-9]. In co-delivery, this dual-compartment action produces broad, persistent cytoprotection.

Histological changes closely mirrored biochemical redox improvements. The strong positive correlation between MDA and composite injury scores (r = 0.82) establishes lipid peroxidation as a proximal driver of embryonic cytopathology [10]. Reduction in cytoplasmic vacuolation reflects mitochondrial membrane preservation. Enhanced PAS reactivity indicates preserved glycolytic metabolism, while a 26% increase in blastocyst cell count and optimal ICM/TE ratio (0.34) support enhanced implantation and post-transfer developmental potential [11,12].

Supplementation concentrations (5 µg/mL SeNPs and 2 µg/mL G-AgNPs) remained strictly within the non-cytotoxic therapeutic window established by MTT assays [15,19]. Findings align with single-nanoparticle IVP studies in bovine and porcine models [8-10,18], with the dual regimen demonstrating clear synergistic superiority. Limitations include the murine model scope and lack of post-transfer gestation tracking.

CONCLUSION

SeNPs and G-AgNPs, especially when combined, greatly reduce oxidative stress, enhance developmental competence, and maintain a normal histoarchitectural profile of murine blastocysts. These histological improvements, confirmed by apoptotic and immunohistochemical indices, confirm biochemical redox restoration and emphasize the complementary mechanisms of both nanoparticles, offering a valid foundation for dual-nanoparticle antioxidant strategies in assisted reproduction.

Abbreviations & Declarations

ABBREVIATIONS

CAT: Catalase

DLS: Dynamic Light Scattering

FTIR: Fourier-Transform Infrared Spectroscopy

G-AgNPs: Green-Synthesized Silver Nanoparticles

GPx: Glutathione Peroxidase

GSH: Reduced Glutathione

GSSG: Oxidized Glutathione

H&E: Hematoxylin and Eosin

ICM: Inner Cell Mass

IVF: In-Vitro Fertilization

IVP: In-Vitro Embryo Production

KSOM: Potassium Simplex Optimized Medium

MDA: Malondialdehyde

PAS: Periodic Acid-Schiff

PDI: Polydispersity Index

ROS: Reactive Oxygen Species

SeNPs: Selenium Nanoparticles

SOD: Superoxide Dismutase

TE: Trophectoderm

TEM: Transmission Electron Microscopy

TUNEL: Terminal Deoxynucleotidyl Transferase dUTP Nick-End Labeling

ACKNOWLEDGMENTS

The author acknowledges the College of Science, University of Wasit, Iraq, for providing the facilities to conduct this research. No external funding was received for this study.

DECLARATIONS

Ethical Approval: Approved by the Institutional Animal Care and Use Committee (IACUC-Ref: 232/2024) and performed in accordance with ARRIVE 2.0 guidelines.

Conflicts of Interest: The author declares that there are no competing interests.

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