Complementary Biotransformation of Antimicrobial Triclocarban Obviously Mitigates Nitrous Oxide Emission toward Sustainable Microbial Denitrification.

Sustainable nitrogen cycle is an essential biogeochemical process that ensures ecosystem safety and byproduct greenhouse gas nitrous oxide reduction. Antimicrobials are always co-occurring with anthropogenic reactive nitrogen sources. However, their impacts on the ecological safety of microbial nitrogen cycle remain poorly understood. Here, a denitrifying bacterial strain Paracoccus denitrificans PD1222 was exposed to a widespread broad-spectrum antimicrobial triclocarban (TCC) at environmental concentrations. The denitrification was hindered by TCC at 25 µg L-1 and was completely inhibited once the TCC concentration exceeded 50 µg L-1. Importantly, the accumulation of N2O at 25 µg L-1 of TCC was 813 times as much as the control group without TCC, which attributed to the significantly downregulated expression of nitrous oxide reductase and the genes related to electron transfer, iron, and sulfur metabolism under TCC stress. Interestingly, combining TCC-degrading denitrifying Ochrobactrum sp. TCC-2 with strain PD1222 promoted the denitrification process and mitigated N2O emission by 2 orders of magnitude. We further consolidated the importance of complementary detoxification by introducing a TCC-hydrolyzing amidase gene tccA from strain TCC-2 into strain PD1222, which successfully protected strain PD1222 against the TCC stress. This study highlights an important link between TCC detoxification and sustainable denitrification and suggests a necessity to assess the ecological risks of antimicrobials in the context of climate change and ecosystem safety.

Cathode potential regulates the microbiome assembly and function in electrostimulated bio- dechlorination system.

Mechanism of microbiome assembly and function driven by cathode potential in electro-stimulated microbial reductive dechlorination system remain poorly understood. Here, core microbiome structure, interaction, function and assembly regulating by cathode potential were investigated in a 2,4,6-trichlorophenol bio-dechlorination system. The highest dechlorination rate (24.30 µM/d) was observed under - 0.36 V with phenol as a major end metabolite, while, lower (-0.56 V) or higher (0.04 V or -0.16 V) potentials resulted in 1.3-3.8 times decreased of dechlorination kinetic constant. The lower the cathode potential, the higher the generated CH4, revealing cathode participated in hydrogenotrophic methanogenesis. Taxonomic and functional structure of core microbiome significantly shifted within groups of - 0.36 V and - 0.56 V, with dechlorinators (Desulfitobacterium, Dehalobacter), fermenters (norank_f_Propionibacteriaceae, Dysgonomonas) and methanogen (Methanosarcina) highly enriched, and the more positive interactions between functional genera were found. The lowest number of nodes and links and the highest positive correlations were observed among constructed sub-networks classified by function, revealing simplified and strengthened cooperation of functional genera driven by group of - 0.36 V. Cathode potential plays one important driver controlling core microbiome assembly, and the low potentials drove the assembly of major dechlorinating, methanogenic and electro-active genera to be more deterministic, while, the major fermenting genera were mostly governed by stochastic processes.

The Regulatory Effects of Traditional Chinese Medicine on Ferroptosis.

Traditional Chinese medicine (TCM) has significantly contributed to protecting human health and promoting the progress of world civilization. A total of 2,711 TCMs are included in the 2020 version of the Chinese Pharmacopoeia, which is an integral part of the world's medical resources. Tu Youyou and her team discovered and purified artemisinin. And their contributions made the values and advantageous effects of TCM more and more recognized by the international community. There has been a lot of studies on TCM to treat diseases through antioxidant mechanisms, the reports on the new mechanisms beyond antioxidants of TCM has also increased year by year. Recently, many TCMs appear to have significant effects in regulating ferroptosis. Ferroptosis is an iron-dependent, non-apoptotic, regulated cell death characterized by intracellular lipid peroxide accumulation and oxidative membrane damage. Recently, accumulating studies have demonstrated that numerous organ injuries and pathophysiological process of many diseases are companied with ferroptosis, such as cancer, neurodegenerative disease, acute renal injury, arteriosclerosis, diabetes, and ischemia-reperfusion injury. This work mainly introduces dozens of TCMs that can regulate ferroptosis and their possible mechanisms and targets.

Coupled sulfur and electrode-driven autotrophic denitrification for significantly enhanced nitrate removal.

Elemental sulfur (S0)-based autotrophic denitrification (SAD) has gained intensive attention in the treatment of secondary effluent for its low cost, high efficiency, and good stability. However, in practice, the supplementary addition of limestone is necessary to balance the alkalinity consumption during SAD operation, which increases water hardness and reduces the effective reaction volume. In this study, a coupled sulfur and electrode-driven autotrophic denitrification (SEAD) process was proposed with superior nitrate removal performance, less accumulation of sulfate, and self-balance of acidity-alkalinity capacity by regulating the applied voltage. The dual-channel electron supply from S0 and electrodes made the nitrate removal rate constant k in the SEAD process 3.7-5.1 and 1.4-3.5 times higher than that of the single electrode- and sulfur-driven systems, respectively. The S° contributed to 75.3%-83.1% of nitrate removal and the sulfate yield during SEAD (5.67-6.26 mg SO42-/mg NO3--N) was decreased by 17%-25% compared with SAD. The S0 particle and electrode both as active bio-carriers constructed collaborative denitrification communities and functional genes. Pseudomonas, Ralstonia and Brevundimonas were the dominant denitrifying genera in S0 particle biofilm, while Pseudomonas, Chryseobacterium, Pantoea and Comamonas became dominant denitrifying genera in the cathode biofilm. The narG/Z/H/Y/I/V, nxrA/B, napA/B, nirS/K, norB/C and nosZ were potential functional genes for efficient nitrate reduction during the SEAD process. Metagenomic sequencing indicated that S0 as an electron donor has greater potential for complete denitrification than the electrode. These findings revealed the potential of SEAD for acting as a highly efficient post denitrification process.

Chemical constituents from the stems of Acanthopanax senticosus with their cytotoxic activities.

A new coumestan named 7,5'-dihydroxy-4'-(3''-hydroxy-3''-methyl-trans-isobut-1''-enyl) coumestan (1), together with five known compounds (2-6), was isolated from the EtOAc-soluble extract of the stems of Acanthopanax senticosus. Their structures were elucidated based on extensive spectroscopic analyses. All the isolates were evaluated for in vitro cytotoxic activities against four human cancer cells including HepG2, A549, HeLa and MCF-7. Among them, the new compound 1 was found to exhibit significant cytotoxic activity on HeLa cells with IC50 value of 6.5 µM.

Electroacupuncture promotes the survival and synaptic plasticity of hippocampal neurons and improvement of sleep deprivation-induced spatial memory impairment.

AIMS: This study aimed to investigate whether electroacupuncture (EA) promotes the survival and synaptic plasticity of hippocampal neurons by activating brain-derived neurotrophic factor (BDNF)/tyrosine receptor kinase (TrkB)/extracellular signal-regulated kinase (Erk) signaling, thereby improving spatial memory deficits in rats under SD. METHODS: In vivo, Morris water maze (MWM) was used to detect the effect of EA on learning and memory, at the same time Western blotting (WB), immunofluorescence (IF), and transmission electron microscopy (TEM) were used to explore the plasticity of hippocampal neurons and synapses, and the expression of BDNF/TrkB/Erk signaling. In vitro, cultured hippocampal neurons were treated with exogenous BDNF and the TrkB inhibitor K252a to confirm the relationship between BDNF/TrkB/Erk signaling and synaptic plasticity. RESULTS: Our results showed that EA mitigated the loss of hippocampal neurons and synapses, stimulated hippocampal neurogenesis, and improved learning and memory of rats under SD accompanied by upregulation of BDNF and increased phosphorylation of TrkB and Erk. In cultured hippocampal neurons, exogenous BDNF enhanced the expression of synaptic proteins, the frequency of the postsynaptic currents, and the phosphorylation of TrkB and Erk; these effects were reversed by treatment with K252a. CONCLUSIONS: Electroacupuncture alleviates SD-induced spatial memory impairment by promoting hippocampal neurogenesis and synaptic plasticity via activation of BDNF/TrkB/Erk signaling, which provided evidence for EA as a therapeutic strategy for countering the adverse effects of SD on cognition.

Electrical selection for planktonic sludge microbial community function and assembly.

Electrostimulated hydrolysis acidification (eHA) has been used as an efficient biological pre-treatment of refractory industrial wastewater. However, the effects of electrostimulation on the function and assembly of planktonic anaerobic sludge microbial communities are poorly understood. Using 16S rRNA gene and metagenomic sequencing, we investigated planktonic sludge microbial community structure, composition, function, assembly, and microbial interactions in response to electrostimulation. Compared with a conventional hydrolysis acidification (HA) reactor, the planktonic sludge microbial communities selected by electrostimulation promoted biotransformation of the azo dye Alizarin Yellow R. The taxonomic and functional structure and composition were significantly shifted upon electrostimulation with azo dyes degraders (e.g. Acinetobacter and Dechloromonas) and electroactive bacteria (e.g. Pseudomonas) being enriched. More microbial interactions between fermenters and decolorizing and electroactive bacteria, as well as fewer interactions between different fermenters evolved in the eHA microbial communities. Moreover, the decolorizing bacteria were linked to the higher abundance of genes encoding for azo- and nitro-reductases and redox mediator (e.g. ubiquinone) biosynthesis involved in the transformation of azo dye. Microbial community assembly was more driven by deterministic processes upon electrostimulation. This study offers new insights into the effects of electrostimulation on planktonic sludge microbial community function and assembly, and provides a promising strategy for the manipulation of anaerobic sludge microbiomes in HA engineering systems.

Efficient treatment of alizarin yellow R contained wastewater in an electrostimulated anaerobic-oxic integrated system.

An electrostimulated anaerobic-oxic integrated system was constructed for treating alizarin yellow R (AYR) containing wastewater. In electro-stimulated anaerobic unit, AYR decolorization efficiency improved from 51.2% to 96.6%. Two amination metabolites, p-phenylenediamine and 5-aminosalicylic acid, went through oxidation, ammonification and mineralization in oxic unit. Electro-stimulation promoted denitrification and COD removal efficiencies by 15.5% and 8.6%, respectively. A 20% improved nitrification efficiency was observed in oxic unit, due to elimination of AYR toxicity inhibition. No corrosion of heat-treated stainless steel occurred during the 60 days of continuous operation. Electrons sunk in denitrification and decolorization accounted for 34.4-36.8% of those released from COD removal, and 7.3% increase of removed nitrogen in nitrogenous compounds (AYR, nitrate and ammonia) was found. Electro-stimulated anaerobic unit predominated with fermentation and denitrification genera (Propionispira, Rhodocyclus, etc.) and aboundance of electro-active decolorization genus (Desulfovibrio, etc.) increased. Ammonia-oxidizing genus, Comamonas, was the most abundant in aerobic unit. Compared to the suspension, the electrostimulation could increased the abundance of electro-active genera in cathodic biofilm. This study revealed the feasibility of applying electro-stimulation and the conversion laws of nitrogenous organics in secondary bio-treatment system for treating toxic nitrogenous organics-contained wastewater.

Development and validation of a rapid and sensitive LC-MS/MS method for the determination of polyphyllin II in rat plasma and its application in a pharmacokinetic study.

Polyphyllin II, a major steroidal saponin isolated from Paris polyphylla, exhibits significant pharmacological activities. In this study, a rapid and sensitive liquid chromatography-tandem mass spectrometry method was established and validated for the determination of polyphyllin II in plasma. Polyphyllin II and polyphyllin VII (internal standard) were separated on a Waters Acquity™ HSS T3 column and the mass analysis was performed in a triple quadrupole mass spectrometer equipped with an electrospray ionization ion source. Results showed that the method was sensitive (lower limit of quantitation 0.5 ng/ml), precise (<15%) and linear in the range of 0.5-500 ng/ml (r > 0.99). Interestingly, the sensitivity in current study was ~10 times higher than that in the previous study. The results of the pharmacokinetic study of polyphyllin II in rats suggested that polyphyllin II was poorly absorbed into blood and reached its highest concentration at ~3.67-5.00 h with a slow elimination half-life of 8.34-13.37 h. The bioavailability was 6.1-8.2%. The results indicated that the absorption of polyphyllin II may primarily occur via passive diffusion in rats. This study provides valuable information that can be used as a reference for the pharmacokinetic investigation of other steroidal saponins.

Accelerated microbial reductive dechlorination of 2,4,6-trichlorophenol by weak electrical stimulation.

Microbial reductive dechlorination of chlorinated aromatics frequently suffers from the long dechlorination period and the generation of toxic metabolites. Biocathode bioelectrochemical systems were verified to be effective in the degradation of various refractory pollutants. However, the electrochemical and microbial related working mechanisms for bio-dechlorination by electro-stimulation remain poorly understood. In this study, we reported the significantly improved 2,4,6-trichlorophenol dechlorination activity through the weak electro-stimulation (cathode potential of -0.36 V vs. SHE), as evidenced by the 3.1 times higher dechlorination rate and the complete dechlorination ability with phenol as the end dechlorination product. The high reductive dechlorination rate (20.8 µM/d) could be maintained by utilizing electrode as an effective electron donor (coulombic efficiency of 82.3 ±â€¯4.8%). Cyclic voltammetry analysis of the cathodic biofilm gave the direct evidences of the cathodic respiration with the improved and positive-shifted reduction peaks of 2,4,6-TCP, 2,4-DCP and 4-CP. The optimal 2,4,6-TCP reductive dechlorination rate (24.2 µM/d) was obtained when a small amount of lactate (2 mM) was added, and the generation of H2 and CH4 were accompanied due to the biological fermentation and methanogenesis. The electrical stimulation significantly altered the cathodic biofilm structure and composition with some potential dechlorinators (like Acetobacterium) predominated. The microbial interactions in the ecological network of cathodic biofilm were more simplified than the planktonic community. However, some potential dechlorinators (Acetobacterium, Desulfovibrio, etc.) shared more positive interactions. The co-existence and possible cooperative relationships between potential dechlorinators and fermenters (Sedimentibacter, etc.) were revealed. Meanwhile, the competitive interrelations between potential dechlorinators and methanogens (Methanomassiliicoccus) were found. In the network of plankton, the fermenters and methanogens possessed the more positive interrelations. Electro-stimulation at the cathodic potential of -0.36 V selectively enhanced the dechlorination function, while it showed little influence on either fermentation or methanogenesis process. The study gave suggestions for the enhanced bioremediation of chlorinated aromatics, in views of the electro-stimulation capacity, efficiency and microbial interrelations related microbial mechanism.

Bioremediation of contaminated urban river sediment with methanol stimulation: Metabolic processes accompanied with microbial community changes.

The intense pollution of urban river sediments with rapid urbanization has attracted considerable attention. Complex contaminated sediments urgently need to be remediated to conserve the ecological functions of impacted rivers. This study investigated the effect of using methanol as a co-substrate on the stimulation of the indigenous microbial consortium to enhance the bioremediation of petroleum hydrocarbons (PHs) and polycyclic aromatic hydrocarbons (PAHs) in an urban river sediment. After 65 days of treatment, the PAHs degradation efficiencies in the sediment adding methanol were 4.87%-40.3% higher than the control. The removal rate constant of C31 was 0.0749 d-1 with 100 mM of supplied methanol, while the corresponding rate was 0.0399 d-1 in the control. Four-ring PAHs were effectively removed at a degradation efficiency of 65%-69.8%, increased by 43.3% compared with the control. Sulfate reduction and methanogenesis activity were detected, and methane-producing archaea (such as Methanomethylovorans, with a relative abundance of 25.87%-58.53%) and the sulfate-reducing bacteria (SRB, such as Desulfobulbus and Desulfobacca) were enriched. In addition, the chemolithoautotrophic sulfur-oxidizing bacteria (SOB, such as Sulfuricurvum, with a relative abundance of 34%-39.2%) were predominant after the depletion of total organic carbon (TOC), and markedly positively correlated with the PHs and PAHs degradation efficiencies (P < 0.01). The SRB and SOB populations participated in the sulfur cycle, which was associated with PHs and PAHs degradation. Other potential functional bacteria (such as Dechloromonas) were also obviously enriched and significantly positively correlated with the TOC concentration after methanol injection (P < 0.001). This study provides a new insight into the succession of the indigenous microbial community with methanol as a co-substrate for the enhanced bioremediation of complexly contaminated urban river sediments.

Electroacupuncture Facilitates the Integration of Neural Stem Cell-Derived Neural Network with Transected Rat Spinal Cord.

The hostile environment of an injured spinal cord makes it challenging to achieve higher viability in a grafted tissue-engineered neural network used to reconstruct the spinal cord circuit. Here, we investigate whether cell survival and synaptic transmission within an NT-3 and TRKC gene-overexpressing neural stem cell-derived neural network scaffold (NN) transplanted into transected spinal cord could be promoted by electroacupuncture (EA) through improving the microenvironment. Our results showed that EA facilitated the cell survival, neuronal differentiation, and synapse formation of a transplanted NN. Pseudorabies virus tracing demonstrated that EA strengthened synaptic integration of the transplanted NN with the host neural circuit. The combination therapy also promoted axonal regeneration, spinal conductivity, and functional recovery. The findings highlight EA as a potential and safe supplementary therapeutic strategy to reinforce the survival and synaptogenesis of a transplanted NN as a neuronal relay to bridge the two severed ends of an injured spinal cord.

[Effects of Rhodiola on the Expression of iNOS mRNA in Severe Acute Pancreatitis Associated Re- nal Injury Rats].

OBJECTIVE: To explore the effect of Rhodiola on the expression of iNOS mRNA in severe acute pancreatitis (SAP) associated renal injury rats. METHODS: A total of 72 healthy rats were randomly divided into the sham-operated group (S), the SAP associated renal injury group (M), and the Rhodiola-treated group (RHO), 24 in each group. Rats in S and M groups were peritoneally injected with 10 mL/kg saline 3h before modeling, while rats in the RHO group were peritoneally injected with 10 mL/kg Rhodiola Injection 3 h before modeling. The peripheral ligament of pancreas was bluntly dissociated in rats of M and RHO groups. The head of pancreas was occlused by nontraumatic blood vessel forceps 3 h later to establish the model. Eight rats were randomly selected from each group at 12, 24, and 36 h after modeling to detect levels of serum amylase, creatinine, and blood urea nitrogen. Serum levels of interleukin 1ß (IL-1ß) and interleukin 10 (IL-10) were detected by enzyme-linked immunosorbent assay (ELISA). Pathological changes of the left kidney were observed under light microscope. The expression of inducible nitric oxide synthase (iNOS) mRNA in the right kidney was detected with real time polymerase chain reaction (RT-PCR). RESULTS: Compared with the S group, serum levels of amylase, creatinine (Cr), blood urea nitrogen (BUN), IL-1ß, IL-10, and iNOS mRNA expression significantly increased in the M group (P < 0.01). The function of kidney and pancreas were obviously improved in the RHO group than in the M group. Levels of IL-1ß and iNOS significantly decreased, but IL-10 levels significantly increased in the RHO group with statistical difference (P < 0.05). CONCLUSION: Rhodiola had better protective effect on SAP associated renal injury, which might be achieved through inhibiting the expression of IL-1ß, stimulating the expression of IL-10, down-regulating iNOS mRNA expression, reducing the generation of oxygen free radicals and NO damage to cells, and improving hypoxia tolerance capabilities of the kidney.

Polyphasic characterization of two microbial consortia with wide dechlorination spectra for chlorophenols.

Two soil-free anaerobic dechlorinating cultures (3-CP and 35-DCP) were enriched from a pentachlorophenol (PCP)-to-phenol dechlorinating soil-dependent culture, using 3-chlorophenol (3-CP) and 3,5-dichlorophenol (3,5-DCP) as specific respective substrates, and characterized polyphasically. Physiological characterization indicated that the 3-CP and 35-DCP cultures had similar features, but with some variations. Both cultures utilized formate or acetate preferably as optimum electron donors for reductive dechlorination, and they shared similar patterns of dechlorination spectra for chlorophenols ranging from mono-CPs to a tetra-CP, with preferred dechlorination pathways in the ortho and meta positions. Alternative electron acceptors such as NO(3)(-) but not SO(4)(2-) inhibited the dechlorination activity in both cultures, while amorphous iron oxides (FeOOH) suppressed dechlorination activity only in the 35-DCP culture. Complete inhibition of dechlorination was observed in both cultures supplemented with chloramphenicol and vancomycin. The addition of 2-bromoethanesulfonate resulted in delayed dechlorination activity in the 35-DCP culture but not in the 3-CP culture; molybdate did not exert any inhibitory effect in either culture. Phylogenetic analysis based on 16S rRNA genes confirmed that the two cultures exhibited similar bacterial species but with varied responsible dechlorinators. Dehalobacter spp. were the likely dechlorinators in the 3-CP culture versus Sulfurospirillum spp. in the 35-DCP culture, with Clostridium and Clostridium-like spp. as candidate dechlorinators in both cultures.

Endocrine traits of polycystic ovary syndrome in prenatally androgenized female Sprague-Dawley rats.

Although hyperandrogenism is an important condition and is considered the possible pathogenesis behind polycystic ovary syndrome (PCOS), data supporting this is still scarce. We sought to determine whether or not prenatal androgen exposure leads to PCOS and the possible cellular mechanisms involved. To induce prenatal androgen exposure, pregnant rats were treated with daily subcutaneous injections of free testosterone (T) or dihydrotestosterone (DHT) from embryonic days 16 to 19, and their female offspring were studied as adults. The mRNA expression of the progesterone receptor (PR) in the preoptic area (POA) hypothalamus was higher in the experimental groups than in the control group after ovariectomy and stimulation with estradiol benzoate. The levels of T, P, leutinizing hormone (LH), and estradiol were higher in the experimental groups than in the control groups. The frequency and magnitude of LH secretion was increased in experimental rats as compared with the control group. The anogenital distance of the experimental groups was prolonged and the nipple number was lower than that of the control group. Almost all experimental rats had prolonged or irregular estrous cycles. The experimental groups had fewer corpus luteum and preovulatory follicles and more preantral follicles and antral follicles than the controls. Our findings are consistent with the hypothesis that excess androgen during the prenatal period may cause PCOS. Additionally, we show that hyperandrogenic interference in the release of preovulatory LH surges is mediated by the suppressive effects of androgens on PR expression in POA-hypothalamic tissue.

Protective effects of ascorbate and catalase on human spermatozoa during cryopreservation.

Human semen cryopreservation in the clinical management of male infertility is complicated by cryodamage to spermatozoa. We aimed to clarify the full pattern of cryodamage and evaluate the protective effects of ascorbate and catalase on cryopreserved spermatozoa. Semen samples were collected from 30 fertile males. Each sample was divided into 6 groups: fresh semen, cryopreserved semen without treatment, and samples cryopreserved with ascorbate (300 or 600 µM) or catalase (200 or 400 IU/mL). Spermatozoa were examined for their viability, motility, reactive oxygen species (ROS) generation, mitochondrial membrane potential (MMP), apoptosis (positive for annexin V and negative for propidium iodide [ie, Ann(+)/PI(-)]), and DNA damage (Olive tail moment [OTM]) in the presence or absence of ascorbate or catalase during cryopreservation. In comparison with the fresh spermatozoa, there was a significant decrease in the viability, motility, and MMP but increase in Ann(+)/PI(-) and OTM in the cryopreserved spermatozoa (P < .01 and P < .05, respectively). Concurrently, ROS levels in the postthaw spermatozoa also increased significantly, and this elevation was well correlated with the quality variations of postthaw spermatozoa (P < .01 for all). Ascorbate (300 µM) and catalase (200 and 400 IU/mL) reduced the ROS levels in postthaw spermatozoa significantly, compared with those in the control (P < .05). Furthermore, these antioxidants also prevented those characteristics from being adversely affected (P < .05). This study demonstrated that cryopreservation results in cryodamage to human spermatozoa, possibly through the mechanism of ROS. Appropriate ascorbate or catalase supplementation of cryoprotective medium restrains ROS levels and the resultant cryodamage.

[Reducing oxidative DNA damage by adding antioxidants in human semen samples undergoing cryopreservation procedure].

OBJECTIVE: To evaluate the protective effects of oxidative DNA damage by adding antioxidants: ascorbate, catalase (CAT), and superoxide dismutase (SOD) in human semen samples undergoing cryopreservation procedure. METHODS: Semen sample form 30 fertile men were mixed with modified cryoprotectant and divided into six groups according to the category and concentration of antioxidants: ascorbate 300 micromol/L, ascorbate 600 micromol/L, CAT 200 U/ml, CAT 400 U/ml, SOD 200 U/ml, and SOD 400 U/ml. Comet assay was conducted to measure the percentage of comet cells, and the nuclear DNA damaged parameters: tail DNA percentage (TD%) and Olive tail moment (OTM). Flow cytometry was used to detect the reactive oxidative species (ROS). The motility (a + b grade), viable recovery rate, nuclear DNA integrity and reactive oxidative species (ROS) of all groups were analyzed before and/or after freeze-thawing. RESULTS: (After cryopreservation, compared with the control group, the a + b grade sperm rates of the ascorbate 300 micromol/L, CAT 200 U, and CAT 400 U groups were all higher than that of the control group (all P < 0.05), however, the levels of reactive oxygen species (ROS) of the ascorbate 300 micromol/L, CAT 200 U, and CAT 400 U groups were 30 +/- 13, 30 +/- 11, and 30 +/- 11 respectively, all significantly lower than that of the control group (37 +/- 17 , all P < 0.05). The viable recovery rates of the ascorbate 300 micromol/L , CAT 200 U, and CAT 400 U groups were 67% +/- 14%, 68% +/- 14%, and 69% -/+ 15% respectively, all significantly higher than that of the control group (59% +/- 10%, all P < 0.05). (2) The TD% levels of the ascorbate 300 micromol/L, CAT 200 U, and CAT 400 U groups were 41% +/- 4%, 40% +/- 7%, 40% +/- 6%, all similar to that of the raw semen (all P > 0.05), but significantly lower than that of the control group (46% +/- 6%, all P < 0.01). The OTM levels of the ascorbate 300 micromol/ L, CAT 200 U, and CAT 400 U groups were 7.7 +/- 1.2, 7.5 +/- 1.6, and 7.8 +/- 1.9, all similar to that of the raw semen (all P > 0.05), but significantly lower than that of the control group (10.1 +/- 3.1, all P < 0.01) too. The TD% and OTM levels of the other groups were all significantly higher than that of the raw semen (all P < 0.01), but not significantly different from those of the control group (all P > 0.05). (3) ROS was significantly negatively correlated with the motility in all groups (P < 0.05 or P < 0.01). Apart from the ascorbate 600 micromol/L group, the TD% and OTM of the other groups were all significantly positively correlated with the ROS (P < 0.05 or P < 0.01). CONCLUSION: Supplementation of ascorbate or CAT reduces the level of ROS that induces sperm nuclear DNA damage, and improves the human sperm quality in the process of freeze-thawing.