Identification of deoxynivalenol, 3-acetyldeoxynivalenol and zearalenone in the galactose oxidase-producing fungus Dactylium dendroides

The galactose oxidase-producing fungus Dactylium dendroides was re-identified as a Fusarium species. Fungi of this genus are well known for the production of mycotoxins. Verification of growth of this fungus on rice, corn and liquid medium described for the production of galactose oxidase is provided to determine whether the fungus could produce Fusarium toxins, namely, moniliformin, fusaric acid, fumonisin, zearalenone and the trichothecenes, deoxynivalenol, 3-acetyldeoxynivalenol, fusarenone, nivalenol, diacetoxyscirpenol, neosolaniol, and toxin T-2. Under the culture conditions used, deoxynivalenol, 3-acetyldeoxynivalenol and zearalenone were detected in the fungal culture medium. The finding is consistent with the hypothesis that the fungus is in fact a Fusarium species. This revised version was published online in July 2006 with corrections to the Cover Date.     

Vulnerability of polarised intestinal porcine epithelial cells to mycotoxin deoxynivalenol depends on the route of application

Background and Aims

Deoxynivalenol (DON) is a Fusarium derived mycotoxin, often occurring on cereals used for human and animal nutrition. The intestine, as prominent barrier for nutritional toxins, has to handle the mycotoxin from the mucosa protected luminal side (apical exposure), as well as already absorbed toxin, reaching the cells from basolateral side via the blood stream. In the present study, the impact of the direction of DON exposure on epithelial cell behaviour and intestinal barrier integrity was elucidated.

Methods

A non-transformed intestinal porcine epithelial cell line (IPEC-J2), cultured in membrane inserts, serving as a polarised in vitro model to determine the effects of deoxynivalenol (DON) on cellular viability and tight junction integrity.

Results

Application of DON in concentrations up to 4000 ng/mL for 24, 48 and 72 hours on the basolateral side of membrane cultured polarised IPEC-J2 cells resulted in a breakdown of the integrity of cell connections measured by transepithelial electrical resistance (TEER), as well as a reduced expression of the tight junction proteins ZO-1 and claudin 3. Epithelial cell number decreased and nuclei size was enlarged after 72 h incubation of 4000 ng/mL DON from basolateral. Although necrosis or caspase 3 mediated apoptosis was not detectable after basolateral DON application, cell cycle analysis revealed a significant increase in DNA fragmentation, decrease in G0/G1 phase and slight increase in G2/M phase after 72 hours incubation with DON 2000 ng/mL.

Conclusions

Severity of impact of the mycotoxin deoxynivalenol on the intestinal epithelial barrier is dependent on route of application. The epithelium appears to be rather resistant towards apical (luminal) DON application whereas the same toxin dose from basolateral severely undermines barrier integrity.     

First survey on the natural occurrence of Fusarium mycotoxins in Bulgarian wheat

Wheat for human consumption (140 samples) was collected after harvest from all regions of Bulgaria. The 1995 crop year was characterized by heavy rainfall in the spring and summer months. The internal mycoflora of wheat samples was dominated by Fusarium spp. and Alternaria spp., and storage fungi were rarely present. The samples were analysed for contamination with Fusarium mycotoxins deoxynivalenol (DON), 3-acetyldeoxynivalenol (3-AcDON), 15-acetyldeoxynivalenol (15-AcDON), T-2 Toxin (T-2), diacetoxyscirpenol (DAS), and zearalenone (ZEA), using enzyme immunoassay methods. DON and ZEA were the predominant toxins, with a contamination frequency of 67% and 69%, respectively. The average levels of these toxins in positive samples were 180 g/kg (DON) and 17 g/kg (ZEA), maximum concentrations were 1800 g kg^–1 and 120 g kg^–1, respectively. Acetyl derivatives of DON, namely 3-AcDON and 15-AcDON, were found in 2.1 % and 0.7% of the samples, at at maximum level of about 100 g kg^–1. Only one sample was positive for T-2 (55 g/kg), DAS was not detected. This is the first report about the natural occurrence of a range of Fusarium mycotoxins in wheat for human consumption in Bulgaria.Abbreviations 3-AcDON 3-acetyldeoxynivalenol - 15-AcDON 15-acetyldeoxynivalenol - DAS diacetoxyscirpenol - DON deoxynivalenol - EIA enzyme immunoassay - T-2 T-2 toxin - ZEA zearalenone     

Angiopoietin-1 Regulates Brain Endothelial Permeability through PTPN-2 Mediated Tyrosine Dephosphorylation of Occludin

Objective

Blood brain barrier (BBB) breakdown and increased endothelial permeability is a hallmark of neuro-vascular inflammation. Angiopoietin-1 (Ang-1), a Tie-2 receptor agonist ligand, is known to modulate barrier function of endothelial cells; however the molecular mechanisms related to Ang-1 mediated repair of Tight Junctions (TJs) in brain endothelium still remain elusive. In this study, we investigated a novel role of non-receptor protein tyrosine phosphatase N-2 (PTPN-2) in Ang-1 mediated stabilization of tight junction proteins.

Method and Result

To study the barrier protective mechanism of Ang-1, we challenged human brain microvascular endothelial cells in-vitro, with a potent inflammatory mediator thrombin. By using confocal microscopy and transwell permeability assay, we show that pretreatment of brain endothelial cells with Ang-1 diminish thrombin mediated disruption of TJs and increase in endothelial permeability. We also found that Ang-1 inhibits thrombin induced tyrosine phosphorylation of Occludin and promote Occludin interaction with Zona Occludens-1 (ZO-1) to stabilize TJs. Interestingly, our study revealed that depletion of PTPN-2 by siRNAs abolishes Ang-1 ability to promote tyrosine dephosphorylation of Occludin, resulting Occludin disassociation from ZO-1 and endothelial hyperpermeability.

Summary

Collectively, our findings suggest that in brain endothelial cells blocking PTPN-2 mediated tyrosine phosphorylation of Occludin is a novel mechanism to maintain BBB function, and may offer a key therapeutic strategy for neuro-inflammatory disorders associated with BBB disruption.     

In Vitro Induction of Endothelial Apoptosis of the Post-Hypoxic Blood-Brain Barrier by Isoflurane but Not by Sevoflurane and Midazolam

Background

The effects of anesthetics on the injured brain continue to be the subject of controversial discussion. Since isoflurane has recently been shown to induce apoptosis of cerebral endothelial cells, this study compared different anesthetic compounds regarding their potential to induce cerebro-vascular apoptosis.

Methods

The in vitro model of the blood-brain barrier used in this study consisted of astrocyte-conditioned human umbilical vein endothelial cells (AC-HUVEC) has been used. After 24 h of deep hypoxia and reoxygenation or control treatment, AC-HUVEC were exposed to 0, 0.5, 1.0, or 2.0 times the minimum alveolar concentration of isoflurane or sevoflurane, or 0, 75, 150, or 300 nM of midazolam for 2 h. After 24 h, AC-HUVEC were harvested, and the degree of apoptosis was assessed by means of Western blots for the Bax and Bcl-2 ratio and, for controls and the highest concentration groups, terminal deoxynucleotidyl-mediated dUTP-biotin nick end labeling (TUNEL).

Results

Without hypoxic pretreatment, 2.0 MAC of isoflurane slightly increased TUNEL intensity compared to control and sevoflurane, but without any significant changes in the Bax and Bcl-2 ratio. After hypoxic pretreatment, exposure to isoflurane led to a multifold increase in the Bax and Bcl-2 ratio in a dose dependent manner, which was also significantly higher than the ratio observed in the 2 MAC sevoflurane group. TUNEL intensity in the post-hypoxic 2 MAC isoflurane group was increased by a factor of 11 vs. control and by 40 vs. sevoflurane. Sevoflurane and midazolam did not significantly alter these markers of apoptosis, when compared to the control group.

Conclusions

Isoflurane administered after hypoxia elevates markers of apoptosis in endothelial cells transdifferentiated to the cerebro-vascular endothelium. Endothelial apoptosis may be a previously underestimated mechanism of anesthetic neurotoxicity. Administration of high concentrations of isoflurane in experimental settings may have negative effects on the blood-brain barrier.     

Natural occurrence of deoxynivalenol, 3-acetyl-deoxynivalenol, 15-acetyl-deoxynivalenol,nivalenol, 4,7-dideoxynivalenol,and zearalenone in polish wheat

Three wheat samples collected in 1987 in Central Poland and naturally infected withFusarium spp were analyzed for the presence ofFusarium spp andFusarium toxins. Heads were separated into three fractions: kernels with visibleFusarium damage, healthy looking kernels, and chaff + rachis. The samples contained deoxynivalenol (2.0 – 40.0μg/g), nivalenol (O.O1μg/g), 4,7-dideoxynivalenol (0.10 – 0.15μg/g). 15-acetyldeoxynivalenol (0.10–2.00 μg/g), 3-acetyldeoxynivalenol (O/1Oμg/g), and zearalenone (0.01–2.00μg/g). This is the first report about 15 - acetyldeoxynivalenol in European wheat and the co-occurrence of 3 - acetyldeoxynivalenol and 15-acetyldeoxynivalenol in the same sample of contaminated cereals.     

Production of zearalenone,moniliformin and trichothecenes in intact sugar beets under laboratory conditions

Five toxigenic isolates of Fusarium species were tested for the production of zearalenone, moniliformin and trichothecenes (deoxynivalenol, 15-acetyldeoxynivalenol, T-2, HT-2 and neosolaniol) when grown on solid sugar beet slices in the laboratory for thirty days. The isolates were also grown on a solid rice medium for comparison. High zearalenone and trichothecene-producing isolates originally obtained from corn and corn-based feedstuff were compared with isolates obtained from sugar beets. One moniliformin-producing isolate from wheat was included in the study. With the exception of moniliformin, all toxins were produced on both substrates; however, the rice medium yielded the greater concentrations except for HT-2 which was produced on sugar beets in equal or greater concentrations. Zearalenone production on rice reached 729–1943 gmg/g whereas on sugar beet it reached 72–193 gmg/g. The moniliformin-producing isolate grew well on both substrates; however, moniliformin was produced only on the rice substrate. This study demonstrates for the first time that Fusarium species can produce both zearalenone and the trichothecenes on a sugar beet substrate.     

Gliotoxin penetrates and impairs the integrity of the human blood-brain barrier in vitro

Cerebral fungal infections represent an important public health concern, where a key element of pathophysiology is the ability of the fungi to cross the blood-brain barrier (BBB). Yet the mechanism used by micro-organisms to cross such a barrier and invade the brain parenchyma remains unclear. This study investigated the effects of gliotoxin (GTX), a mycotoxin secreted by Aspergillus fumigatus, on the BBB using brain microvascular endothelial cells (BMECs) derived from induced pluripotent stem cells (iPSCs). We observed that both acute (2 h) and prolonged (24 h) exposure to GTX at the level of 1 μM or higher compromised BMECs monolayer integrity. Notably, acute exposure was sufficient to disrupt the barrier function in iPSC-derived BMECs, resulting in decreased transendothelial electrical resistance (TEER) and increased fluorescein permeability. Further, our data suggest that such disruption occurred without affecting tight junction complexes, via alteration of cell-matrix interactions, alterations in F-actin distribution, through a protein kinase C-independent signaling. In addition to its effect on the barrier function, we have observed a low permeability of GTX across the BBB. This fact can be partially explained by possible interactions of GTX with membrane proteins. Taken together, this study suggests that GTX may contribute in cerebral invasion processes of Aspergillus fumigatus by altering the blood-brain barrier integrity without disrupting tight junction complexes.     

Production of zearalenone,nivalenol, moniliformin,and wortmannin from toxigenic cultures ofFusarium obtained from pasture soil samples collected in New Zealand

One culture ofF avenaceum, 4 cultures ofF oxysporum, and 11 cultures of Fsambucinum were isolated from soil samples of pasture in New Zealand in 1987. All cultures, when grown on rice media and fed to rats caused a weight loss in rats as well as toxic signs including hemorrhaging and congestion, uterine enlargement, and hematuria. 6 out of 16 cultures caused death in rat feeding tests.F oxysporum #1 killed rats (feeding test) within 5-12hrs. 10 cultures produced zearalenone (19 to 8,849 ppm), 8 cultures produced nivalenol (32 to 117 ppm), 1 culture,F sambucinum #8, produced wortmannin (40 ppm), and 5 cultures produced moniliformin (19 to 9,000ppm). We report for the first time the co-occurrence of zearalenone, nivalenol, and moniliformin produced byF sambucinum #3 in culture.F avenaceum #1 andF oxysporum cultures (nos 1, 2, and 3) produced moniliformin alone.F oxysporum #4 produced zearalenone alone as well.F sambucinum #5 caused erythema in the small intestine of rats and 100% mortality and did not produce any known toxin(s). Nivalenol when administered to the stomach of rats orally at levels 10, 20, and 40mg/kg body weight caused inflammation in the intestines, coma, and death. The mycotoxins T-2 toxin, HT-2 toxin, T-2 tetraol, diacetoxyscirpenol (DAS), monoacetoxyscirpenol (MAS), deoxynivalenol (DON), 3-acetyl-and 15-acetyldeoxynivalenol, depoxynivalenol, fusarenon-X, alpha-and beta-zearalenone, and fusarochromanone (TDP-1) were not detected in the extracts of these cultures.     

Fusarium Species from Nepalese Rice and Production of Mycotoxins and Gibberellic Acid by Selected Species

Infection of cereal grains with Fusarium species can cause contamination with mycotoxins that affect human and animal health. To determine the potential for mycotoxin contamination, we isolated Fusarium species from samples of rice seeds that were collected in 1997 on farms in the foothills of the Nepal Himalaya. The predominant Fusarium species in surface-disinfested seeds with husks were species of the Gibberella fujikuroi complex, including G. fujikuroi mating population A (anamorph, Fusarium verticillioides), G. fujikuroi mating population C (anamorph, Fusarium fujikuroi), and G. fujikuroi mating population D (anamorph, Fusarium proliferatum). The widespread occurrence of mating population D suggests that its role in the complex symptoms of bakanae disease of rice may be significant. Other common species were Gibberella zeae (anamorph, Fusarium graminearum) and Fusarium semitectum, with Fusarium acuminatum, Fusarium anguioides, Fusarium avenaceum, Fusarium chlamydosporum, Fusarium equiseti, and Fusarium oxysporum occasionally present. Strains of mating population C produced beauvericin, moniliformin, and gibberellic acid, but little or no fumonisin, whereas strains of mating population D produced beauvericin, fumonisin, and, usually, moniliformin, but no gibberellic acid. Some strains of G. zeae produced the 8-ketotrichothecene nivalenol, whereas others produced deoxynivalenol. Despite the occurrence of fumonisin-producing strains of mating population D, and of 8-ketotrichothecene-producing strains of G. zeae, Nepalese rice showed no detectable contamination with these mycotoxins. Effective traditional practices for grain drying and storage may prevent contamination of Nepalese rice with Fusarium mycotoxins.     

Occurrence of moniliformin,deoxynivalenol, and zearalenone in durum wheat (Triticum durum Desf.)

Forty-eight durum wheat samples from 5 locations in Austria were examined forFusarium infection andFusarium toxin content.F.gramlnearum andF.avenaceum were by far the prevailingFusarium species In durum wheat kernels, followed byEpoae, F.culmorum, andF.equlsetl. Ion-paired HPLC analyses of the samples showed moniliformin contents of kernels up to 0.88 mg/kg. All moniliformin contaminated samples also contained high levels of deoxynivalenol (up to 8.2 mg/kg) and lower levels of zearalenone (<0.33 mg/kg). The levels of zearalenone in naturally contaminated durum wheat samples did not correspond to the high yields of zearalenone found in cultures of the fusaria isolated from the durum wheat kernels. These conflicting results as well as some toxicologlcal aspects of the carry over ofFusarium toxins from durum wheat kernels into pasta are discussed.     

Fusarium species associated with banana fruit rot and their potential toxigenicity

Banana fruits exhibiting signs of decay were collected from markets in the United States and Italy. Fungi isolated from the lesions on the banana fruits wereFusarium moniliforme, F subglutinans, andF. semitectum var.majus. When the fungal strains were cultivated on maize kernels, the cultures did not produce zearalenone (ZON), zearalenols (á-, â-ZOH), and trichothecenes [deoxynivalenol (DON), 3-acetyldeoxynivalenol (3-AcDON), T-2 toxin (T-2), diacetoxyscirpenol (DAS)]. Fumonisins and fusarin C (FUS-C) were not detected naturally nor in bananas purchased in the U.S. and artificially infected withFusarium. Moniliformin (M) (up to 267 mg/kg) was detected in maize kernel cultures ofF. subglutinans from bananas. No mycotoxins were detected in naturally infected fruits. Although no mycotoxins were detected in the extracts from corn cultures ofF. semitectum var.majus, the extracts were toxic to brine shrimp and mice.     

Hyperthermic Laser Ablation of Recurrent Glioblastoma Leads to Temporary Disruption of the Peritumoral Blood Brain Barrier

Background

Poor central nervous system penetration of cytotoxic drugs due to the blood brain barrier (BBB) is a major limiting factor in the treatment of brain tumors. Most recurrent glioblastomas (GBM) occur within the peritumoral region. In this study, we describe a hyperthemic method to induce temporary disruption of the peritumoral BBB that can potentially be used to enhance drug delivery.

Methods

Twenty patients with probable recurrent GBM were enrolled in this study. Fourteen patients were evaluable. MRI-guided laser interstitial thermal therapy was applied to achieve both tumor cytoreduction and disruption of the peritumoral BBB. To determine the degree and timing of peritumoral BBB disruption, dynamic contrast-enhancement brain MRI was used to calculate the vascular transfer constant (K^trans) in the peritumoral region as direct measures of BBB permeability before and after laser ablation. Serum levels of brain-specific enolase, also known as neuron-specific enolase, were also measured and used as an independent quantification of BBB disruption.

Results

In all 14 evaluable patients, K^trans levels peaked immediately post laser ablation, followed by a gradual decline over the following 4 weeks. Serum BSE concentrations increased shortly after laser ablation and peaked in 1–3 weeks before decreasing to baseline by 6 weeks.

Conclusions

The data from our pilot research support that disruption of the peritumoral BBB was induced by hyperthemia with the peak of high permeability occurring within 1–2 weeks after laser ablation and resolving by 4–6 weeks. This provides a therapeutic window of opportunity during which delivery of BBB-impermeant therapeutic agents may be enhanced.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT01851733","term_id":"NCT01851733"}}NCT01851733     

Production of Beauvericin, Moniliformin, Fusaproliferin, and Fumonisins B1, B2, and B3 by Fifteen Ex-Type Strains of Fusarium Species

Fifteen Fusarium species were analyzed by high-performance liquid chromatography for the production of six mycotoxins in corn grits cultures. Production of mycotoxins ranged from 66 to 2,500 μg/kg for fumonisin B₁, 0.6 to 1,500 μg/g for moniliformin, 2.2 to 720 μg/g for beauvericin, and 12 to 130 μg/g for fusaproliferin. Fumonisin B₂ (360 μg/kg) was produced by two species, fumonisin B₃ was not detected in any of the 15 species examined, and Fusarium bulbicola produced none of the six mycotoxins that we analyzed.     

Expression of Aquaporin 4 and Breakdown of the Blood-Brain Barrier after Hypoglycemia-Induced Brain Edema in Rats

Background

Hypoglycemia-induced brain edema is a severe clinical event that often results in death. The mechanisms by which hypoglycemia induces brain edema are unclear.

Methods

In a hypoglycemic injury model established in adult rats, brain edema was verified by measuring brain water content and visualizing water accumulation using hematoxylin and eosin staining. Temporal expression of aquaporin 4 (AQP4) and the integrity of the blood-brain barrier (BBB) were evaluated. We assessed the distribution and expression of AQP4 following glucose deprivation in astrocyte cultures.

Results

Brain edema was induced immediately after severe hypoglycemia but continued to progress even after recovery from hypoglycemia. Upregulation of AQP4 expression and moderate breakdown of the BBB were observed 24 h after recovery. In vitro, significant redistribution of AQP4 to the plasma membrane was induced following 6 h glucose deprivation.

Conclusion

Hypoglycemia-induced brain edema is caused by cytotoxic and vasogenic factors. Changes in AQP4 location and expression may play a protective role in edema resolution.     

Uptake mechanism of ApoE-modified nanoparticles on brain capillary endothelial cells as a blood-brain barrier model

Background

The blood-brain barrier (BBB) represents an insurmountable obstacle for most drugs thus obstructing an effective treatment of many brain diseases. One solution for overcoming this barrier is a transport by binding of these drugs to surface-modified nanoparticles. Especially apolipoprotein E (ApoE) appears to play a major role in the nanoparticle-mediated drug transport across the BBB. However, at present the underlying mechanism is incompletely understood.

Methodology/Principal Findings

In this study, the uptake of the ApoE-modified nanoparticles into the brain capillary endothelial cells was investigated to differentiate between active and passive uptake mechanism by flow cytometry and confocal laser scanning microscopy. Furthermore, different in vitro co-incubation experiments were performed with competing ligands of the respective receptor.

Conclusions/Significance

This study confirms an active endocytotic uptake mechanism and shows the involvement of low density lipoprotein receptor family members, notably the low density lipoprotein receptor related protein, on the uptake of the ApoE-modified nanoparticles into the brain capillary endothelial cells. This knowledge of the uptake mechanism of ApoE-modified nanoparticles enables future developments to rationally create very specific and effective carriers to overcome the blood-brain barrier.     

Effects of Sertraline and Fluoxetine on P-Glycoprotein at Barrier Sites: In Vivo and In Vitro Approaches

Background and Purpose

Retention of substances from systemic circulation in the brain and testes are limited due to high levels of P-glycoprotein (P-gp) in the luminal membranes of brain and testes capillary endothelial cells. From a clinical perspective, P-gp rapidly extrudes lipophilic therapeutic agents, which then fail to reach efficacious levels. Recent studies have demonstrated that acute administration of selective serotonin reuptake inhibitors (SSRI) can affect P-gp function, in vitro and in vivo. However, little is known concerning the time-course of these effects or the effects of different SSRI in vivo.

Experimental Approach

The P-gp substrate, tritiated digoxin ([³H] digoxin), was co-administered with fluoxetine or sertraline to determine if either compound increased drug accumulation within the brains and testes of mice due to inhibition of P-gp activity. We undertook parallel studies in endothelial cells derived from brain microvessels to determine the dose-response and time-course of effects.

Key Results

In vitro, sertraline resulted in rapid and potent inhibition of P-gp function in brain endothelial cells, as determined by cellular calcein accumulation. In vivo, a biphasic effect was demonstrated. Brain accumulation of [³H] digoxin was increased 5 minutes after treatment with sertraline, but by 60 minutes after sertraline treatment, brain accumulation of digoxin was reduced compared to control. By 240 minutes after sertraline treatment brain digoxin accumulation was elevated compared to control. A similar pattern of results was obtained in the testes. There was no significant effect of fluoxetine on P-gp function, in vitro or in vivo.

Conclusions and Implications

Acute sertraline administration can modulate P-gp activity in the blood-brain barrier and blood-testes barrier. This clearly has implications for the ability of therapeutic agents that are P-gp substrates, to enter the brain when co-administered with SSRI.     

The Effect of Low-Level Laser Irradiation on Sperm Motility,and Integrity of the Plasma Membrane and Acrosome in Cryopreserved Bovine Sperm

Background and Objective

Freezing changes sperm integrity remarkably. Cryopreservation involves cooling, freezing, and thawing and all these contribute to structural damage in sperm, resulting in reduced fertility potential. Low-level laser irradiation (LLLI) could increase energy supply to the cell and cause reactive oxygen species reduction (ROS), contributing to the restoration of oxygen consumption and adenosine triphosphate synthesis (ATP) in the mitochondria. Our goal was to analyze the effects of low-level laser irradiation on sperm motility and integrity of the plasma membrane and acrosome in cryopreserved bovine sperm.

Study Design/Materials and Methods

We analyzed 09 samples of bull semen (Bos taurus indicus), divided into three groups: a control group without laser irradiation, a 4J group subjected to a laser irradiation dose of 4 joules, and a 6J group subjected to dose of 6 joules. Samples were divided for the analysis of cell viability and acrosomal membrane integrity using flow cytometry; another portion was used for motion analysis. Irradiation was performed in petri dishes of 30 mm containing 3 ml of semen by an aluminum gallium indium phosphide laser diode with a wavelength of 660 nm, 30 mW power, and energy of 4 and 6 joules for 80 and 120 seconds respectively. Subsequently, the irradiated and control semen samples were subjected to cryopreservation and analyzed by flow cytometry (7AAD and FITC-PSA) using the ISAS - Integrated Semen Analysis System.

Results

Flow cytometry showed an increase in the percentage of live sperm cells and acrosome integrity in relation to control cells when subjected to irradiation of low-power laser in two different doses of 4 and 6 joules (p < 0.05). In the analysis of straightness, percentage of cell movement, and motility, a dose of 4 joules was more effective (p < 0.05).

Conclusion

We conclude that LLLI may exert beneficial effects in the preservation of live sperm. A dose of 4 joules prior to cryopreservation was more effective than a dose of 6 joules in preserving sperm motility.