Cinnamyl Schiff bases: synthesis, cytotoxic effects and antifungal activity of clinical interest.

The aim of this study was to synthesize and investigate the in vitro antifungal properties of 23 cinnamyl Schiff bases. In addition, cytotoxic effects of such cinnamyl Schiff bases against human lung, kidney or red blood cells were also checked. The compounds were synthesized in a single-step, 2 min of reaction under microwave irradiation produced up to 97% yield. Six of the 23 cinnamyl Schiff bases possessed antifungal activities against strains of Candida, Aspergillus, Fonsecaea and, particularly, Cryptococcus species. Indeed, cinnamyl Schiff bases 1 and 23 exhibited minimum inhibitory concentration (MIC) values more than twofold lower than fluconazole (FCZ) against all the Cryptococcus neoformans strains (MIC = 1·33, 1·4 and 5·2 µg ml-1 , respectively) and Cryptococcus gattii strains (MIC = 5·3, 2·8 and 9·2 µg ml-1 , respectively) (12 strains of each species) while cinnamyl Schiff base 11 was as potent as FCZ against all strains from both Cryptococcus species. No significant cytotoxic effects were observed for Schiff bases against human lung, kidney or red blood cells, all presenting selective indexes higher than 10. In conclusion, this study revealed cinnamyl Schiff bases, especially 1 and 23, as new lead anticryptococcal agents for the discovery of novel antifungal drugs. SIGNIFICANCE AND IMPACT OF THE STUDY: The occurrence and severity of fungal infections have increased in recent decades due to resistance to available antifungal drugs and the appearance of new emerging pathogens. Thus, the search for new antifungal agents is mandatory. From a series of 23 cinnamyl Schiff bases, two compounds (1 and 23) were interrogated as new anticryptococcal agents without significant cytotoxicity against human lung, kidney or red blood cells. In turns, these new Schiff bases are lead compounds for the discovery of novel antifungal drugs.

Effects of whole-body vibration on muscle strength, bone mineral content and density, and balance and body composition of children and adolescents with Down syndrome: a systematic review.

The aim of this study is to verify the effects of whole-body vibration (WBV) training on the muscle strength of children and adolescents with Down syndrome. We searched MEDLINE, Cochrane, SciELO, Lilacs and PUBMED databases and included manual searches to identify randomised controlled trials to investigate the effects of WBV on the structure and body function of children and adolescents with Down syndrome. Two reviewers independently selected the studies and performed statistical analysis. In total, five studies with 171 patients that compared WBV with exercise and/or control were included. Two studies demonstrated a significant difference between the muscle strength of children and adolescents with Down syndrome who received WBV training and that of those who did not receive the intervention. The studies included in this systematic review showed that WBV training has positive effects on bone mineral density (BMD), body composition and balance. Results of this study showed that WBV training improves muscle strength, BMD, body composition and balance of children and adolescents with Down syndrome, and a more in-depth analysis of its effects on other variables in this population is required, as well as of parameters to be used.

Flow Cytometric Chromosomal Sex Sorting of Stallion Spermatozoa Induces Oxidative Stress on Mitochondria and Genomic DNA.

To date, the only repeatable method to select spermatozoa for chromosomal sex is the Beltsville sorting technology using flow cytometry. Improvement of this technology in the equine species requires increasing awareness of the modifications that the sorting procedure induces on sperm intactness. Oxidative stress is regarded as the major damaging phenomenon, and increasing evidence regards handling of spermatozoa - including sex sorting - as basic ground for oxidative damage. The aim of this study was to disclose whether the flow cytometric sorting procedure increases the production of reactive oxygen species (ROS), and to identify if ROS production relates to DNA damage in sorted spermatozoa using specific flow cytometry-based assays. After sorting, oxidative stress increased from 26% to 33% in pre- and post-incubation controls, to 46% after sex sorting (p < 0.05). Proportions of DNA fragmentation index post-sorting were approximately 10% higher (31.3%); an effect apparently conduced via oxidative DNA damage as revealed by the oxyDNA assay. The probable origin of this increased oxidative stress owes the removal of enough seminal plasma due to the unphysiological sperm extension, alongside a deleterious effect of high pressure on mitochondria during the sorting procedure.

Effect of overnight staining on the quality of flow cytometric sorted stallion sperm: comparison with tradtitional protocols.

Flow cytometry is considered the only reliable method for the separation of X and Y chromosome bearing spermatozoa in equines. The MoFlo SX DP sorter is highly efficient, allowing the production of foals of the desired sex. However, to achieve acceptable pregnancy rates the currently used protocol requires working with fresh semen obtained close to, or at, the sorting facility. An alternative protocol was tested during two consecutive breeding seasons. Fresh stallion semen was cooled for 20 h, during which staining with Hoechst 33342 took place. On the following day, this sample was flow sorted and compared with spermatozoa from the same ejaculate that had been sexed on the previous day. All sperm parameters evaluated remained unchanged when fresh sorted and refrigerated sorted semen were compared. Pre-sorting storage at 5°C did not alter sperm velocities nor kinetics, viability or membrane permeability, production of reactive oxygen species, mitochondrial membrane potential or DNA fragmentation index of the sorted sample. The findings open for the possibility of using semen from stallions housed far from the sorting facilities. Processed and stained sperm could be shipped refrigerated on the previous day, sorted and inseminated on the next day.

Hydroxyaldimines as potent in vitro anticryptococcal agents.

UNLABELLED: Cryptococcosis, a fungal infection that affects both immunocompromised and immunocompetent individuals, contributes to increasing indices of mortality and morbidity. The development of resistance by Cryptococcus spp., the limited number of commercial antifungal drugs and the various side effects of these drugs cause the treatment of cryptococcosis to be a challenge. The in vitro anticryptococcal activity of nine hydroxyaldimines was evaluated against 24 strains of Cryptococcus spp. Antifungal susceptibility was evaluated using a broth microdilution assay following the Clinical and Laboratory Standards Institute guidelines, using fluconazole as a positive control. Parameters such as the minimum inhibitory concentration and the minimum fungicidal concentration (MIC and MFC, respectively) were also determined. Antiproliferative activity on the normal cell line VERO was assessed 48 h post-compound exposure to determine the selectivity index (SI) of the hydroxyaldimines and fluconazole. All hydroxyaldimines were active against Cryptococcus spp. strains. Compounds 3A9 and 3B7 were the most potent against the Cryptococcus gattii and Cryptococcus neoformans strains. Selectivity indices also revealed that 3B10, 3C3, 3D3 and 3D9 are good candidates for in vivo studies. The in vitro anticryptococcal activity of hydroxyaldimines against various strains of C. gattii and C. neoformans indicates the potential of this class of molecules as lead compound for the development of selective and efficient anticryptococcal agents. SIGNIFICANCE AND IMPACT OF THE STUDY: The effectiveness of hydroxyaldimines for inhibition of Cryptococcus spp. growth and their low toxicity against healthy monkey kidney epithelial cells makes them promising lead compounds for the design of new anticryptococcal agents.

Photoinactivation of Aedes aegypti larvae using riboflavin as photosensitizer.

More than half of the global population lives in areas where the Aedes aegypti mosquito is present. Efforts have been made to deal with the population of this mosquito in the larval and adult stages to prevent outbreaks of diseases (Dengue, Zika, Chikungunya, and Yellow Fever). In this scenario, photodynamic inactivation may be an effective alternative method to control this vector population. To evaluate the efficacy of the riboflavin - B2 vitamin - as photosensitizer (PS) in the photodynamic inactivation of Ae. aegypti larvae, different concentrations (0; 0.005; 0.010; 0.025; 0.050; 0.075 and 0.100 mg mL-1) were evaluated under white light from RGB LEDs at a light dose of 495.2  J cm-2. The results reveal that riboflavin can be successfully applied as a PS agent to photoinactivate Ae. aegypti larvae, showing its potential to deal with the larvae population.

Emotional and behavioral problems in children and adolescents with lower urinary tract dysfunction: a population-based study.

BACKGROUND: An association has been found between lower urinary tract dysfunction (LUTD) and emotional and behavioral problems, particularly in cases of urinary incontinence. Other associated symptoms and the coexistence of functional constipation require further investigation. OBJECTIVE: To assess whether emotional and behavioral problems are more common in children and adolescents with LUTD. STUDY DESIGN: A multicenter, cross-sectional, population-based study conducted in public places. Parents answered questions on urinary and psychological symptoms in their children aged 5-14 years. Children/adolescents with neurological problems or anatomical urinary tract abnormalities were excluded. The Dysfunctional Voiding Scoring System was used for assessing urinary symptoms, the Rome III Diagnostic Criteria for evaluating bowel symptoms, and the Strengths and Difficulties Questionnaire (SDQ) for evaluating emotional and behavioral problems. RESULTS: Of the 806 children/adolescents included, 53% were female. The mean age was 9.1 ± 2.7 years. The prevalence of LUTD was 16.4%. Overall, 26.2% had abnormal scores in the overall SDQ scale, 29.2% in the emotional problems subscale, and 30% in the conduct problems subscale. Of the children with LUTD, 40.5% screened positive for emotional/behavioral problems, with a significant association being found for the overall SDQ scale (P < 0.001) and for the emotional problems (P < 0.001), conduct problems (P < 0.001), and hyperactivity (P = 0.037) subscales. Urinary urgency, urinary incontinence, and voiding postponement were significantly associated with a greater prevalence of abnormalities in the overall SDQ score (P = 0.05; P = 0.004, and P = 0.012, respectively). Bladder and bowel dysfunction was an aggravator of emotional and behavioral problems, with more intense symptoms, both in the overall SDQ scale and in the subscales. In the multivariate analysis, the factors independently associated with the presence of emotional and behavioral problems were LUTD (odds ratio [OR] = 1.91), constipation (OR = 1.7), studying in a government-funded school (OR = 2.2), and poor education of the head of the family (OR = 1.9). CONCLUSIONS: Children and adolescents with LUTD have more emotional and behavioral problems, with bladder and bowel dysfunction being an aggravating factor for this association.

Plasma hydroxy-metronidazole/metronidazole ratio in hepatitis C virus-induced liver disease.

It has been suggested that the measurement of metronidazole clearance is a sensitive method for evaluating liver function. The aim of this study was to evaluate the usefulness of plasma hydroxy-metronidazole/metronidazole ratios as indicators of dynamic liver function to detect changes resulting from the various forms of chronic hepatitis C virus (HCV) infection. A total of 139 individuals were studied: 14 healthy volunteers, 22 healthy, asymptomatic, consecutive anti-HCV-positive HCV-RNA negative subjects, 81 patients with chronic hepatitis C (49 with moderate/severe chronic hepatitis and 34 with mild hepatitis), and 20 patients with cirrhosis of the liver. HCV status was determined by the polymerase chain reaction. Plasma concentrations of metronidazole and its hydroxy-metabolite were measured by reverse-phase high-performance liquid chromatography with ultraviolet detection in a blood sample collected 10 min after the end of a metronidazole infusion. Anti-HCV-positive HCV-RNA-negative individuals demonstrated a significantly reduced capacity to metabolize intravenously infused metronidazole compared to healthy individuals (0.0478 +/- 0.0044 vs 0.0742 +/- 0.0232). Liver cirrhosis patients also had a reduced plasma hydroxy-metronidazole/metronidazole ratio when compared to the other groups of anti-HCV-positive individuals (0.0300 +/- 0.0032 vs 0.0438 +/- 0.0027 (moderate/severe chronic hepatitis) vs 0.0455 +/- 0.0026 (mild chronic hepatitis) and vs 0.0478 +/- 0.0044 (anti-HCV-positive, HCV-RNA-negative individuals)). These results suggest an impairment of the metronidazole metabolizing system induced by HCV infection that lasts after viral clearance. In those patients with chronic hepatitis C, this impairment is paralleled by progression of the disease to liver cirrhosis.

Molecular cloning and characterization of actin genes from Echinococcus granulosus.

An Echinococcus granulosus genomic library has been screened with a mouse beta-actin cDNA probe. Two clones carrying DNA fragments of about 15 kb, possibly derived from the same genome region, have been isolated. This 15-kb genomic region includes 2 actin-related sequences (EgactI and EgactII) separated by about 4 kb. The nucleotide sequences of both genes were determined. The EgactI sequence presents no introns, but an intron of 591 bp was observed in the EgactII sequence. The genes potentially encode 375 and 376 amino-acid-long actins, respectively, with a homology of 85.3%. The deduced amino acid sequences from both genes were compared to the actin sequences from other organisms, showing similarities ranging from 63.5% to 90.6%. The nucleotide sequence of a partial actin cDNA clone has been determined. The deduced amino acids sequence showed a homology of 90.3% and 88.0% in relation to the EgactI and EgactII sequences respectively, suggesting the existence of at least one more actin gene in E. granulosus. This hypothesis is reinforced by the number of bands detected in the Southern blot analysis. Experiments based on the amplification of DNA segments using 3'-specific actin primers indicate that the EgactI gene is transcribed in protoscoleces.

Addition of bismuth subnitrate to omeprazole plus amoxycillin improves eradication of Helicobacter pylori.

OBJECTIVE: To evaluate whether the addition of bismuth subnitrate to a dual oral therapy regimen with omeprazole plus amoxycillin could improve Helicobacter pylori eradication. METHODS: Fifty consecutive Helicobacter pylori-positive patients were randomly enrolled to receive either (A) bismuth subnitrate (300 mg q.d.s.), omeprazole (20 mg b.d.) and amoxycillin (500 mg q.d.s.), or (B) omeprazole (20 mg b.d.) and amoxycillin (500 mg q.d.s.). Both groups (n=25 each) received the medication for 14 days. H. pylori status was reassessed 30 days after completion of the therapy in order to evaluate eradication rates. RESULTS: Six patients were lost to follow-up and therefore excluded from the study (three patients from each group). One patient from Group B withdrew from the study because of side-effects. The addition of bismuth subnitrate to omeprazole and amoxycillin significantly improved its efficacy in eradicating H. pylori, with 72% (18/25) eradication in Group A and 52% (13/25) in Group B (P=0.027). The addition of bismuth subnitrate to dual oral therapy was also capable of improving the healing of peptic ulcers when compared with dual oral therapy alone (100%, 8/8 vs. 58%, 4/7; P=0.021). CONCLUSION: Our results demonstrate that the addition of bismuth subnitrate to dual oral therapy enhances H. pylori eradication, and improves healing of peptic ulcers.

Acid suppression by omeprazole does not affect orally administered metronidazole bioavailability and metabolism in healthy male volunteers.

BACKGROUND: The addition of omeprazole to classical triple therapy for eradication of H. pylori may enhance compliance through reducing ulcer symptoms and side-effects. The aim of this study was to investigate the effects of a 5-day administration of omeprazole on metronidazole pharmacokinetics. METHODS: Fourteen healthy male volunteers were selected. The study had an open, randomized, two-period crossover design with a 21-day washout period between the phases. Plasma concentrations of metronidazole and its hydroxy-metabolite were measured by reversed-phase HPLC with ultraviolet detection. RESULTS: Administration of omeprazole did not affect the pharmacokinetic parameters of orally administered metronidazole. CONCLUSION: Our results indicate that short-term treatment with omeprazole in healthy volunteers does not alter the extent or the rate of metronidazole absorption, and does not affect metronidazole clearance.

Plasma hydroxy-metronidazole/metronidazole ratio can detect early changes in hepatic function in ethanol-induced liver injury.

AIMS: To evaluate the usefulness of plasma hydroxy-metronidazole/metronidazole (OH-MET/MET) ratios as a dynamic liver function test in ethanol abusers with or without liver cirrhosis. METHODS: Metronidazole was administered intravenously for 20 min to healthy volunteers, and to patients with alcohol-induced, non-cirrhotic hepatopathy and liver cirrhosis. Plasma concentrations of metronidazole and hydroxy-metronidazole were measured by high performance liquid chromatography in samples collected 5, 10, 20 and 30 min after the metronidazole infusion. RESULTS: Patients with non-cirrhotic alcoholic hepatopathy had significantly elevated aminotransferase levels compared to healthy volunteers and Child A patients. Child-Pugh C patients had significantly prolonged prothrombin times when compared to healthy volunteers and patients with non-cirrhotic hepatopathy. Metronidazole metabolism, as measured by the OH-MET/MET ratio following the intravenous administration of 500 mg of the drug, was significantly impaired in all ethanol-abusing individuals, including patients with non-cirrhotic alcoholic hepatopathy. CONCLUSIONS: Metronidazole metabolism was impaired in ethanol abusers, even in the absence of liver cirrhosis, indicating that ethanol was capable of affecting liver function in the early stages of alcohol-induced liver disease.

Characterization of beta-galactosidase in leukocytes and fibroblasts of GM1 gangliosidosis heterozygotes compared to normal subjects.

OBJECTIVES: Characterization of beta-galactosidase in leukocytes and fibroblasts of heterozygotes for GM1 type I. DESIGN AND METHODS: Leukocyte and fibroblast beta-galactosidase activity was determined fluorimetrically using 4-methylumbelliferyl-beta-D-galactoside as an artificial substrate. Optimum pH, Km, Vmax and thermostability of the enzyme at 42 degrees C were determined. RESULTS: The leukocyte and fibroblast enzyme of heterozygotes have an optimum pH of 4.0 and 4.2, respectively. In normal subjects, the optimum pH was 4.2 in both cells, according to previous studies. The Km of the enzyme of heterozygotes was determined to be 0.65 mM in leukocytes and 0.59 mM in fibroblasts. The Vmax was determined in 167.21 nmol/h/mg of protein in heterozygotes leukocytes and 541.2 nmol/h/mg of protein in heterozygotes fibroblasts compared to 291.7 and 1768.1 nmol/h/mg of protein in controls leukocytes and fibroblasts, respectively. When leukocyte and fibroblast heterozygote beta-galactosidase was preincubated at 42 degrees C, after 80 min the residual activity was determined to be 25 to 30% of the initial activity. These results are similar to the control group. CONCLUSIONS: We have found significant differences between the two groups in some investigated parameters. Both fibroblasts and leukocytes showed a virtually similar level of reliability as source of enzyme for the detection of heterozygotes.

Short-term sucralfate administration does not alter the absorption of metronidazole in healthy male volunteers.

BACKGROUND: An oral triple therapy using sucralfate instead of a bismuth to eradicate Helicobacter pylori has yielded worse results than those obtained with conventional oral triple therapies. To date, the effect of sucralfate on the pharmacokinetics of nitroimidazolic compounds used in triple therapy such as with metronidazole is unknown. The aim of this study was to investigate the effect of a 5-day administration period of sucralfate (2 g b.i.d.) on metronidazole pharmacokinetics. METHODS: Fourteen healthy male volunteers were selected. The study had an open randomized 2-period crossover design with a 14-day washout period between the phases. The plasma concentration of metronidazole and its hydroxy-metabolite were measured by reverse-phase HPLC with ultraviolet detection. RESULTS: No statistically significant difference was observed in any of the pharmacokinetic parameters studied in the absence and presence of sucralfate. CONCLUSION: Our results clearly indicate that short-term treatment with sucralfate in healthy volunteers does not alter the extent or the rate of metronidazole absorption, and does not affect metronidazole clearance.

[Evaluation of orientation and serological support centers in the Brazilian Northeast]. / Avaliação dos Centros de Orientação e Apoio Sorológico/CTA/Coas da Região Nordeste do Brasil.

This article presents the results of an evaluation of Orientation and Serological Support Centers, or Anonymous HIV Testing Centers, in the Northeast of Brazil. Methodological triangulation was used to evaluate these health programs and services, including qualitative and quantitative methodology and pointing to the insufficiency of single-discipline reasoning to encompass phenomena in social organizations. The article also shows results from this triangulation experience, seeking to combine Social Sciences and Epidemiology. It describes the structure and dynamics of these services, analyzes the relationships, perceptions, and representations of the actors, presents a self-evaluation by the professionals, expounds on some quantitative results, and discusses some limits and problems, as well as proposals to overcome them.

[Analysis of hospital morbidity by motor vehicle accidents in public hospitals in Rio de Janeiro, Brazil]. / Análise da morbidade hospitalar por acidentes de trânsito em hospitais públicos do Rio de Janeiro, RJ, Brasil.

INTRODUCTION: Morbidity due to motor vehicle accidents was analysed in a descriptive study based on reports of hospitals with the objective to characterize their victims, types of injuries, and the health care provided. METHODS: The study was carried out in two municipal hospitals of Rio de Janeiro, Brazil. Motor vehicle accidents were defined as a run over, a collision, and "other traffic accidents". Data was collected from cases seen in May (Hospital 1) and June (Hospital 2) of 1996 by 7 teams that alternated in shifts of 12 hours, covering the collection 24 hours per day. The events were classified according to the victim or, when it was impossible, to the person who helped them or accompanied them to the hospital. RESULTS: In the study period, 320 cases were seen in Hospital 1 and 290 in Hospital 2. Most of the victims were men (69.3%) in both of hospitals and the most affected group was young adults between 20 to 39 years (60.5% in Hospital 1 and 47.5% in Hospital 2). Of all 610 cases, the main cause of injury was being run over (49.3%), followed by collisions (35.6%), and "other traffic accidents" (15.1%). CONCLUSIONS: The analysis of the cases distributions and the type of care required corroborates with the need of a better organization of hospital care teams, more completeness of hospital reports and emphasis on some aspects in prevention campaigns.

During cooled storage the extender influences processed autophagy marker light chain 3 (LC3B) of stallion spermatozoa.

To investigate the role of the processed autophagy marker light chain 3 (LC3B) protein in sperm survival in stallion semen processing during cooled storage, split ejaculates were diluted in two different extenders, KMT and INRA 96, and LC3B processing and sperm quality evaluated during incubation at 5°C for five days. After 3 days of incubation there was a drop in total motility in both extenders, although the percentage of progressive motile sperm was greater (P<0.05) in samples extended in INRA96. On Day 5 of cooled storage all sperm parameters decreased significantly independent of the extender, however, samples extended in INRA 96 maintained motility values while those extended in KMT had a further decrease in motility compared with data collected on Day 3 of incubation. The percentage of live sperm decreased over the time of incubation, but only in samples incubated in KMT. The extender had a marked effect in LC3B processing during cooled storage. Spermatozoa maintained in KMT extender did not exhibit LC3B processing, while in spermatozoa incubated in INRA96 there was an increase (P<0.01) in LC3B processing after 5 days of cooled storage. Stallion spermatozoa experience LC3B turnover during cooled storage, however, the extent depends on the extender used. Apparently LC3B turnover is associated with enhanced survival.

Effect of sex sorting on stallion spermatozoa: Heterologous oocyte binding, tyrosine phosphorylation and acrosome reaction assay.

The interest on sex sorting by flow cytometry on the equine industry has been increasing over the years. In this work, three different tests were performed in order to evaluate the membrane status of sorted stallion spermatozoa: assessment of binding ability to porcine oocytes, evaluation of acrosome integrity after stimulation with A23187, and detection of tyrosine phosphorylation. These evaluations were made after incubation for 0h, 1.5h and 3h in a capacitating medium. Sorted stallion spermatozoa attached similarly to the porcine oocytes, when compared with control samples. Sorted spermatozoa were more prone to undergo acrosome reaction (P<0.05), at the beginning and after 1.5h and 3h of incubation, and also had higher tyrosine phosphorylation of the tail (P<0.001), only at the beginning of the incubation period. Apparently sex sorted stallion spermatozoa are in a more advanced status of membrane destabilization, which could be associated with capacitation, although similar binding ability to porcine oocytes is maintained.

Sex sorting increases the permeability of the membrane of stallion spermatozoa.

At present, the only repeatable means of selecting the sex of offspring is the Beltsville semen sorting technology using flow cytometry (FC). This technology has reached commercial status in the bovine industry and substantial advances have occurred recently in swine and ovine species. In the equine species, however, the technology is not as well developed. To better understand the changes induced in stallion spermatozoa during the sorting procedure, pooled sperm samples were sorted: sperm motility and kinematics were assessed using computer assisted sperm analysis, sperm membrane integrity was assessed using the YoPro-1 assay, while plasmalemmal stability and lipid architecture were assessed using Merocyanine 540/SYTOX green and Annexin-V, respectively. Lipid peroxidation was also investigated with the probe Bodipy(581/591)-C11. All assays were performed shortly after collection, after incubation and after sex sorting using FC. In order to characterize potential molecular mechanisms implicated in sperm damage, an apoptosis protein antibody dot plot array analysis was performed before and after sorting. While the percentage of total motile sperm remained unchanged, sex sorting reduced the percentages of progressive motile spermatozoa and of rapid spermatozoa as well as curvilinear velocity (VCL). Sperm membranes responded to sorting with an increase in the percentage of YoPro-1 positive cells, suggesting the sorted spermatozoa had a reduced energy status that was confirmed by measuring intracellular ATP content.

Toxicity of glycerol for the stallion spermatozoa: effects on membrane integrity and cytoskeleton, lipid peroxidation and mitochondrial membrane potential.

Glycerol is, to date, the most widely used cryoprotectant to freeze stallion spermatozoa at concentrations between 2% and 5%. Cryoprotectant toxicity has been claimed to be the single most limiting factor for the success of cryopreservation. In order to evaluate the toxic effects of the concentrations of glycerol used in practice, stallion spermatozoa were incubated in Biggers Whitten and Whittingham (BWW) media supplemented with 0%, 0.5%, 1.5%, 2.5%, 3.5%, and 5% glycerol. In two additional experiments, a hyposmotic (75 mOsm/kg) and a hyperosmotic (900 mOsm/kg) control media were included. Sperm parameters evaluated included cell volume, membrane integrity, lipid peroxidation, caspase 3, 7, and 8 activation, mitochondrial membrane potential, and integrity of the cytoskeleton. Glycerol exerted toxicity at concentrations ≥ 3.5% and the maximal toxicity was observed at 5%. The actin cytoskeleton was especially sensitive to glycerol presence, inducing rapid F actin depolymerization at concentrations over 1.5%. The sperm membrane and the mitochondria were other structures affected. The toxicity of glycerol is apparently related to osmotic and nonosmotic effects. In view of our results the concentration of glycerol in the freezing media for stallion spermatozoa should not surpass 2.5%.