Analysis of the relationship between drug susceptibility of Cryptococcus neoformans isolates and mortality in HIV-negative cryptococcal meningitis.

OBJECTIVES: The relationship between antifungal susceptibility and mortality of cryptococcal meningitis (CM) in HIV-negative patients is poorly understood. METHODS: We conducted a retrospective analysis of 1-year follow-up of 200 HIV-negative CM patients with an initial cerebrospinal fluid (CSF) culture for Cryptococcus neoformans. According to the cut-off values of minimum inhibitory concentration (MIC), two groups of five antifungal agents were classified: amphotericin B (AmB), ≤0.5 µg/mL, >0.5 µg/mL; 5-flucytosine (5-FC), ≤4 µg/mL, >4 µg/mL; fluconazole (FLU), ≤4 µg/mL, >4 µg/mL; itraconazole (ITR), ≤0.125 µg/mL, >0.125 µg/mL; and voriconazole (VOR), <0.25 µg/mL, ≥0.25 µg/mL. Comparisons were performed to analyse clinical features, laboratory, modified Rankin Scale (mRS) scores, and CSF findings under different prognosis outcomes in 1-year. RESULTS: All of Cryptococcus neoformans isolates were sensitive to AmB and VOR, most of them were sensitive to 5-FC and FLU (95.5% and 90.5%, respectively) while only 55.0% of them were susceptible to ITR. Minimum inhibitory concentrations of ITR and VOR were significantly related to baseline mRS scores. All-cause mortality was not significantly related to MICs in Cryptococcus neoformans strains. The combination of actual antifungal agents and two groups of the MICs values for antifungal agents had no significant effects on all-cause mortality. CONCLUSION: Most Cryptococcus neoformans isolates were sensitive to AmB, VOR, 5-FC, and FLU. Because of the small number of deaths, we are not able to comment on whether MIC is associated with mortality of CM in HIV-negative patients.

Ultrasonic-Enabled Nondestructive and Substrate-Independent Liquid Metal Ink Sintering.

Printing or patterning particle-based liquid metal (LM) ink is a good strategy to overcome poor wettability of LM for its circuits' preparation in flexible and printed electronics. Subsequently, a crucial step is to recover conductivity of LM circuits consisting of insulating LM micro/nano-particles. However, most widely used mechanical sintering methods based on hard contact such as pressing, may not be able to contact the LM patterns' whole surface conformally, leading to insufficient sintering in some areas. Hard contact may also break delicate shapes of the printed patterns. Hereby, an ultrasonic-assisted sintering strategy that can not only preserve original morphology of the LM circuits but also sinter circuits on various substrates of complex surface topography is proposed. The influencing factors of the ultrasonic sintering are investigated empirically and interpreted with theoretical understanding by simulation. LM circuits encapsulated inside soft elastomer are successfully sintered, proving feasibility in constructing stretchable or flexible electronics. By using water as energy transmission medium, remote sintering without any direct contact with substrate is achieved, which greatly protect LM circuits from mechanical damage. In virtue of such remote and non-contact manipulation manner, the ultrasonic sintering strategy would greatly advance the fabrication and application scenarios of LM electronics.

Erratum: A crack compensation strategy for highly stretchable conductors based on liquid metal inclusions.

[This corrects the article DOI: 10.1016/j.isci.2022.105495.].

A crack compensation strategy for highly stretchable conductors based on liquid metal inclusions.

Crack control strategies have been proven very useful for enhancing the stretchability of metal film-based stretchable conductors. However, existing strategies often suffer from the drawbacks of complicated preparation and predefined effective directions. Here, we propose a crack compensation strategy for preparing conductors featured with high stretchability by using liquid metal microparticles (LMMPs)-embedded polydimethylsiloxane (PDMS) as the substrate with a thin film of gold (Au) sputtered on the surface. LMMPs can be elongated to connect the cracked Au film upon stretching, which can form a conductive "island-tunnel" (IT) architecture to compensate for the cracks and maintain the conductivity. The high performance of the stretchable conductor is demonstrated by using it as electrodes to record surface electromyography of human brachioradialis and monitor electrocorticography signals of a rat in normal and epileptic states. The developed strategy shows the potential to provide a new perspective for the fabrication of flexible electronics.

Chitosan nanoparticles attenuate hydrogen peroxide-induced stress injury in mouse macrophage RAW264.7 cells.

This study was carried out to investigate the protective effects of chitosan nanoparticles (CNP) against hydrogen peroxide (H2O2)-induced oxidative damage in murine macrophages RAW264.7 cells. After 24 h pre-incubation with CNP (25-200 µg/mL) and chitosan (CS) (50-200 µg/mL, as controls), the viability loss in RAW264.7 cells induced by H2O2 (500 µM) for 12 h was markedly restored in a concentration-dependent manner as measured by MTT assay (P < 0.05) and decreased in cellular LDH release (P < 0.05). Moreover, CNP also exerted preventive effects on suppressing the production of lipid peroxidation such as malondialdehyde (MDA) (P < 0.05), restoring activities of endogenous antioxidant including superoxide dismutase (SOD), and glutathione peroxidase (GSH-Px) (P < 0.05), along with increasing total antioxidant capacity (T-AOC) (P < 0.05). In addition, pre-incubation of CNP with RAW264.7 cells for 24 h resulted in the increase of the gene expression level of endogenous antioxidant enzymes, such as MnSOD and GSH-Px (P < 0.05). At the same concentration, CNP significantly decreased LDH release and MDA (P < 0.05) as well as increased MnSOD, GSH-Px, and T-AOC activities (P < 0.05) as compared to CS. Taken together, our findings suggest that CNP can more effectively protect RAW264.7 cells against oxidative stress by H2O2 as compared to CS, which might be used as a potential natural compound-based antioxidant in the functional food and pharmaceutical industries.

Changes in small intestinal morphology and digestive enzyme activity with oral administration of copper-loaded chitosan nanoparticles in rats.

The experiment was conducted to evaluate the effect of copper-loaded chitosan nanoparticles on the small intestinal morphology and activities of digestive enzyme and mucosal disaccharase in rats. Forty male Sprague-Dawley rats, with average body weight of 82 g, were randomly allotted to five groups (n = 8). All rats were received a basal diet (control) or the same basal diet added with 80 mg/kg BW CuSO(4), 80 mg/kg BW chitosan (CS-I), 80 mg/kg BW copper-loaded chitosan nanoparticles (CSN-I), 160 mg/kg BW copper-loaded chitosan nanoparticles (CSN-II), respectively. The experiment lasted 21 days. The results showed that the villus heights of the small intestinal mucosa in groups CSN-I and CSN-II were higher than those of the control, group CuSO(4) or CS-I. The crypt depth of duodenum and ileum mucosa in group CSN-I or CSN-II was depressed. Compared with the control, there were no significant effects of CuSO(4) or CS-I on the villus height and crypt depth of small intestinal mucosa. Supplementation with CSN improved the activities of trypsin, amylase and lipase in the small intestinal contents and maltase, sucrase and lactase of duodenum, jejunum, and ileum mucosa while there were no significant effects of CuSO(4) on the digestive enzyme activities of the small content compared with the control. The results indicated that intestinal morphology, activities of digestive enzyme in digesta and mucosal disaccharase were beneficially changed by treatment of copper-loaded chitosan nanoparticles.

Chitosan nanoparticles act as an adjuvant to promote both Th1 and Th2 immune responses induced by ovalbumin in mice.

The study was conducted to investigate the promoted immune response to ovalbumin in mice by chitosan nanoparticles (CNP) and its toxicity. CNP did not cause any mortality or side effects when mice were administered subcutaneously twice with a dose of 1.5 mg at 7-day intervals. Institute of Cancer Research (ICR) mice were immunized subcutaneously with 25 µg ovalbumin (OVA) alone or with 25 µg OVA dissolved in saline containing Quil A (10 µg), chitosan (CS) (50 µg) or CNP (12.5, 50 or 200 µg) on days 1 and 15. Two weeks after the secondary immunization, serum OVA-specific antibody titers, splenocyte proliferation, natural killer (NK) cell activity, and production and mRNA expression of cytokines from splenocytes were measured. The serum OVA-specific IgG, IgG1, IgG2a, and IgG2b antibody titers and Con A-, LPS-, and OVA-induced splenocyte proliferation were significantly enhanced by CNP (P < 0.05) as compared with OVA and CS groups. CNP also significantly promoted the production of Th1 (IL-2 and IFN-γ) and Th2 (IL-10) cytokines and up-regulated the mRNA expression of IL-2, IFN-γ and IL-10 cytokines in splenocytes from the immunized mice compared with OVA and CS groups. Besides, CNP remarkably increased the killing activities of NK cells activity (P < 0.05). The results suggested that CNP had a strong potential to increase both cellular and humoral immune responses and elicited a balanced Th1/Th2 response, and that CNP may be a safe and efficacious adjuvant candidate suitable for a wide spectrum of prophylactic and therapeutic vaccines.

Effects of montmorillonite on alleviating dietary Cd-induced oxidative damage in carp (Carassius auratus).

The present study was designed to investigate the effects of montmorillonite (MMT) on dietary Cd-induced oxidative damage in liver and kidney of carp (Carassius auratus). One hundred eighty carp were randomly divided into four groups and fed with a basal diet, a basal diet supplemented with 0.5% MMT, Cd-comtaminated basal diet (120 mg Cd/kg dry weight) and Cd-contaminated basal diet supplemented with 0.5% MMT, respectively. After 60 days, fish were sacrificed to measure malondialdehyde (MDA) content and antioxidative indices in liver and kidney. The results showed that the exposure of carp to dietary Cd caused decreases in glutathione peroxidase activity, catalase activity, superoxide dismutase activity, glutathione content and total antioxidant capacity level, while MMT supplemented in diet compensated Cd-induced decreases in above antioxidant indices to some extent in liver and kidney. As compared with the control group, increases in MDA content were observed in both measured tissues of carp exposed to dietary Cd, while MDA content decreased in carp exposed to Cd-contaminated basal diet supplemented with MMT in comparison with the Cd-contaminated group. It was suggested that MMT, when co-administered with Cd in diet, could alleviate dietary Cd-induced oxidative damage in liver and kidney of carp.

Chemical composition and microstructure of uroliths associated with the feeding of high-level cottonseed meal diet to sheep.

The chemical composition and microstructure of five urolith samples (4 bladder stones and one kidney stone) associated with the feeding of high level of cottonseed meal (CSM) diet to Chinese merino fine wool sheep (Junken breed, Xinjiang) were examined by optical microscope, X-ray diffraction, X-ray energy dispersive spectrometry (EDS), scanning electron microscopy (SEM), and infrared spectroscopy analysis. The bladder stone samples appeared yellow or white, small powder and loose mass, and as finely granular under the optical microscope. However, the kidney stone samples from a experimental sheep were found as small brown mass, higher hardness, and as a cracklike structure. Oxygen, phosphorus, potassium and magnesium were found as four major elements in these uroliths by X-ray energy dispersive spectrometry (EDS). Potassium magnesium phosphate (MgKPO(4)) and potassium magnesium phosphate hexahydrate (MgKPO(4)·6H(2)O) were major components in the bladder stones, while less magnesium ammonium phosphate hexahydrate (MgNH(4)PO(4)·6H(2)O) examined by X-ray diffraction and infrared spectroscopy analysis. However, the newly found prismatic crystals, which were rich in magnesium and pyrophosphate, were identified as magnesium pyrophosphate (Mg(2)P(2)O(7)) in the kidney stone. The bladder stone samples appeared irregular mass and balls, cracked under SEM with low magnification, while appeared cracked, irregular layer-like, honeycomb-like or tiny balls under high magnification. The kidney stone samples were observed as cone, irregular block or layered crystal structures.

Adsorptive property of Cu(2+)-loaded montmorillonite clays for Escherichia coli K88 in vitro.

The adsorption properties of Cu(2+)-loaded montmorillonite clays (MMT-Cu) for Escherichia coli K88 as a function of time, bacteria concentrations, pH, ionic strength and temperature were investigated. The results showed that the bacteria adsorption onto MMT-Cu surface reached equilibrium after 90 min. The percentages of E. coli K88 adsorbed onto the surfaces of MMT-Cu and montmorillonite clays (MMT) at equilibrium were 88.9% and 56.5%, respectively. Scanning electron microscopy revealed that a lot of E. coli K88 adhered to the surface of MMT-Cu. The zeta potential of MMT-Cu was relatively high as compared to that of MMT. The adsorptive ability of MMT-Cu for E. coli K88 was higher than that of MMT (P < 0.05). Moreover, pH, ionic strength and temperature produced a strong influence on the extent of E. coli K88 adsorption to surface of MMT-Cu and MMT. The mechanism of adsorption of E. coli onto MMT-Cu may involve electrostatic attraction and physiochemical properties of bacterial cell walls and minerals surfaces.

Effects of montmorillonite on Pb accumulation, oxidative stress, and DNA damage in tilapia (Oreochromis niloticus) exposed to dietary Pb.

In order to investigate the effects of montmorillonite (MMT) on reducing dietary lead (Pb) toxicity to tilapia (Oreochromis niloticus), 240 fish were randomly divided into four treatments denominated as follows: control treatment (fed with a basal diet), MMT treatment (fed with a basal diet added with 0.5% MMT), Pb treatment (fed with a basal diet added with 100 mg Pb per kilogram dry weight (dw)), and Pb + MMT treatment (fed with a basal diet added with 100 mg Pb per kilogram dw and 0.5% MMT). Changes in Pb accumulation, oxidative stress, and DNA damage in tilapia were measured after 60 days. DNA damage was assessed using comet assay. The results showed that MMT supplemented in diet significantly reduced Pb accumulation in kidney and blood of tilapia exposed to dietary Pb (P < 0.05). Malondialdehyde level decreased insignificantly while levels of total antioxidant capacity and glutathione (GSH), activities of glutathione peroxidase, and superoxide dismutase increased insignificantly in kidney of tilapia in Pb + MMT treatment as compared to Pb treatment (P > 0.05). Significant decreases in tail length, tail DNA, tail moment, and Olive tail moment of peripheral blood cells in Pb + MMT treatment were observed when compared with Pb treatment (P < 0.05). The results indicated that dietary MMT supplementation could alleviate dietary Pb toxicity to tilapia effectively.

Immune responses of Fenneropenaeus chinensis against white spot syndrome virus after oral delivery of VP28 using Bacillus subtilis as vehicles.

The protective efficacy of oral administration of VP28 using Bacillus subtilis as vehicles (rVP28-bs) in shrimp, Fenneropenaeus chinensis, upon challenge with white spot syndrome virus (WSSV) was investigated. The calculated relative percent survival (RPS) value of rVP28-bs fed shrimp was 83.3% when challenged on the 14th day post-administration, which is significantly higher (p < 0.001) than that of the group administered recombinant Escherichia coli over-expressing rVP28 (rVP28-e21). After immunization, activities of phenoloxidase (PO), superoxide dismutase (SOD) and inducible nitric oxide synthase (iNOS) in hemolymph were analyzed. It was found that the supplementation of rVP28-bs into shrimp food pellets resulted in the most pronounced increase of iNOS activity (p < 0.001), but had the least influence on activities of PO and SOD. Besides, in the shrimp orally administered with rVP28-bs, the caspase-3 activity was one-fifth that of the control, though the signs of apoptosis (chromatin margination, nuclear fragmentation and apoptotic bodies) could not be observed by transmission electron microscope (TEM). These results suggest that by oral delivery of rVP28-bs, shrimp showed significant resistance to WSSV and an effect on the innate immune system of shrimp. The remarkably enhanced level of iNOS after rVP28-bs administration might be responsible for antiviral defense in shrimp.

Influence of Cu2+-loaded silicate on the growth performance and microflora of crucian carp Carassius auratus.

We investigated the effects of Cu2+-loaded silicate (CLS) on the growth performance, microflora of skin, gill and intestine, and intestinal morphology of crucian carp Carassius auratus. A total of 225 native wild crucian carp, with an average initial body weight of 20 g, were randomly divided into 5 treatment groups using 3 replicate tanks of 15 fish per group. The dietary treatments were (1) basal diet, (2) basal diet + CuSO4, (3) basal diet + silicate, (4) basal diet + 0.5% CLS and (5) basal diet + 50 mg kg(-1) chlortetracycline (CTC, purity 98.8%). The trial lasted for 60 d. We found that body weight increased slightly while feed conversion ratio decreased in the CLS-treated group compared with the control groups. The total number of aerobic bacteria counted in the intestine of carp fed the diet supplemented with the CLS (i.e. Vibrio sp. and E. coli), was significantly lower (p = 0.05) compared with the control groups and the CTC-treated fish, while lactobacillus counts were significantly higher (p = 0.05). Lactobacilli counts of the intestine increased significantly (p = 0.05). However, the microflora of the skin and gill was not affected by the addition of CuSO4, silicate, CLS or CTC. The height of the villi in the proximal, mid and distal intestine mucosa of the silicate- and CLS-treated groups was found to be longer (p = 0.05) compared with the villi of the control or the CTC-treated fish. Supplementation with CuSO4 had no effect on the microflora and the intestinal morphology (p = 0.05). These results indicate that CLS had an antibacterial activity in vivo, which may help protect the intestinal mucosa from invasion of pathogenic bacteria and their toxins.

Mechanism of anti-angiogenic activities of chitooligosaccharides may be through inhibiting heparanase activity.

Metastatic disease is the primary cause of death for most cancer patients. Angiogenesis is the formation of a new capillary network from pre-existing vessels and required for tumor vasculature. Heparanase, a beta-endoglucuronidase, assistants tumor invasion, metastasis and angiogenesis. Chitooligosaccharides (COS) is obtained by hydrolysis of chitosan. COS has been proved to be anti-angiogenesis activity. The mechanism of COS inhibits angiogenesis is not very clear, COS is hypothesized by author to be an inhibitor of heparanase.

Protection of Procambarus clarkii against white spot syndrome virus using inactivated WSSV.

White spot syndrome virus (WSSV) is a highly pathogenic and prevalent virus infecting shrimp and other crustaceans. The potentiality of binary ethylenimine (BEI)-inactivated WSSV against WSSV in crayfish, Procambarus clarkii, was investigated in this study. Efficacy of BEI-inactivated WSSV was tested by vaccination trials followed by challenge of crayfish with WSSV. The crayfish injected with BEI-inactivated WSSV showed a better survival (P<0.05) to WSSV on the 7th and 21st day post-vaccination (dpv) compared to the control. Calculated relative percent survival (RPS) values were 77% and 60% on the 7th and 21st dpv for 2mM BEI-inactivated WSSV, and 63%, 30% on 7th and 21st dpv for 3mM BEI-inactivated WSSV. However, heat-inactivated WSSV did not provide protection from WSSV even on 7th dpv. In the inactivation process WSSV especially their envelope proteins maybe changed as happened to 3mM BEI and heat-inactivated WSSV particles. These results indicate the protective efficacy of BEI-inactivated WSSV lies on the integrity of envelope proteins of WSSV and the possibility of BEI-inactivated WSSV to protect P. clarkii from WSSV.

Chitosan nanoparticles inhibit the growth of human hepatocellular carcinoma xenografts through an antiangiogenic mechanism.

Chitosan nanoparticles (CNP) have demonstrated anticancer activity in vitro and in vivo by a few recent researches. However, the mechanisms involved in their potential anticancer activity remain to be elucidated. In this study, the effects of CNP on tumor growth were investigated using a model of nude mice xenografted with human hepatocellular carcinoma (HCC) (BEL-7402) cells. The results demonstrated that the treatment of these nude mice with CNP significantly inhibited tumor growth and induced tumor necrosis. Furthermore, microvessel density (MVD) determination by counting immunohistologically stained tumor microvessels suggested that CNP dose-dependent tumor suppression was correlated with the inhibition of tumor angiogenesis. Mechanistically, immunohistochemical and quantitative real-time reverse transcription-polymerase reaction assays provided evidence that CNP-mediated inhibition of tumor angiogenesis was linked to impaired levels of vascular endothelial growth factor receptor 2 (VEGFR2). Due to their low or non-toxicity, CNP and their derivatives may represent a novel class of anti-cancer drug.

Effects of dietary Pb on accumulation, histopathology, and digestive enzyme activities in the digestive system of tilapia (Oreochromis niloticus).

With the increasing occurrence of dietary lead (Pb) contamination in aquatic environment, threat of the dietary Pb toxicity to aquatic organisms attracted more attention. In this study, after being exposed to dietary Pb at concentrations of 0, 100, 400, and 800-microg/g dry weight for 60 days, the groups of tilapia (Oreochromis niloticus) were sacrificed and sampled to analyze the effects of dietary Pb on accumulation, histopathology, and digestive enzyme activities in tissues of the digestive system. The results showed that the Pb accumulation in tissues increased with the dietary Pb concentrations. Moreover, Pb accumulated in sampled tissues in the following order: intestine > stomach > liver. By observation of liver histological sections in optical microscope, lesions could be detected in the Pb-contaminated groups. It was also demonstrated that the inhibitory effect of dietary Pb on digestive enzyme activities was dietary Pb concentration dependent. Different degrees of inhibition of enzyme activities were exhibited in sampled tissues. It was indicated that digestive enzyme activities in the digestive system might be considered as the potential biomarkers of dietary Pb contamination in tilapia.

Changes in growth performance, metabolic enzyme activities, and content of Fe, Cu, and Zn in liver and kidney of tilapia (Oreochromis niloticus) exposed to dietary Pb.

Tilapia (Oreochromis niloticus) were exposed to 0, 100, 400, and 800 microg/g concentrations of Pb in diet for 60 days, and changes in growth performance, metabolic enzyme activities, and essential trace elements (Fe, Cu, and Zn) content in liver and kidney were investigated. Daily weight gain, feed conversation ratio, and survival of tilapia were not significantly affected by dietary Pb. Alanine transaminase (ALT), aspartate transaminase (AST), and lactate dehydrogenase (LDH) activities in liver and kidney were affected by dietary Pb in a dissimilar way: Pb concentration-related decreases in ALT, AST, and LDH activities were observed in kidney, while these enzyme activities in liver were stimulated in a Pb concentration-dependent manner. It was demonstrated that the inhibitory effects of dietary Pb on alkaline phosphatase, Na, K-adenosine triphosphatase (ATPase), Ca, and Mg-ATPase activities in both liver and kidney were Pb concentration-dependent. It was also indicated that the content of Fe, Cu, and Zn in liver and kidney decreased with the increasing dietary Pb concentrations. The results suggested that long-term dietary Pb exposure could affect metabolic enzyme activities and the content of Fe, Cu, and Zn in liver and kidney, whereas growth impairment was not observed in tilapia.

High-level secretion of a chimeric thermostable lichenase from Bacillus subtilis by screening of site-mutated signal peptides with structural alterations.

A chimeric gene mHG (669 bp) was constructed by substitution of Clostridium thermocellum ZJL4 lichenase (CG) N-terminal fragment (except its signal sequence) for the counterpart of Bacillus sp. A3 lichenase (BG). To acquire high-level secretion of the chimeric lichenase (mHG) in Bacillus subtilis, a series of site-mutated signal peptides were designed. The activity of mHG, which was directed by an artificial hydrophobic signal peptide H1 (MMARKIAGMATSLLVIFSSSAVA) from cytoplasm into growth medium, reached 80.56 U/ml after 22-h incubation, indicating that signal peptide hydrophobicity appears to be critical for early stages in mHG export. By purification of the mHG (approximately 25.3 kDa) from cultures of B. subtilis (pBSG-H1), enzymatic property assays showed that the common optima for mHG were 70 degrees C and pH 5.0. The residual activity of mHG at 90 degrees C for 10 min was 83.45% of its maximum activity, which was almost similar to that of CG (90 degrees C, 10 min, 84.33%). This constructed shuttle expression vector with a novel signal peptide exhibited its applicability for high-level production of heterologous proteins from B. subtilis. Moreover, the high-level secreted mHG with relatively high thermostability could be a potential candidate for feed industrial applications.

Effects of different activation protocols on preimplantation development, apoptosis and ploidy of bovine parthenogenetic embryos.

The objective of this study was to optimize the protocols for bovine oocytes activation through comparing the effectiveness of different treatments on the activation and subsequent development of oocytes and examining the effects of two combined activation treatments on the blastocyst apoptosis and ploidy. Cumulus-oocyte complexes (COCs) were recovered from abattoir-derived ovaries and matured in vitro. After maturation, cumulus-free oocytes were activated according to the experiment designs. Activated oocytes were cultured in vitro in modified synthetic oviductal fluid (mSOF) medium and assessed for pronuclear formation (15-16 h), cleavage (46-48 h) and development to the blastocyst stage. In Experiment 1, the matured oocytes were treated with single activation agents, including ionomycin (5 microM for 5 min), ethanol (7% for 7 min), calcium ionophore A23187 (5 microM for 5 min) or strontium (10mM for 5h). The pronuclear formation and cleavage rate were higher significantly in ionomycin (39.0 and 30.7%) and ethanol (41.5 and 28.1%) treatment alone compared to other treatments (9.7-25.2 and 11.3-23.7%, respectively, P<0.05). Very low blastocyst rates (3.9-5.3%) resulted which were not significantly different among treatments (P>0.05). For the combined activation treatment (Experiment 2), the same concentrations of ionomycin and ethanol as in Experiment 1 were used in combination with either 6-dimethylaminopurine (6-DMAP, 2.0 mM for 3 h) or cycloheximide (CHX)+cytochalasin B (CB, 10 microg/ml for 3 h). The pronuclear formation, cleavage rate, blastocyst rate and cell number of blastocyst were higher significantly (P<0.05) in ionomycin+6-DMAP treatment (67.1, 69.2, 28.0 and 91.3%, respectively) and ethanol+CHX+CB treatment (68.9, 70.2, 25.5 and 89.3%, respectively) compared to other treatments (11.7-58.1, 10.2-47.1, 1.5-24.2 and 34.2-62.7%, respectively). In Experiment 3, the parthenogenetic blastocysts produced by activation with ionomycin+6-DAMP and ethanol+CHX+CB and in vitro fertilized blastocysts (control group) were examined for apoptosis using a terminal deoxynucleotidyl transferase mediated deoxyuridine 5-triphosphate nick-end labeling (TUNEL) assay. The ethanol+CHX+CB treatment (7.0%) showed significantly lower blastocyst apoptosis index compared to ionomycin+6-DAMP treatment (9.1%, P<0.05). Furthermore, the chromosomal composition in the parthenotes embryos differed (P<0.05) among treatments. The percentage of haploid parthenotes was higher in ionomycin+6-DMAP treatment than ethanol+CHX+CB treatment. These results suggested that ethanol+CHX+CB treatment was more favorable protocol for parthenogenesis of bovine oocytes.