Evaluation Of Membrane Integrity Research Articles

Amine reactive dyes: An alternative to estimate membrane integrity in fish sperm cells

Fluorescent dyes that binds irreversibly to cellular amines, come in several available emission spectra, and do not poses health concerns were used to evaluate membrane integrity in fish sperm cells. The objectives of the present study were to determine: (1) a working dye concentration for fish sperm samples, and (2) if the traditional propidium iodide/SYBR-14 staining combination was comparable with the amine reactive dye (ARD) methods at identifying cell populations with intact and compromised membranes after sperm activation, refrigerated storage, and exposure to cryoprotectant and surfactant. Zebrafish (Danio rerio) sperm were obtained by stripping, and pooled samples (in triplicate) were used in all tests. Six dilutions of the amine dye (ranging from 0.625 to 0.02μl/ml) were evaluated, and compared with the traditional staining protocol. A concentration of 0.5μl/ml ARD was selected to be used in subsequent assays.Sperm suspensions were activated with deionized water to simulate urine contamination. After 10s, osmolality was increased to stop activation, and the procedure was repeated in 10-s intervals until the sperm remained activated for 120 consecutive sec; membrane integrity was analyzed at each time interval. For the storage assay, sperm suspensions were prepared in Hanks' balanced salt solution at 302mOsm/kg osmolality (HBSS302), HBSS354 and HBSS402, and evaluated every 2h for 8h, and every 24h for 72h. Cryoprotectant toxicity was tested by diluting sperm suspensions in HBSS340 with methanol at 5, 10 and 15% final concentrations. Surfactant toxicity was tested by diluting sperm suspensions in HBSS354 with Triton X-100 at 0.2, 0.15 and 0.1mM final concentrations. In each toxicity assay, membrane integrity was tested every 20min for 80min. The number of membrane-intact cells significantly decreased across time in all treatments (P<0.05). Significant differences between staining protocols were observed after activation and after exposure to methanol at 10 and 15%, and to Triton X-100 (P<0.05). The average difference, however, was minor (between 1 and 6% in average) in relation to the typical values used for decision making based on this assay. Results showed that this method has the potential to contribute greatly to the standardization of cryopreservation in aquatic species. Statement of relevanceQA-QC for repository development in aquatic species.

Dormancy in Deinococcus sp. UDEC-P1 as a survival strategy to escape from deleterious effects of carbon starvation and temperature.

Dormancy is characterized by low metabolism and absence of protein synthesis and cellular division enabling bacterial cells to survive under stress. The aim was to determine if carbon starvation and low temperature are factors that modify the proportion of dormant/active cells in Deinococcus sp. UDEC-P1. By flow cytometry, RedoxSensor Green (RSG) was used to quantify metabolic activity and Propidium Iodide (PI) to evaluate membrane integrity in order to determine the percentage of dormant cells. Cell size and morphology were determined using scanning electronic microscopy. Under carbon starvation at 30°C, Deinococcus sp. UDEC-P1 increased its proportion of dormant cells from 0.1% to 20%, decreased the count of culturable cells and average cell volume decreased 7.1 times. At 4°C, however, the proportion of dormant cells increased only to 6%, without a change in the count of culturable cells and an average cellular volume decrease of 4.1 times and 3% of the dormant cells were able to be awakened. Results indicate a greater proportion of dormant Deinococcus sp. UDEC-P1 cells at 30ºC and it suggests that carbon starvation is more deleterious condition at 30ºC than 4ºC. For this reason Deinococcus sp. UDEC-P1 cells are more likely to enter into dormancy at higher temperature as a strategy to survive.

Assessment of changes in sperm cell membrane integrity occurring during the storage of semen from genetically different males using two diagnostic methods

Wysokińska, A. and Kondracki, S. 2014. Assessment of changes in sperm cell membrane integrity occurring during the storage of semen from genetically different males using two diagnostic methods. Can. J. Anim. Sci. 94: 601–606. The present study was carried out to assess changes in sperm cell membrane integrity occurring during the storage of semen collected from genetically different domestic male pigs. The study was aimed at assessing differences in the course of changes in the integrity of cell membranes in spermatozoa produced by males with different degrees of genetic diversity (pure-bred males, two-breed hybrids and multi-breed crosses) and testing the usefulness of two methods of sperm cell membrane integrity evaluation, based on material collected from genetically different males. The experiments were conducted on 56 ejaculates collected from 28 domestic male pigs. The examination of sperm cell membrane integrity was performed three times for each ejaculate, i.e., after 1 h, after 24 h and after 48 h from collection. The preparations for analysing cell membrane integrity were made using two methods: the SYBR 14/PI method and the eosin–nigrosin method. It was found that both SYBR 14/PI and eosin–nigrosin staining methods make it possible to successfully assess the integrity of the plasma membrane of domestic pig sperm cells under in vitro conditions. Hybrid pig spermatozoa, especially those from multi-breed crosses, better retain the integrity of their plasmalemmas than the spermatozoa of pure-bred boars. The ejaculates of Hypor cross-breed boars assessed after 1, 24 and 48 h of storage contain more spermatozoa with intact cell membranes than the ejaculates of pure-bred Duroc and Pietrain boars. The ejaculates of Hypor boars also show fewer decaying spermatozoa than those produced by pure-bred boars.

Assessment of changes in sperm cell membrane integrity occurring during the storage of semen from genetically different males using two diagnostic methods

Wysokinska, A. and Kondracki, S. 2014. Assessment of changes in sperm cell membrane integrity occurring during the storage of semen from genetically different males using two diagnostic methods. Can. J. Anim. Sci. 94: 601-606. The present study was carried out to assess changes in sperm cell membrane integrity occurring during the storage of semen collected from genetically different domestic male pigs. The study was aimed at assessing differences in the course of changes in the integrity of cell membranes in spermatozoa produced by males with different degrees of genetic diversity (pure-bred males, two-breed hybrids and multi-breed crosses) and testing the usefulness of two methods of sperm cell membrane integrity evaluation, based on material collected from genetically different males. The experiments were conducted on 56 ejaculates collected from 28 domestic male pigs. The examination of sperm cell membrane integrity was performed three times for each ejaculate, i.e., after 1 h, after 24 h and after 48...

Growth inhibition of bloom forming cyanobacterium Microcystis aeruginosa by green route fabricated copper oxide nanoparticles.

The cyanobacterium Microcystis aeruginosa can potentially proliferate in a wide range of freshwater bionetworks and create extensive secondary metabolites which are harmful to human and animal health. The M. aeruginosa release toxic microcystins that can create a wide range of health-related issues to aquatic animals and humans. It is essential to eliminate them from the ecosystem with convenient method. It has been reported that engineered metal nanoparticles are potentially toxic to pathogenic organisms. In the present study, we examined the growth inhibition effect of green synthesized copper oxide nanoparticles against M. aeruginosa. The green synthesized copper oxide nanoparticles exhibit an excitation of surface plasmon resonance (SPR) at 270 nm confirmed using UV-visible spectrophotometer. The dynamic light scattering (DLS) analysis revealed that synthesized nanoparticles are colloidal in nature and having a particle size of 551 nm with high stability at -26.6 mV. The scanning electron microscopy (SEM) analysis shows that copper oxide nanoparticles are spherical, rod and irregular in shape, and consistently distributed throughout the solution. The elemental copper and oxide peak were confirmed using energy dispersive x-ray analysis (EDAX). Fourier-transform infrared (FT-IR) spectroscopy indicates the presence of functional groups which is mandatory for the reduction of copper ions. Besides, green synthesized copper oxide nanoparticles shows growth inhibition against M. aeruginosa. The inhibition efficiency was 31.8 % at lower concentration and 89.7 % at higher concentration of copper oxide nanoparticles, respectively. The chlorophyll (a and b) and carotenoid content of M. aeruginosa declined in dose-dependent manner with respect to induction of copper oxide nanoparticles. Furthermore, we analyzed the mechanism behind the cytotoxicity of M. aeruginosa induced by copper oxide nanoparticles through evaluating membrane integrity, reactive oxygen species (ROS), and mitochondrial membrane potential (Δψm) level. The results expose that there is a loss in membrane integrity with ROS formation that leads to alteration in the Δψm, which ends up with severe mitochondrial injury in copper oxide nanoparticles treated cells. Hence, green way synthesized copper oxide nanoparticles may be a useful selective biological agent for the control of M. aeruginosa.

Influences of a diet supplemented with linseed oil and antioxidants on quality of equine semen after cooling and cryopreservation during winter

Seasonal changes in the reproductive physiology of stallions contribute to a decrease in the quality of frozen-thawed semen during late winter. Changes in the lipid composition of the sperm plasma membrane may contribute to this phenomenon. In the present study, we have, therefore, investigated the effects of adding linseed oil (LO) in combination with antioxidants to the diet of breeding stallions on the motility and membrane integrity of cooled–stored and cryopreserved semen. Starting in November, the diet of LO stallions (n = 6) but not control (C) stallions (n = 5) was supplemented with LO (100 mL once daily) plus an antioxidant (Myostem Protect; Audevard, Clichy, France) for a total of 84 days. Before (November) and at the end of this period (February), ejaculates were processed for cryopreservation (n = 3 ejaculates per stallion) and cooled shipping at 5 °C. Frozen-thawed and cooled–shipped semen was sent to the laboratory for computer-assisted semen analysis of total motility, progressive motility, and velocity parameters (average path velocity [VAP], curved line velocity [VCL], and straight-line velocity [VSL]) and evaluation of membrane integrity. The quality of frozen-thawed semen decreased (P < 0.05) from November (e.g., total motility LO 69 ± 3% and C 67 ± 3%) to February (total motility: LO 55 ± 4% and C 59 ± 3%) independent of treatment (P > 0.05). A decrease in the velocity parameters VAP, VCL, and VSL was more pronounced in LO stallions than in C stallions (e.g., VSL: November LO 67 ± 1 μm/s, C 64 ± 2 μm/s; February LO 59 ± 2 μm/s, C 63 ± 2 μm/s; interaction month by treatment, P < 0.05). In cooled–stored semen, total motility, progressive motility, and membrane integrity were lower in February than in November (P < 0.001 for all parameters). Supplementation of the diet with LO and antioxidants attenuated this decrease (e.g., Day 1 of cooled storage = 24 hours after semen collection: total motility in November LO 88 ± 1% and C 87 ± 3%; in February LO 83 ± 2% and C 73 ± 11%; interaction month by treatment: P < 0.05). Velocity parameters VAP, VCL, and VSL were significantly lower in February than in November (P < 0.001), but this decrease was not affected by treatment. In summary, dietary supplementation of stallions with LO plus antioxidants attenuated a decline in motility and membrane integrity of cooled–stored stallion semen during winter. This may improve the fertility of cooled–shipped semen. In contrast, the treatment did not counteract the decrease in quality of frozen-thawed semen that occurs in late winter.

Semen quality assessments and their significance in reproductive technology

Semen quality assessment methods are very important in predicting the fertilizing ability of persevered spermatozoa and to improve animal reproductive technology. This review discusses some of the current laboratory methods used for semen quality assessments, with references to their relevance in the evaluation of male fertility and semen preservation technologies. Semen quality assessment methods include sperm motility evaluations, analyzed with the computer-assisted semen analysis (CASA) system, and plasma membrane integrity evaluations using fluorescent stains, such as Hoechst 33258 (H33258), SYBR-14, propidium iodide (PI), ethidium homodimer (EthD) and 6-carboxyfluorescein diacetate (CFDA), and biochemical tests, such as the measurement of malondialdehyde (MDA) level. This review addresses the significance of specific fluorochromes and ATP measurements for the evaluation of the sperm mitochondrial status. Laboratory methods used for the evaluation of chromatin status, DNA integrity, and apoptotic changes in spermatozoa have been discussed. Special emphasis has been focused on the application of proteomic techniques, such as two-dimensional (2-D) gel electrophoresis and liquid chromatography mass spectrometry (LC-MS/MS), for the identification of the properties and functions of seminal plasma proteins in order to define their role in the fertilization-related processes.

The effect of the chamber on kinetic results in cryopreserved bull spermatozoa

In this study, the effect of the chamber used for the automated analysis of sperm motility by a computer-assisted semen analysis system on sperm kinematics was evaluated, and the cause of this effect was also verified. Twenty-three bull semen batches were thawed, and semen was diluted, aliquoted and analysed with six different chambers, (three capillary-loaded chambers and three droplet (DR)-loaded chambers). For each chamber type, each sample was analysed in quadruplicate, and the reliability of the analysis was tested using an intraclass correlation coefficient (ICC). Furthermore, sperm membrane integrity (MI) was evaluated, for each sample and chamber, in 12 randomly selected central and 12 edge fields. The ICC analysis showed that some parameters could have a significant variability related to the chamber. High stability of results was detected in Leja 4-chamber slide. Furthermore, as previously reported in other studies, capillary-loaded chambers seemed to affect the total and progressive motility and sperm velocities (average path velocity, straight line velocity and curvilinear velocity). These findings were corroborated by the evaluation of sperm MI that was significantly higher in the DR-loaded chambers. This study confirms that the chamber used for the objective kinetic evaluation of bull-thawed spermatozoa significantly affects the result. These differences could be present also in other species, even if the specific effect on the sperm kinematics should be verified. The Makler chamber seemed to give reliable results with negligible effects on sperm kinematics.

Boar sperm changes after sorting and encapsulation in barium alginate membranes

A routine use of boar-sexed semen is limited by the long sorting time necessary to obtain an adequate number of sexed spermatozoa for artificial insemination and by the high susceptibility of spermatozoa of this species to damages induced by sorting procedure and subsequent cryopreservation. The aim of this work was to study the impact of encapsulation in barium alginate membrane on sorted boar spermatozoa by evaluating membrane integrity, chlortetracycline staining patterns, protein tyrosine phosphorylation, and Hsp70 immunolocalization during storage over 72 hours in liquid or encapsulated form. The encapsulation procedure significantly (P < 0.05) decreased the overall membrane integrity of control unsorted semen (81.8 vs. 57.4, CTR vs. CPS), but did not negatively affect the overall viability and the chlortetracycline staining patterns of sorted encapsulated cells. Moreover, encapsulation significantly decreased (P < 0.05) the overall phosphotyrosin A pattern cell percentage in unsorted (98.4 vs. 92.6, CTR vs. CPS) but not in sorted semen (64.0 vs. 74.2; SORT CTR vs. SORT CPS). As for Hsp70, the overall percentage of cells displaying the different patterns was significantly influenced (P < 0.05) by treatment but not by storage time. The sorting procedure seems to induce the major changes, whereas encapsulation tends to exert a protective effect on sorted semen by increasing the percentage of spermatozoa displaying the T pattern (2.8 vs. 24.3; SORT CTR vs. SORT CPS). In conclusion, our data confirm that the damaging impact of the encapsulation in barium alginate capsules seems to be limited when compared with that of the sorting procedure and, moreover, the association of the two procedures does not result in an algebraic sum of the negative effects. These results suggest the possibility of a future utilization of the encapsulation technology in order to store sorted spermatozoa and permit their controlled release in the female genital tract.

Challenge tests with virus surrogates: an accurate membrane integrity evaluation system?

Abstract The use of membrane filtration in drinking water treatment has significantly increased in the last decades due to its advantages, including its capacity to produce water of high quality with a high level of pathogens rejection. However, if membrane integrity is compromised, this feature cannot be guaranteed, increasing the associated microbial risk of the treated water. This study has focused on the development and application of a protocol based on virus surrogates challenge tests applicable to the three existing ultrafiltration (UF) configurations. The operational conditions have been defined, and the tests have been conducted successfully. The selected micro-organisms, PDR-1, MS-2, GA and Bacillus spores, present different characteristics providing complementary information of membrane integrity and its status. In particular, PDR-1 and Bacillus spores, due to their larger size, are mainly removed by size exclusion and low removal rates may indicate membrane impairment. MS-2 and GA, 25 nm in si...

Avaliação de diferentes crioprotetores intra e extracelulares na criopreservação de sêmen de touros

Em um delineamento experimental usando o fatorial 3x2, três crioprotetores internos, glicerol (GLI), etilenoglicol (EG) e dimetilformamida (DMF), e dois externos, gema de ovo (GEMA) e lipoproteína de baixa densidade (LDL), avaliaram-se a motilidade ao descongelamento de GLI-GEMA 53,9±1,96, sendo superior aos demais tratamentos (P&lt;0,05). Na avaliação de morfologia ao descongelamento, não houve diferença (P&gt;0,05) entre os tratamentos EG-GEMA 68,3±1,58, EG-LDL 72,2±2,39 e DMF-GEMA 68,7±1,67 que foram mais altos que os demais (P&lt;0,05). A avaliação de integridade de membrana por fluorescência ao descongelamento GLI-GEMA 34,2±2,28 e EG-GEMA 30,9±1,32 não diferiram entre si (P&gt;0,05), mas foram mais elevados que os demais (P&lt;0,05), enquanto que a HOST dos tratamentos DMF-GEMA 13,6±1,30 e DMF-LDL 9,8±0,78 diferirem entre si (P&lt;0,05) e foram mais baixas que as demais (P&lt;0,05). O uso de etilenoglicol associado à gema de ovo pode ser uma alternativa ao uso de glicerol nos protocolos de congelamento de sêmen de touros.

Bacterial and extracellular polysaccharide content of brine‐wetted snow over Arctic winter first‐year sea ice

AbstractDuring freeze‐up and consolidation, sea ice rejects to its surface brine of marine origin that is incorporated into overlying snow. To evaluate the transport of biological components in brines from ice to snow, vertical profiles of temperature, salinity, bacterial abundance, and extracellular polysaccharide substances (EPS) were obtained through snow and first‐year sea ice (Barrow, AK) in consecutive winters (2010, 2011). Snow profiles showed strong interannual variation, with 2010 presenting higher values and wider ranges in salinity (0.3–30.9, practical salinity), bacterial abundance (2.8 × 102–1.5 × 104 cells mL− 1), and particulate EPS (pEPS, 0.04–0.23 glucose equivalents (glu‐eq) mg L− 1) than 2011 (0–11.9, 2.7 × 103–4.2 × 103 cells mL− 1 and 0.04–0.09 glu‐eq mg L− 1, respectively). Surface ice also differed interannually, with 2010 presenting again higher salinity (19.4, n = 1), bacterial abundance (5.4 × 104–9.6 × 104 cells mL− 1) and pEPS (0.13–0.51 glu‐eq mg L− 1) than 2011 (7.7–11.9, 1.7 × 104–2.2 × 104 cells mL− 1, and 0.01–0.09 glu‐eq mg L− 1, respectively). Transport of bacteria and pEPS from sea‐ice brines into snow was evident in 2010 but not 2011, a year with more extreme winter conditions of colder temperature, thinner snow, and stronger wind. By size fraction, the smallest EPS (&lt; 0.1 µm) dominated (&gt; 80%) total EPS in both ice and snow; the &gt; 3 µm fraction of EPS in snow appeared to have an atmospheric source. Evaluation of membrane integrity by Live/Dead stain revealed a high percentage (85%) of live bacteria in saline snow, identifying this vast environment as a previously unrecognized microbial habitat.

79 A COMBINATION OF ETHYLENE GLYCOL AND PROPYLENE GLYCOL IS SUPERIOR TO INDIVIDUAL CRYOPROTECTANTS FOR THE VITRIFICATION OF IMMATURE PORCINE OOCYTES

We compared the feasibility of ethylene glycol (EG) and propylene glycol (PG) for the vitrification of immature porcine cumulus–oocyte complexes (COC). Porcine COC collected from 3- to 6-mm follicles of slaughterhouse-derived ovaries were subjected to solid-surface vitrification (Somfai et al. 2010 Theriogenology 73, 147–156) either in 35% (v/v) EG or 35% (v/v) PG or in the mixture of 17.5% (v/v) EG and 17.5% (v/v) PG. After warming, the COC were subjected to in vitro maturation, IVF, and embryo culture according to Kikuchi et al. (2002 Biol. Reprod. 66, 1033–1041). Oocyte survival and maturation rates were assessed after in vitro maturation by evaluating membrane integrity and the extrusion of the first polar body. All live oocytes were subjected to IVF and in vitro culture. Cleavage and blastocyst rates were calculated from the total number of oocytes subjected to IVF on Day 2 (Day 0 = IVF) and Day 7, respectively. Total-cell (blastomeres) numbers in blastocysts were recorded on Day 7 after staining with Hoechst 33342. In Experiment 1, competence parameters of oocytes vitrified either in EG-based (EG group; n = 310) or a PG-based (PG group; n = 265) vitrification media were compared with those in the nonvitrified control (n = 160). The experiment was replicated 4 times. In Experiment 2, the competence parameters of oocytes vitrified with the combination of 17.5% EG and 17.5% PG (EG+PG group; n = 397) were compared with those in nonvitrified control (n = 245) and toxicity control (TC, exposed to cryoprotectants without cooling; n = 245) groups. Five replications were performed. Results were analyzed by ANOVA. Differences with P &lt; 0.05 were considered significant. In Experiment 1, the mean survival rate of vitrified oocytes was significantly higher (P &lt; 0.05) in 35% PG compared with that in 35% EG (73.3 and 25.9%, respectively). Maturation rates of surviving oocytes did not differ among vitrified (PG and EG) and nonvitrified control groups (71.1, 62.4, and 64.0%, respectively). After IVF of surviving oocytes, blastocyst formation rate in the group vitrified in EG was higher (P &lt; 0.05) compared with that vitrified in PG but was lower (P &lt; 0.05) compared with control (10.8, 2.0, and 25.0%, respectively). Mean cell numbers in blastocysts did not differ among EG, PG, and control groups (50.5, 47.7, and 48.7, respectively). In Experiment 2, survival of immature oocytes in the EG+PG group was 42.6%. After IVF, 10.7% of oocytes developed to the blastocyst stage in the EG+PG group, which was lower (P &lt; 0.05) than those of the control (18.1%) and TC (23.3%) groups. Blastocyst rates in the control and TC groups were not statistically different. Mean cell numbers in blastocysts did not differ significantly among the EG+PG, control, and TC groups (61.6, 59.3, and 53.3, respectively). In conclusion, 35% PG provided a higher oocyte survival rate after vitrification compared with 35% EG. However, presumably due to toxic effects, 35% PG greatly reduced the development competence of oocytes. The combination of 17.5% EG and 17.5% PG yielded higher survival rates than did 35% EG, without any toxic effect on oocytes.

Efeito da utilização de diferentes diluidores para a produção in vitro de embriões bovinos

Objetivou-se avaliar as características morfológica e funcional do sêmen bovino congelado comparando-se a eficácia de dois diferentes diluidores. O ejaculado de quatro touros foi dividido em duas partes iguais, uma submetida ao diluidor Tris e gema de ovo (A) e outra ao diluidor à base de lecitina de soja (Andromed®) (B). No experimento I, cinco palhetas dos diluidores A e B de cada touro foram descongeladas e avaliadas quanto à motilidade, vigor, concentração, morfologia espermática e teste de termor-resistência lento. Foram feitas, ainda, avaliação da integridade de membranas, por meio da associação das sondas iodeto de propídio, isotiocionato de fluoresceína - Pisum sativum e carbocianina catiônica lipofílica, e avaliação funcional da membrana plasmática com teste hiposmótico. A avaliação da integridade da cromatina foi realizada pelo método de coloração com laranja de acridina. No experimento II, o sêmen com os diferentes diluidores foi utilizado na fecundação in vitro, sendo observadas taxas de clivagem e desenvolvimento embrionário in vitro. Em relação aos resultados obtidos, apenas a porcentagem de espermatozoides no sêmen congelado foi discretamente maior com o diluidor A, concluindo-se que o diluidor composto por lecitina de soja pode substituir o composto por Tris e gema de ovo, respeitando-se as variações individuais de cada touro utilizado no presente experimento.

Development of a short-term assay based on the evaluation of the plasma membrane integrity of the alga Pseudokirchneriella subcapitata

Membrane integrity has been used as a criterion for the definition of cell viability. In the present work, staining conditions (time and dye concentration) for the evaluation of membrane integrity in a fluorescence microplate reader, using the membrane-impermeant nucleic-acid dye SYTOX Green, were optimized. Incubating Pseudokirchneriella subcapitata algal cells with 0.5μmol/l SYTOX Green for 40min allowed a clear discrimination between live (intact plasma membrane) and dead cells (with compromised plasma membrane). Algal cell suspensions, labelled with SYTOX Green, exhibited a green fluorescence proportional to the fraction of the cells with a permeabilized plasma membrane. The optimized staining conditions were used to assess the toxicity of 1-pentanol on P. subcapitata in a short-term exposure (6h) assay. The loss of membrane integrity in the cell population increased with the concentration of 1-pentanol. The 6-h EC(10) and EC(50) values were 7,617mg/l 1-pentanol (95% confidence limits 4,670-9,327) and 12,818mg/l 1-pentanol (95% confidence limits 10,929-15,183), respectively. The developed microplate-based short-term assay can be useful in the high-throughput screening of toxics or environmental samples using the alga P. subcapitata.

AVALIAÇÃO DA INTEGRIDADE DE MEMBRANA EM ESPERMATOZÓIDE BOVINO CRIOPRESERVADO PARA PREVER O ÍNDICE DE PRENHEZ

Semen of twelve Tabapuã young bulls, which were according to patterns established by CBRA (1998) after cryopreservation, was submitted to fast and slow thermoresistence tests, hypoosmotic swelling test, induced acrosome reaction by heparin, sperm viability test by Trypan blue post-freezing and post-incubation with heparin, and pregnancy rates at first artificial insemination of Nellore heifers. Continuous variable averages of the mentioned tests were compared by Student–Newman–Keuls test and subjective variables were compared by Kruskal-Wallis non-parametric test (p&lt;0.05). Simple correlation among all variables, and multiple regression models to predict pregnancy rate of Nellore heifers, using semen of Tabapuã bulls, were estimated. It was shown that none of the variables studied to evaluate membrane integrity in frozen semen was isolately effective (p&gt;0.05) to preview pregnancy rate, even if using the multiple regression model that included all variables. One can conclude that the model proposed in this study was not effective to predict pregnancy rate. Hypoosmotic Swelling Test can be suggested to evaluate functional and structural integrities of plasma membrane and the capacity of spermatozoa going through acrosome reaction.KEYWORDS: hypoosmotic swelling test; induced acrosome reaction; semen; sperm membrane; thermoresistence test.

Aerosolized ZnO Nanoparticles Induce Toxicity in Alveolar Type II Epithelial Cells at the Air-Liquid Interface

The majority of in vitro studies characterizing the impact of engineered nanoparticles (NPs) on cells that line the respiratory tract were conducted in cells exposed to NPs in suspension. This approach introduces processes that are unlikely to occur during inhaled NP exposures in vivo, such as the shedding of toxic doses of dissolved ions. ZnO NPs are used extensively and pose significant sources for human exposure. Exposures to airborne ZnO NPs can induce adverse effects, but the relevance of the dissolved Zn(2+) to the observed effects in vivo is still unclear. Our goal was to mimic in vivo exposures to airborne NPs and decipher the contribution of the intact NP from the contribution of the dissolved ions to airborne ZnO NP toxicity. We established the exposure of alveolar type II epithelial cells to aerosolized NPs at the air-liquid interface (ALI) and compared the impact of aerosolized ZnO NPs and NPs in suspension at the same cellular doses, measured as the number of particles per cell. By evaluating membrane integrity and cell viability 6 and 24 h post-exposure, we found that aerosolized NPs induced toxicity at the ALI at doses that were in the same order of magnitude as doses required to induce toxicity in submersed cultures. In addition, distinct patterns of oxidative stress were observed in the two exposure systems. These observations unravel the ability of airborne ZnO NPs to induce toxicity without the contribution of dissolved Zn(2+) and suggest distinct mechanisms at the ALI and in submersed cultures.

Production of F1Offspring with Vitrified Sperm from a Live-Bearing Fish, the Green SwordtailXiphophorus hellerii

This study reports the first production of offspring with vitrified sperm from a live-bearing fish Xiphophorus hellerii. The overall goal of this study was to develop streamlined protocols for integration into a standardized approach for vitrification of aquatic species germplasm. The objectives were to (1) estimate acute toxicity of cryoprotectants, (2) evaluate vitrification solutions, (3) compare different thawing methods, (4) evaluate membrane integrity of post-thaw sperm vitrified in different cryoprotectants, and (5) evaluate the fertility of vitrified sperm. Nine cryoprotectants and two commercial vitrification additives were tested for acute toxicity and glass forming ability, alone and in combination. Two vitrification solutions, 40% glycerol (Gly) and 20% Gly+20% ethylene glycol (EG) in 500 mOsmol/kg Hanks' balanced salt solution (HBSS), were selected for vitrification of 10 μL sperm samples using inoculating loops plunged into liquid nitrogen. Samples were thawed at 24°C (one loop in 5 μL of HBSS or three loops in 500 μL of HBSS). Samples thawed in 500 μL were concentrated by centrifugation (1000 g for 5 min at 4°C) into 5 μL for artificial insemination. Offspring were produced from virgin females inseminated with sperm vitrified with 20% Gly+20% EG and concentrated by centrifugation.

Prediction of maturational competence of feline oocytes using supravital staining of cumulus cells by propidium iodide

We examined the relationship between integrity of cumulus cells and nuclear maturation rate after in vitro culture to determine a non-invasive prediction of the maturational competence of feline oocytes. Feline cumulus-oocyte complexes (COCs) were collected from either small (400-800 μm) or large (≥800 μm) follicles. Immediately after collection, cumulus cells were evaluated morphologically (thickness of cumulus cell layers) and stained with propidium iodide (PI), which penetrates only non-viable cells. Cumulus cells without PI staining were judged as having good membrane integrity. After evaluation, COCs were cultured for 30 h and their nuclear maturation rate was determined. The nuclear maturation rate of oocytes derived from large follicles (89.8%) was higher (p < 0.05) than that from small follicles (60.8%). There was no difference in the maturation rate of oocytes from follicles with the same size regardless of cumulus morphology. In contrast, oocytes that had cumulus cells with good membrane integrity showed a higher maturation rate (93.8%) than oocytes with poor cumulus integrity (76.9%) in large follicles (p < 0.05). We conclude that evaluation of membrane integrity of cumulus cells by propidium iodide staining can be used to predict the maturational competence of oocytes.

The effects of acrylamide on sperm parameters and membrane integrity of epididymal spermatozoa in mice

Objective Acrylamide is a chemically reactive substance used in various industries. Recently, the discovery of acrylamide in a variety of human foods including heat-processed starchy foods such as potato chips and bread has been reported. Acrylamide is also known as a carcinogen and cytotoxic material. The aim of this study was to evaluate the detrimental effects of acrylamide on membrane integrity and sperm parameters in mice. Study design This experimental study was conducted on thirty male NMRI mice, aged 8–10 weeks and weighing 25–30 g. They were randomly allotted into three equal groups. Group I (low dose) and group II (high dose) were fed on water solutions containing acrylamide 5 and 10 mg/kg/day, respectively, for 2 months, while the third group received fresh water as the control group. Sperm analysis was done for parameters as well as evaluation of membrane integrity by Hypoosmotic Swelling Test (HOS-test) for sperm tails and Eosin-Y staining for sperm heads. Results Total sperm motility and progressive motility (fast and slow) in both groups, I and II, decreased significantly ( P = 0.00), but no significant change was observed in non-progressive motility ( P > 0.05). The total motile sperm percentage decreased significantly only in group II ( P = 0.01). Sperm morphology did not significantly change in the experimental groups compared to the controls ( P > 0.05). In sperm membrane integrity evaluation, functional intact membrane of sperm tail in both groups I and II had a significant decrease ( P = 0.00), but membrane integrity of the sperm head decreased significantly only in group II ( P = 0.00). Conclusion These results indicate that acrylamide, through effects on membrane integrity, decreased sperm vitality as well as causing abnormal sperm parameters in progressive motility and total motility.