The protective effect of zinc oxide nanoparticles on boar sperm during preservation at 17 °C

Effects of reactive oxygen species on sperm function

The effect of semen storage duration on motility parameters and ATP content of rabbit spermatozoa were investigated. Ejaculates were collected from 9 New Zealand White male rabbits and diluted with a commercial rabbit semen extender Galap. Semen was stored at 15 degrees C for 3 days. On each day of storage sperm motility and intracellular ATP content were evaluated. Sperm motility parameters were assessed using the computer-assisted sperm analysis (CASA) system and ATP content using the bioluminescence method. The time of storage had a significant effect on sperm motility parameters (except straight-line velocity) and ATP content. A significant correlation was observed between motility parameters and sperm ATP content. The motility parameters most strongly correlated with ATP content were total motile spermatozoa (r = 0.6364), progressively motile spermatozoa (r = 0.529), amplitude of lateral head displacement (r = 0.4178), curvilinear velocity (r = 0.4111) and average path velocity (r = 0.3743). Results show that motility parameters determined using the CASA system and intracellular ATP content are sensitive indicators of sperm quality during in vitro storage and may be useful for estimation of in vivo fertilizing ability of rabbit semen.

The widespread use of zinc oxide nanoparticles (ZnO NPs) has resulted in their release to the environment. There has been concern about the ecotoxicity of ZnO NPs, but little is known about their toxic mechanisms. In the present study, we conducted acute toxicity tests to show that ZnO NPs are more toxic to the freshwater crustacean Daphnia pulex compared to bulk ZnO or ZnSO4·7H2O. To provide an integrated and quantitative insights into the toxicity of ZnO NPs, we conducted isobaric tags for relative and absolute quantitation (iTRAQ) proteomic analysis, which detected 262, 331, and 360 differentially expressed proteins (DEPs) in D. pulex exposed to ZnO NPs, bulk ZnO, and ZnSO4·7H2O, respectively. Among the DEPs, 224 were shared among the three treatments. These proteins were related to energy metabolism, oxidative stress, and endoplasmic reticulum stress. The three forms of Zn all caused D. pulex to downregulate Chitinase expression, disrupt Ca2+ homeostasis, and reduce expression of digestive enzymes. Nevertheless, 29 proteins were expressed only in the ZnO NP treatment. In particular, histone (H3) and ribosomal proteins (L13) were obviously influenced under ZnO NP treatment. However, increased expression levels of h3 and l13 genes were not induced only in ZnO NP treatment, they were sensitive to Zn ions under the same exposure concentration. These results indicate that the three zinc substances have a similar mode of action and that released zinc ions are the main contributor to ZnO NP toxicity to D. pulex under a low concentration. Further investigation is needed to clarify whether a small proportion of DEPs or higher bioavailability cause ZnO NPs to be more toxic compared to bulk ZnO or ionic zinc.

Nutrient strengthening of winter wheat by foliar ZnO and Fe3O4 NPs: Food safety, quality, elemental distribution and effects on soil bacteria

The effects of three preservation methods (ultrasound, ZnO NPs, and ultrasound combined with ZnO NPs) on the odor, microstructure, and edible quality of fresh-cut lettuce were investigated in this study. When stored for 8 days, significant improvements were observed in the following when using ultrasound combined with ZnO NP treatment to better preserve fresh-cut lettuce (and were reduced when compared with the control group): the color (L* value (34.53); a* value (−5.89); b* value (15.00); browning index (40.63); firmness (25.66); propectin (2.12%); chlorophyll (2.75 mg/100 g); cellulose (20.35%); total phenolic content (0.95 mg/100 g); PAL activity (54.91 U·h−1·g−1); CAT activity (41.78 U·min−1·mg−1); ABTS free-radical scavenging ability (137.62 µmol/L); FRAP total reducing ability (94.42 µmol/L) (p < 0.05), PPO activity (0.85 U·min−1·g−1); MDA (1.97 µmol/g); and H2O2 (54.26 µmol/g). In addition, the results of the volatile components indicated that the use of ultrasound combined with ZnO NP treatment decreased the production of adverse flavor compounds by inhibiting the generation of aldehydes and ketones, as well as by promoting the generation of olefins, nitriles, and quinolines, and the contents of nitriles and quinolines were 20.07% and 2.07% of the total components, respectively. The resultant microstructure indicated that the microchannels generated by ultrasound allowed for the ZnO NPs to enter the intracellular cavity of the fresh-cut lettuce more efficiently; such a finding could serve as a basis for a hypothesis on the mechanism of ultrasound combined with ZnO NP treatment. The results of fresh-cut lettuce preservation when using ultrasound combined with ZnO NPs were better than those that were obtained when using ultrasound and ZnO NP treatment alone. And, using ultrasound combined with ZnO NP treatment as a new preservation method for fresh-cut lettuce provides a promising preservation idea for other fresh-cut fruits and vegetables.

Soil salinization severely restricts the growth and development of crops globally, especially in the northwest Loess Plateau, where apples constitute a pillar industry. Nanomaterials, leveraging their unique properties, can facilitate the transport of nutrients to crops, thereby enhancing plant growth and development under stress conditions. To investigate the effects of nano zinc oxide (ZnO NP) on the growth and physiological characteristics of apple self-rooted rootstock M9-T337 seedlings under saline alkali stress, one-year-old M9-T337 seedlings were used as experimental materials and ZnO NPs were used as donors for pot experiment. Six treatments were set up: CK (normal growth), SA (saline alkali stress,100 mmol/L NaCl + NaHCO3), T1 (saline alkali stress + 50 mg/L ZnO NPs), T2 (saline alkali stress + 100 mg/L ZnO NPs), T3 (saline alkali stress + 150 mg/L ZnO NPs) and T4 (saline alkali stress + 200 mg/L ZnO NPs). The results were found to show that saline alkali stress could significantly inhibit the growth and development of M9-T337 seedlings, reduce photosynthetic characteristics, and cause ion accumulation to trigger osmotic regulation system, endogenous hormone and antioxidant system imbalances. However, the biomass, plant height, stem diameter, total leaf area and leaf perimeter of M9-T337 seedlings were significantly increased after ZnO NP treatment. Specifically speaking, ZnO NPs can improve the photosynthetic capacity of M9-T337 by increasing the content of photosynthetic pigment, regulating photosynthetic intensity and chlorophyll fluorescence parameters. ZnO NPs can balance the osmotic adjustment system by increasing the contents of soluble protein (SP), soluble sugar (SS), proline (Pro) and starch, and can also enhance the activities of enzymatic (SOD, POD, and CAT) and non-enzymatic antioxidant enzymes (APX, AAO, GR, and MDHAR) to enhance the scavenging ability of reactive oxygen species (H2O2, O2•-), ultimately reducing oxidative damage; ZnO NPs promoted the growth of M9-T337 seedlings under saline alkali stress by synergistically responding to auxin (IAA), gibberellin (GA3), zeatin (ZT) and abscisic acid (ABA). Additionally, the Na+/K+ ratio was reduced by upregulating the expression of Na+ transporter genes (MdCAX5, MdCHX15, MdSOS1, and MdALT1) and downregulating the expression of K+ transporter genes (MdSKOR and MdNHX4). After comprehensive analysis of principal components and correlation, T3 (150 mg/L ZnO NPs) treatment possessed the best mitigation effect. In summary, 150 mg/L ZnO NPs(T3) can effectively maintain the hormone balance, osmotic balance and ion balance of plant cells by promoting the photosynthetic capacity of M9-T337 seedlings, and enhance the antioxidant defense mechanism, thereby improving the saline alkaline tolerance of M9-T337 seedlings.

Previously we found that live, fresh or thawed boar sperm show little tendency to accumulate ROS spontaneously, but live sperm accumulated ROS during a 30-min incubation with xanthine and xanthine oxidase and showed marked reduction in motility. High mitochondrial transmembrane potential (MMP) is required to drive the F0/F1 ATPase responsible for producing ATP in most cell types, and ATP is required for sperm motility. This experiment was conducted to investigate the effects of menadione (disrupter of electron transport at Complex I) on sperm motility, MMP, and ATP content. The incidence of cells with high MMP was determined by measuring the fluorescence of JC-1 aggregates bound to the inner mitochondrial membrane using flow cytometry. Computer-assisted motion analysis was conducted using the IVOS version 12 (Hamilton Thorne Research, Beverly, MA, USA), and ATP (pmoles/106 sperm) was determined using the luciferin-luciferase assay. Sperm from six boars were individually Percoll washed to eliminate seminal plasma and incubated at 40 � 106/mL with 0, 1, 10, or 30 �M menadione for 5, 30, 60, and 120 min at 38�C in a modified Tyrode's medium containing 0.1% polyvinyl alcohol with no bicarbonate or BSA. The formation of ROS was confirmed by measuring the red fluorescence developed by the oxidation of hydroethidine to ethidium using flow cytometry. Whereas the basal level of ethidium fluorescence in the absence of menadione was low (2% ethidium-positive cells at 5 min), 10 and 30 �M menadione increased (P < 0.05) the percentage of ethidium-positive cells to 47 and 87%, respectively, at 30 min. Sperm motility did not decrease significantly (79-83%) during the 120 min incubation with no menadione, but menadione caused a significant dose-related decrease (P < 0.05) over time, with 10 and 30 �M menadione decreasing motility to 60 and 40%, 51 and 7%, and 10 and 1% at 30, 60, and 120 min, respectively. JC-1 aggregate fluorescence intensity decreased (P < 0.05) by 45-60% in a dose-related fashion at 120 min compared to the same doses at 5 min. Sperm viability, as measured by number of propidium iodide negative cells, averaged 93.6% during the incubation and was not significantly affected by treatment. The effect of menadione on ATP content was much less than that on motility or JC-1 fluorescence intensity. Mean ATP content averaged 63 pmoles through 60 min at all menadione doses; at 120 min only 30 �M menadione decreased (P < 0.05) ATP to 43 pmoles, compared to all other treatments. Menadione caused an increase in ROS formation and a decline in MMP, which suggested uncoupling of the respiratory chain and oxidative phosphorylation. However, sperm ATP content was not highly correlated with motility. This suggests that ATP content was maintained by the activity of other intermediary metabolism pathways. The reduction in motility may have been due to a ROS induced lesion in ATP utilization or in the contractile apparatus of the cell.

Sperm cryosurvival rates are not optimal for most species. Therefore, new cryopreservation strategies are needed with the objective of increasing the number of surviving sperm and the quality of those sperm after thawing. Cholesterol plays important roles in many sperm functions, including effects on membrane properties. One of these effects is to stabilize membranes at low temperatures. Thus, species that produce sperm which possess high membrane cholesterol : phospholipid ratios are more resistant to cold shock than sperm with low cholesterol : phospholipid ratios. Therefore, increasing the cholesterol content of sperm membranes may be a strategy that can improve sperm quality after freeze-thawing. In this review, information is presented related to using cyclodextrins pre-loaded with cholesterol for cryopreserving sperm from different species. The topics discussed include both in vitro and in vivo assessments of sperm quality after cryopreservation, as well as how increasing sperm cholesterol content affects other sperm functions.

Sperm viability, acrosome integrity, motility, and swimming velocity are determinants of male fertility and exhibit an extreme degree of variation among closely related species. Many of these sperm parameters are associated with sperm ATP content, which has led to predictions of trade-offs between ATP content and sperm motility and velocity. Selective pressures imposed by sperm competition have been proposed as evolutionary causes of this pattern of diversity in sperm traits. Here, we examine variation in sperm viability, acrosome integrity, motility, swimming velocity, and ATP content over time, among 18 species of closely related muroid rodents, to address the following questions: (a) Do sperm from closely related species vary in ATP content after a period of incubation? (b) Are these differences in ATP levels related to differences in other sperm traits? (c) Are differences in ATP content and sperm performance over time explained by the levels of sperm competition in these species? Our results revealed a high degree of interspecific variability in changes in sperm ATP content, acrosome integrity, sperm motility and swimming velocity over time. Additionally, species with high sperm competition levels were able to maintain higher levels of sperm motility and faster sperm swimming velocity when they were incubated under conditions that support sperm survival. Furthermore, we show that the maintenance of such levels of sperm performance is correlated with the ability of sperm to sustain high concentrations of intracellular ATP over time. Thus, sperm competition may have an important role maximizing sperm metabolism and performance and, ultimately, the fertilizing capacity of spermatozoa.

Previous studies recorded positive impact of ZnO NPs on plants stressed with salinity. The current work was performed to study the effect of two different concentrations of biosynthesized ZnO NPs (50 and 100 mg L−1) on faba bean plants under salinity stress. The zinc oxide nanoparticles (ZnO NPs) were synthesized using Mentha extract, and their shape and size were characterized using X-ray diffraction and transmission electron microscope while diffuse reflectance spectra were measured using UV–Vis spectrophotometer. The generated ZnO NPs were spherical with a particle size 9.4 nm and had a rod form with particle size 15.2 in length and 3.5 nm in width. The response of faba been plants to the foliar spray of ZnO NPs concentrations (0, 50, and 100 mg L−1) alone and in combination with salt stress at 150 mM NaCl was studied. Salinity induced reduction in faba bean root and shoot length and dry/fresh weights, while an enhancement was recorded in response to foliar treatment with ZnO NPs at 50 and 100 mg L−1 either in presence or absence of salinity stress. The highest amounts of chlorophyll a, b, carotenoids, and total pigments were recorded in plants received 50 mg L−1 ZnO NPs compared to the alternative control. Secondary metabolites (phenols, flavonoids, and tannins) were accumulated in salinity-stressed plants and further accumulation in response to ZnO NPs treatment was noticed. Amino acids, proline, glycine betaine, and total soluble sugars, as well as enzymatic and non-enzymatic antioxidant contents, increased almost onefold in salinity-stressed plants as compared to control plants while the 50 mg L−1 ZnO NPs treatment resulted in higher accumulation of the previously mentioned substances. In contrast, plants oxidative stress was reduced in response to ZnO NPs treatments. The nitrogen, phosphorus, potassium, calcium, zinc, and iron contents of faba bean plants were recorded under salinity stress and in response to the two applied concentrations of ZnO NPs. Faba bean plants stressed with 150 MN NaCl showed growth decline that may be attributed to osmotic stress and low water availability imposed by salinity. The treatment of stressed plants with 50 mg L−1 ZnO NPs induced an enhancement in plant growth as well as an accumulation of antioxidants, osmolytes, and secondary metabolites that could help plants overcome the negative effects of salinity.

Zinc oxide nanoparticles (ZnO NPs) exhibit diverse applications, including antimicrobial, UV-blocking, and catalytic properties, due to their unique structure and properties. This study focused on the characterization of zinc oxide nanoparticles (ZnO NPs) synthesized from Juglans regia leaves and their application in mitigating the impact of simultaneous infection by Meloidogyne arenaria (root-knot nematode) and Macrophomina phaseolina (root-rot fungus) in cowpea plants. The characterization of ZnO NPs was carried out through various analytical techniques, including UV–visible spectrophotometry, Powder-XRD analysis, FT-IR spectroscopy, and SEM-EDX analysis. The study confirmed the successful synthesis of ZnO NPs with a hexagonal wurtzite structure and exceptional purity. Under in vitro conditions, ZnO NPs exhibited significant nematicidal and antifungal activities. The mortality of M. arenaria juveniles increased with rising ZnO NP concentrations, and a similar trend was observed in the inhibition of M. phaseolina mycelial growth. SEM studies revealed physical damage to nematodes and structural distortions in fungal hyphae due to ZnO NP treatment. In infected cowpea plants, ZnO NPs significantly improved plant growth parameters, including plant length, fresh mass, and dry mass, especially at higher concentrations. Leghemoglobin content and the number of root nodules also increased after ZnO NP treatment. Additionally, ZnO NPs reduced gall formation and egg mass production by M. arenaria nematodes and effectively inhibited the growth of M. phaseolina in the roots. Furthermore, histochemical analyses demonstrated a reduction in oxidative stress, as indicated by decreased levels of reactive oxygen species (ROS) and lipid peroxidation in ZnO NP-treated plants. These findings highlight the potential of green-synthesized ZnO NPs as an eco-friendly and effective solution to manage disease complex in cowpea caused by simultaneous nematode and fungal infections.

This study aimed to assess the apoptosis-inducing mechanism of zinc oxide nanoparticles (ZnO NPs) stabilized by Solanum xanthocarpum plant extract in human osteosarcoma MG63 cells. In the present study, we synthesized ZnO NPs from S. xanthocarpum extract and evaluated its anticancer mechanism on MG 63 cells. The synthesized ZnO NPs were characterized by ultraviolet spectroscopy, X-ray crystallography, transmission electron microscopy, energy dispersive X-ray, and Fourier-transform infrared spectroscopy analysis. The mean size of the synthesized ZnO NPs was 21.62 ± 7.45 nm and spherical in shape. The cytotoxicity of ZnO NPs on MG63 cells was determined by MTT assay. The Western blot analysis was carried out to examine the expression of apoptotic and autophagy-related proteins in MG63 cells. The findings of the study reveal that ZnO NPs treatment showed concentration-dependent cytotoxicity, increased lipid peroxidation, decreased antioxidant activity, increased reactive oxygen species generation, and increased DNA damage. In addition, ZnO NPs treatment increased the expression of apoptotic members such as p53, Bax, caspase-3, -8, and -9 while downregulating Bcl-2 expression in MG63 cells. Furthermore, ZnO NPs treatment suppressed the P13K/AKT/mTOR signaling pathway and increased the expression of LC3 and beclin-1 in MG63 cells. The present study demonstrated that ZnO NPs induced apoptosis and autophagy in MG63 cells through modifying apoptotic and autophagy-related proteins.

Numerous studies have shown that Astragalus polysaccharide (APS) has strong antioxidant effects and high practical value for preserving semen at low temperatures in vitro. However, to date, little attention has been paid to the precise mechanism of APS in sperm preservation at 4 °C. Thus, to gain further insight into the protective effects of APS, the present study was performed to assess the changes in sperm quality parameters, antioxidant capacity, ATP content, and protein phosphorylation levels. Here, we demonstrated that supplementation with APS could effectively preserve boar sperm quality parameters such as sperm motility, acrosome integrity, and mitochondrial membrane potential. Moreover, we found that the positive effects of APS on boar sperm quality were mainly due to the elimination of excessive mitochondrial ROS, the improvement of antioxidant capacities and the enhancement of ATP levels. Interestingly, by conducting a series of studies on protein phosphorylation, we also discovered that APS could protect boar sperm from oxidative stress and energy deficiency through inhibiting the protein dephosphorylation caused by ROS via the cAMP-PKA signaling pathway. To our knowledge, this is the first exploration of the molecular mechanism underlying the protective roles of APS toward ROS toxicity from the perspective of energy metabolism and protein modification. This study comprehensively provides novel insights into the action mechanism of the protective effects of antioxidants on sperm stored at 4 °C and reveals the practical feasibility of using APS as a boar semen extender supplement for assisted reproductive technology.

Zinc oxide nanoparticles (ZnO NPs) were synthesized by Carica papaya leaf extract. The nanoparticles were characterized by UV–Vis spectrum, Fourier Transform Infrared spectroscopy (FTIR), X-ray Diffraction (XRD), Dynamic light scattering (DLS) analyser and Energy-dispersive X-ray spectroscopy analysis with a scanning electron microscope (SEM–EDX). The ZnO NPs were assessed using 2,2′-Azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay with varying ZnO NP concentration, showed scavenging activity with the half maximal inhibitory concentration (IC50) = 130.1 and 104.9 µg/mL−1 respectively. Antifungal studies were conducted for ZnO NPs against S. sclerotiorum, R. necatrix and Fusarium species, which demonstrated a higher inhibition rate for S. sclerotiorum (59.7%). Seeds of chickpea were separately treated with various concentrations of ZnO NPs. An exposure to ZnO NPs (25%, 50%, 75% and 100%) and control caused significant changes in seed germination, root length, shoot length and antioxidant enzyme were studied. Compared with control the maximum seed germination, root and plant growth was observed with the treatment of ZnO NPs. Superoxide dismutase and catalase activity increased due to ZnO NPs treatment. This suggest that ZnO NPs may significantly alter antioxidant metabolism during seed germination.

Green synthesis of zinc oxide nanoparticles using Cinnamomum camphora (L.) Presl leaf extracts and its antifungal activity