Effect of Gynostemma Pentaphyllum Polysaccharide on boar spermatozoa quality following freezing–thawing

Gynostemma Pentaphyllum Polysaccharide (GPP) was added at concentrations of 0.25, 0.5, 1.0, 1.5 and 2.0 mg/ml to the extenders used to freeze boar semen and its effects on the quality of frozen–thawed sperm were assessed. The sperm motility was significantly higher in the extenders containing 0.25 and 0.5 mg/ml GPP, as compared to other groups (P < 0.05). The extender supplemented with 0.5 mg/ml GPP favored the highest intact membrane and intact acrosome percentages in comparison with other groups (P < 0.05), respectively. The mitochondrial activity was significantly higher at the concentrations of 0.25, 0.5 and 1.0 mg/ml GPP than that of other treatments, and the control group (P < 0.05). In biochemical assays, the extender supplemented with 0.25 and 0.5 mg/ml GPP significantly improved SOD levels, compared to other groups (P > 0.05). However, the extenders supplemented with GPP did not cause significant differences in levels of CAT and GSH-Px, compared to the control (P > 0.05). In summary, GPP exhibited a dose-related response and the lower concentration produced greater protective effect. According to the standard semen quality parameters and antioxidant activities measured in this study, the concentration of 0.5 mg/ml GPP caused a beneficial cryoprotective effects on the quality of frozen–thawed boar semen. It is proposed that an extender containing 0.5 mg/ml GPP could be used as cryoprotective medium of better efficiency.     

The cryoprotective effect of low-density lipoproteins in extenders on bull spermatozoa following freezing–thawing

Egg low-density lipoprotein (LDL) was added at concentrations of 7–10% to the extenders used to freeze bull semen and its effects on the motility, mitochondria activity, acrosome integrity, membrane integrity and DNA integrity of frozen–thawed sperm were assessed. Analysis of data showed that the motility and characteristics of spermatozoa movement were higher with LDL in the extender, as compared to the extender containing 20% egg yolk. The results indicated that 8% LDL supplementation provided the highest sperm motility (55.8%) and movement characteristics (VSL, straight linear velocity: 33.8 μm/s; VCL, curvilinear velocity: 50.2 μm/s; LIN, linearity index: 56.5%; STR, mean coefficient: 76.7%; VAP, average path velocity: 35.9 μm/s; WOB, wobble coefficient: 63.9%). A concentration of 10% LDL resulted in a significant decline in the VSL, LIN, VAP and WOB values (P < 0.05). Supplementation of LDL at 8% LDL resulted in significantly higher spermatozoa mitochondrial activity, acrosome integrity, membrane integrity and DNA integrity (P < 0.05). According to all measured parameters, the extender containing 8% LDL showed beneficial cryoprotective effects on frozen–thawed bull spermatozoa. In conclusion, our results indicated that the extender containing 8% LDL extracted from egg yolk could be used successfully in the cryopreservation of bull semen with an efficacy that would be greater than present extenders containing 20% egg yolk.     

Effect of freezing–thawing process and quercetin on human sperm survival and DNA integrity

We aimed in the first part of our work to study the effect of cryopreservation on the human sperm DNA integrity and the activation of caspase 3, the main apoptosis indicator. In the second part, we were interested in testing the effect of quercetin, as an antioxidant, in preventing sperm damage during the freeze–thawing process. Seventeen semen samples were obtained from 17 men recruited for infertility investigations. Liquefied sperm was cryopreserved using spermfreeze®. Nine of the used samples were divided into two aliquots; the first one was cryopreserved with spermfreeze only (control) and the second one was cryopreserved with spermfreeze supplemented with quercetin to a final concentration of 50 μM. Sperm motility and viability were assessed according to WHO criteria. We used TUNEL assay and the Oxy DNA assay to assess sperm DNA integrity. Activated caspase 3 levels were measured in spermatozoa using fluorescein-labeled inhibitor of caspase (FLICA). Cryopreservation led to a significant increase in sperm DNA fragmentation, DNA oxidation and caspase 3 activation (p < 0.01). Supplementation of the cryopreservation medium with quercetrin induced a significant improvement in post thaw sperm parameters, compared to those of control, regarding sperm motility (p = 0.007), viability (p = 0.008) and DNA integrity (p = 0.02); however, it had no effect on caspase 3 activation (p = 0.3). We conclude that oxidative stress plays a major role in inducing sperm cryodamage but implication of apoptosis in this impairment requires further investigations. Quercetin could have protective effect during cryopreservation but further research is needed to confirm this effect.     

Is there a relationship between the chromatin status and DNA fragmentation of boar spermatozoa following freezing-thawing?

In this study a radioisotope method, which is based on the quantitative measurements of tritiated-labeled actinomycin D ((3)H-AMD) incorporation into the sperm nuclei ((3)H-AMD incorporation assay), was used to assess the chromatin status of frozen-thawed boar spermatozoa. This study also tested the hypothesis that frozen-thawed spermatozoa with altered chromatin were susceptible to DNA fragmentation measured with the neutral comet assay (NCA). Boar semen was diluted in lactose-hen egg yolk-glycerol extender (L-HEY) or lactose ostrich egg yolk lipoprotein fractions-glycerol extender (L-LPFo), packaged into aluminum tubes or plastic straws and frozen in a controlled programmable freezer. In Experiment 1, the chromatin status and DNA fragmentation were measured in fresh and frozen-thawed spermatozoa from the same ejaculates. There was a significant increase in sperm chromatin destabilization and DNA fragmentation in frozen-thawed semen as compared with fresh semen. The proportions of spermatozoa labeled with (3)H-AMD were concurrent with elevated levels of sperm DNA fragmentation in K-3 extender, without cryoprotective substances, compared with L-HEY or L-LPFo extender. Regression analysis revealed that the results of the (3)H-AMD incorporation assay and NCA for frozen-thawed spermatozoa were correlated. Boars differed significantly in terms of post-thaw sperm DNA damage. In Experiment 2, the susceptibility of sperm chromatin to decondensation was assessed using a low concentration of heparin. Treatment of frozen-thawed spermatozoa with heparin revealed enhanced (3)H-AMD binding, suggesting nuclear chromatin decondensation. The deterioration in post-thaw sperm viability, such as motility, mitochondrial function and plasma membrane integrity, was concurrent with increased chromatin instability and DNA fragmentation. This is the first report to show that freezing-thawing procedure facilitated destabilization in the chromatin structure of boar spermatozoa, resulting in an unstable DNA that was highly susceptible to fragmentation.     

Effects of freezing to −196 °C and thawing on Setaria lutescens seeds

The effects of single and repeated freezing and thawing of Setaria lutescens seeds in liquid nitrogen were investigated. One freeze to ?196 °C followed by a slow thaw, increased seed germination from 40 to 70%, but additional freeze-thaw cycles reduced germination to 30%. Using a scanning electron microscope, evidence was produced that seed coat cracking did not cause either initial increased, or subsequent reduced germination. Observations with a transmission electron microscope revealed that disruption of the integrity of lipid bodies accompanied increased damage from repeated freezing at ?196 °C and thawing. Repeated freezing and thawing of seeds stored in liquid nitrogen should be done with care to avoid loss of the germplasm.     

Effects of different heat–moisture treatments on the physicochemical properties of brown rice flour

We evaluated the effect of heat–moisture treatment (HMT) on the main chemical components, physical properties, and enzyme activities of two types of brown rice flour: high-amylose Koshinokaori and normal-quality Koshiibuki. Five different HMTs using brown rice (moisture content was 12.0%) were assessed: 0.1 MPa/120 °C for 5 or 10 min, 0.2 MPa/134 °C for 5 or 10 min and 0.3 MPa/144 °C for 10 min. HMT, decreased the α-amylase and lipase activities, and fat acidity, and slightly increased the dietary fiber and resistant starch levels. After 2 months’ storage at 35 °C, rice samples that were treated with 0.2 MPa/134 °C or 0.3 MPa/144 °C for 10 min had a lower fat acidity than untreated samples, which would be useful for long-term storage and export of rice flour. And HMT exhibited inhibition of retrogradation in the pasting and physical properties, which is profitable to promote the qualities of the rice products.     

Superoxide dismutase (SOD) in boar spermatozoa: Purification,biochemical properties and changes in activity during semen storage (16 °C) in different extenders

The antioxidant system in semen is composed of enzymes, low-molecular weight antioxidants and seminal plasma proteins. Loss of enzymatic activity of superoxide dismutase (SOD) during semen preservation may cause insufficient antioxidant defense of boar spermatozoa. The aim of this study was to isolate and characterize SOD molecular forms from spermatozoa and to describe changes in SOD activity in boar sperm during preservation at 16 °C. Sperm extracts were prepared from fresh or diluted semen and used for SOD purification or activity measurement. Ion-exchange chromatography and gel filtration was used to purify SOD molecular forms. BTS, Dilu Cell, M III and Vitasem were used as diluents for 5-day storage of semen at +16 °C. The molecular form of SOD released from spermatozoa after cold shock and homogenization had a molecular weight of approximately 67 kDa. The activity of the SOD form was the highest at pH 10 within the temperature range between 20 and 45 °C. The enzymatic activity of form released after cold shock was inhibited by H₂O₂ and diethyldithiocarbamate (DDC; by 65 and 40%, respectively). The SOD form released by homogenization was inhibited by H₂O₂ and DDC (40%). The molecular form released after urea treatment was a 30 kDa protein with maximum activity at 20 °C and pH 10. Enzymatic activity of this form was inhibited by H₂O₂ by 35%, DDC by 80% and 2-mercaptoethanol by 15%. The antigenic determinants of SOD isolated from boar seminal plasma and spermatozoa were similar to each other. Susceptibility of spermatozoa to cold shock increased during storage, but the differences between extenders were statistically non-significant.     

Effects of supercoiling on enhancer–promoter contacts

Using Brownian dynamics simulations, we investigate here one of possible roles of supercoiling within topological domains constituting interphase chromosomes of higher eukaryotes. We analysed how supercoiling affects the interaction between enhancers and promoters that are located in the same or in neighbouring topological domains. We show here that enhancer–promoter affinity and supercoiling act synergistically in increasing the fraction of time during which enhancer and promoter stay in contact. This stabilizing effect of supercoiling only acts on enhancers and promoters located in the same topological domain. We propose that the primary role of recently observed supercoiling of topological domains in interphase chromosomes of higher eukaryotes is to assure that enhancers contact almost exclusively their cognate promoters located in the same topological domain and avoid contacts with very similar promoters but located in neighbouring topological domains.     

Effects of epibiosis on consumer–prey interactions

In many benthic communities predators play a crucialrole in the population dynamics of their prey. Surfacecharacteristics of the prey are important forrecognition and handling by the predator. Because theestablishment of an epibiotic assemblage on thesurface of a basibiont species creates a new interfacebetween the epibiotized organism and its environment,we hypothesised that epibiosis should have an impacton consumer–prey interactions. In separateinvestigations, we assessed how epibionts onmacroalgae affected the susceptibility of the latterto herbivory by the urchin Arbacia punctulataand how epibionts on the blue mussel Mytilusedulis affected its susceptibility to predation bythe shore crab Carcinus maenas.Some epibionts strongly affected consumer feedingbehavior. When epibionts were more attractive thantheir host, consumer pressure increased. Whenepibionts were less attractive than their host or whenthey were repellent, consumer pressure decreased. Insystems that are controlled from the top-down,epibiosis can strongly influence community dynamics.For the Carcinus/Mytilus system that westudied, the in situ distribution of epibiontson mussels reflected the epibiosis-determinedpreferences of the predator. Both direct and indirecteffects are involved in determining theseepibiont-prey–consumer interactions.     

Effects of non-linearity on cell–ECM interactions

Filamentous biopolymers such as F-actin, vimentin, fibrin and collagen that form networks within the cytoskeleton or the extracellular matrix have unusual rheological properties not present in most synthetic soft materials that are used as cell substrates or scaffolds for tissue engineering. Gels formed by purified filamentous biopolymers are often strain stiffening, with an elastic modulus that can increase an order of magnitude at moderate strains that are relevant to cell and tissue deformation in vivo. This review summarizes some experimental studies of non-linear rheology in biopolymer gels, discusses possible molecular mechanisms that account for strain stiffening, and explores the possible relevance of non-linear rheology to the interactions between cell and extracellular matrices.     

Fluxes of nitrous oxide, methane and carbon dioxide during freezing–thawing cycles in an Inner Mongolian steppe

Combined measurements of nitrification activity and N₂O emissions were performed in a lowland and a montane tropical rainforest ecosystem in NE-Australia over a 18 months period from October 2001 until May 2003. At both sites gross nitrification rates, measured by the BaPS technique, showed a strong seasonal pattern with significantly higher rates of gross nitrification during wet season conditions. Nitrification rates at the montane site (1.48?±?0.24–18.75?±?2.38 mg N kg^?1 day^?1) were found to be significantly higher than at the lowland site (1.65?±?0.21–4.54?±?0.27 mg N kg^?1 day^?1). The relationship between soil moisture and gross nitrification rates could be described best by O’Neill functions having a soil moisture optimum of nitrification at app. 65% WFPS. At the lowland site, for which continuous measurements of N₂O emissions were available, nitrification was positively correlated with N₂O emission. Nitrification contributed significantly to N₂O formation during dry season (app.85%) but less (app. 30%) during wet season conditions. In average 0.19‰ of the N metabolized by nitrification was released as N₂O. The N₂O fraction loss for nitrification was positively correlated with changes in soil moisture and varied slightly between 0.15 and 0.22‰. Our results demonstrate that combined N₂O emission and microbial N turnover studies covering prolonged observation periods are needed to clarify and quantify the role of the microbial processes nitrification and denitrification for annual N₂O emissions from soils of terrestrial ecosystems.     

Effects of amyloid-β peptide Aβ25–35 on glycolytic and antioxidant enzymes in erythrocytes of different ages

Amyloid-β peptide Aβ_25–35 was shown to cause lysis of rat erythrocytes of different ages. The toxicity of Aβ_25–35 positively correlated with both the erythrocyte age and the peptide concentration. The activity of glycolytic, antioxidant, and Na⁺/K⁺-ATPase enzymes decreased with erythrocyte aging in vivo. In vitro Aβ_25–35 reduced the activity of hexokinase, phosphofructokinase, pyruvate kinase, glutathione peroxidase, and glutathione transferase and increased Na⁺/K⁺-ATPase activity in aged erythrocytes to a greater degree than in young cells.     

Evaluation of Tris–citric acid,skim milk and sodium citrate extenders for liquid storage of Punjab Urial (Ovis vignei punjabiensis) spermatozoa

The Punjab Urial (Ovis vignei punjabiensis) is an endangered subspecie of ovidae, distributed as small scattered populations in the forest belt of the Himalayan foothills of Pakistan and in the areas enclosed by the Indus and the Jhelum rivers. The present study was conducted to evaluate the liquid storage of Punjab Urial spermatozoa in different extenders for use in future in situ conservation activities. Semen was collected by electro-ejaculation from three captive Punjab Urial rams. Suitable ejaculates of individual animals were pooled and divided into three aliquots for dilution with the experimental extenders (Tris–citric acid, skim milk and sodium citrate) at 37 °C. Extended semen was cooled from 37 °C to 5 °C in 2 h, and stored for three days at 5 °C. Sperm motility (%), viability (%; live/dead), acrosome integrity (%) and plasma membrane integrity (%) were assessed on days 1, 2 and 3 of storage. On day 1, sperm motility, viability as well as acrosome and plasma membrane integrity were similar (p > 0.05) in all three experimental extenders. On day 2, sperm motility, viability, acrosome and plasma membrane integrity were higher (p < 0.05) in Tris–citric acid extender compared to sodium citrate based extender. On day 3 of storage, the values of motility, viability and acrosome integrity were higher (p < 0.05) in Tris–citric acid extender than in skim milk and sodium citrate based extenders. In conclusion, Tris–citric acid extender appears to be a better option compared with skim milk and sodium citrate extenders for liquid storage of Punjab Urial semen.     

Effects of nutritional stress during different developmental periods on song and the hypothalamic–pituitary–adrenal axis in zebra finches

In songbirds, developmental stress affects song learning and production. Altered hypothalamic–pituitary–adrenal (HPA) axis function resulting in elevated corticosterone (CORT) may contribute to this effect. We examined whether developmental conditions affected the association between adult song and HPA axis function, and whether nutritional stress before and after nutritional independence has distinct effects on song learning and/or vocal performance. Zebra finches (Taeniopygia guttata) were raised in consistently high (HH) or low (LL) food conditions until post-hatch day (PHD) 62, or were switched from high to low conditions (HL) or vice versa (LH) at PHD 34. Song was recorded in adulthood. We assessed the response of CORT to handling during development and to dexamethasone (DEX) and adrenocorticotropic hormone (ACTH) challenges during adulthood. Song learning and vocal performance were not affected by nutritional stress at either developmental stage. Nutritional stress elevated baseline CORT during development. Nutritional stress also increased rate of CORT secretion in birds that experienced stress only in the juvenile phase (HL group). Birds in the LL group had lower CORT levels after injection of ACTH compared to the other groups, however there was no effect of nutritional stress on the response to DEX. Thus, our findings indicate that developmental stress can affect HPA function without concurrently affecting song.     

Effects of glutathione on chromium‐induced DNA. Crosslinking and DNA polymerase arrest

Hexavalent chromium (Cr (VI)) is reduced intracellularly to Cr (V), Cr (IV) and Cr (III) by ascorbate (Asc), cysteine and glutathione (GSH). These metabolites induce a spectrum of genomic DNA damage resulting in the inhibition of DNA replication. Our previous studies have shown that treatment of DNA with Cr (III) or Cr (VI) plus Asc results in the formation of DNACrDNA crosslinks (CrDDC) and guaninespecific arrests of both prokaryotic and mammalian DNA polymerases. GSH not only acts as a reductant of Cr (VI) but also becomes crosslinked to DNA by Cr, thus, the focus of the present study was to examine the role of GSH in Crinduced DNA damage and polymerase arrests. Coincubation of Cr (III) with plasmid DNA in the presence of GSH led to the crosslinking of GSH to DNA. GSH cotreatment with Cr (III) also led to a decrease in the degree of Crinduced DNA interstrand crosslinks relative to Cr (III) alone, without affecting total Cr DNA binding. DNA polymerase arrests were observed following treatment of DNA with Cr (III) alone, but were markedly reduced when GSH was added to the reaction mixture. Preformed polymerasearresting lesions (CrDDC) were not removed by subsequent addition of GSH. Treatment of DNA with Cr (VI), in the presence of GSH, resulted in crosslinking of GSH to DNA, but failed to produce detectable DNA interstrand crosslinks or polymerase arrests. The inhibitory effect of GSH on Crinduced polymerase arrest was further confirmed in human genomic DNA using quantitative PCR (QPCR) analysis. Treatment of genomic DNA with Cr (III) resulted in a marked inhibition of the amplification of a 1.6 kb target fragment of the p53 gene by Taq polymerase. This was almost completely prevented by cotreatment with GSH and Cr (III). These results indicate that Crinduced DNA interstrand crosslinks, and not DNACrGSH crosslinks, are the principal lesions responsible for blocking DNA replication. Moreover, the formation of DNACrGSH crosslinks may actually preclude the formation of the polymerase arresting lesions.     

Effects of different disturbance types on butterflyfish communities of Australia’s Great Barrier Reef

The effects of disturbances on coral reef fishes have been extensively documented but most studies have relied on opportunistic sampling following single events. Few studies have the spatial and temporal extent to directly compare the effects of multiple disturbances over a large geographic scale. Here, benthic communities and butterflyfishes on 47 reefs of the Great Barrier Reef were surveyed annually to examine their responses to physical disturbances (cyclones and storms) and/or biological disturbances (bleaching, outbreaks of crown-of-thorns starfish and white syndrome disease). The effects on benthic and butterflyfish communities varied among reefs depending on the structure and geographical setting of each community, on the size and type of disturbance, and on the disturbance history of that reef. There was considerable variability in the response of butterflyfishes to different disturbances: physical disturbances (occurring with or without biological disturbances) produced substantial declines in abundance, whilst biological disturbances occurring on their own did not. Butterflyfishes with the narrowest feeding preferences, such as obligate corallivores, were always the species most affected. The response of generalist feeders varied with the extent of damage. Wholesale changes to the butterflyfish community were only recorded where structural complexity of reefs was drastically reduced. The observed effects of disturbances on butterflyfishes coupled with predictions of increased frequency and intensity of disturbances sound a dire warning for the future of butterflyfish communities in particular and reef fish communities in general.     

Effects of Pyrazinamide on Tryptophan–Niacin Conversion in Rats

In the course of our screening for a new anti-tumor substance, the bisabolane sesquiterpenoid endoperoxide, 3,6-epidioxy-1,10-bisaboladiene (EDBD), was isolated from the edible wild-plant, Cacalia delphiniifolia. EDBD showed cytotoxicity toward human chronic myelogenous leukemia K562 and human prostate carcinoma LNCaP cell lines with IC₅₀ values of 9.1 μM and 23.4 μM, respectively. DNA fragmentation and condensation of chromatin, the hallmarks of apoptosis, appeared in K562 cells after an 18-h treatment with EDBD. α-Curcumene, a bisabolane sesquiterpene that lacks the endoperoxide moiety of EDBD, also showed cytotoxicity toward both K562 and LNCaP cell lines at over a 10-times higher dose than that of EDBD. The results indicate the importance of the endoperoxide structure within EDBD to its anti-tumor activity in vitro.     

Effects of rapid prey evolution on predator–prey cycles

We study the qualitative properties of population cycles in a predator-prey system where genetic variability allows contemporary rapid evolution of the prey. Previous numerical studies have found that prey evolution in response to changing predation risk can have major quantitative and qualitative effects on predator-prey cycles, including: (1) large increases in cycle period, (2) changes in phase relations (so that predator and prey are cycling exactly out of phase, rather than the classical quarter-period phase lag), and (3) "cryptic" cycles in which total prey density remains nearly constant while predator density and prey traits cycle. Here we focus on a chemostat model motivated by our experimental system (Fussmann et al. in Science 290:1358-1360, 2000; Yoshida et al. in Proc roy Soc Lond B 424:303-306, 2003) with algae (prey) and rotifers (predators), in which the prey exhibit rapid evolution in their level of defense against predation. We show that the effects of rapid prey evolution are robust and general, and furthermore that they occur in a specific but biologically relevant region of parameter space: when traits that greatly reduce predation risk are relatively cheap (in terms of reductions in other fitness components), when there is coexistence between the two prey types and the predator, and when the interaction between predators and undefended prey alone would produce cycles. Because defense has been shown to be inexpensive, even cost-free, in a number of systems (Andersson et al. in Curr Opin Microbiol 2:489-493, 1999: Gagneux et al. in Science 312:1944-1946, 2006; Yoshida et al. in Proc Roy Soc Lond B 271:1947-1953, 2004), our discoveries may well be reproduced in other model systems, and in nature. Finally, some of our key results are extended to a general model in which functional forms for the predation rate and prey birth rate are not specified.     

Effects of cotton–maize rotation on soil microbiome structure

Verticillium wilt is a disastrous disease in cotton-growing regions in China. As a common management method, cotton rotation with cereal crops is used to minimize the loss caused by Verticillium dahliae. However, the correlation between soil microbiome and the control of Verticillium wilt under a crop rotation system is unclear. Therefore, three cropping systems (fallow, cotton continuous cropping, and cotton–maize rotation) were designed and applied for three generations under greenhouse conditions to investigate the different responses of the soil microbial community. The soil used in this study was taken from a long-term cotton continuous cropping field and inoculated with V. dahliae before use. Our results showed that the diversity of the soil bacterial community was increased under cotton–maize rotation, while the diversity of the fungal community was obviously decreased. Meanwhile, the structure and composition of the bacterial communities were similar even under the different cropping systems, but they differed in the soil fungal communities. Through microbial network interaction analysis, we found that Verticillium interacted with 17 bacterial genera, among which Terrabacter had the highest correlation with Verticillium. Furthermore, eight fungal and eight bacterial species were significantly correlated with V. dahliae. Collectively, this work aimed to study the interactions among V. dahliae, the soil microbiome, and plant hosts, and elucidate the relationship between crop rotation and soil microbiome, providing a new theoretical basis to screen the biological agents that may contribute to Verticillium wilt control.