Evaluation of succulent plants Echeveria elegans as a biomonitor of heavy metals and radionuclides.

This work evaluates the use of Echeveria elegans as a biomonitor of metals and radionuclides, using semi-urban soils as a study area. The study area is exposed to various trace elements of concern for various social groups in nearby localities. The quantification of metals and radionuclides was performed by X-ray fluorescence spectrometry and gamma spectrometry, respectively. Cumulative frequency distribution curves, descriptive statistics, and multivariate analysis were used to estimate the local geochemical baseline and identify geochemical and anthropogenic patterns of metals and radionuclides from topsoil and E. elegans. The evaluation of contaminants and the contribution of possible exposure routes (topsoil and atmospheric deposition) was performed with the enrichment factor (EF) and the relative concentration factor (CFR). The results suggest that the plant does not present significant physical stress due to the environmental conditions to which it was exposed. Likewise, it can bioaccumulate heavy metals from natural and anthropogenic sources. The quantification of radionuclides in the plant is below the detection limits, indicating a low bioavailability and transfer factor. The CFR and EF results showed that the plant accumulates metals from the topsoil and atmospheric deposition. The bioaccumulation mechanism would be related to the functioning of Crassulaceae Acid Metabolism (CAM). In topsoil, the organic acids of the plant would modify the solubility of the metals present in an insoluble form in the soil, acting as ligands and, subsequently, following the transport route of these metabolites. In atmospheric deposition, the metals deposited in the leaves would be incorporated into the plant through the opening of the stomata because of the capture of CO2 (at night, day, or during environmental stress) by the CAM. Overall, the evidence showed that the succulent can be used as a biomonitor of heavy metals. However, additional studies are required to determine its usefulness as a radionuclide biomonitor.

Trade-offs among transport, support, and storage in xylem from shrubs in a semiarid chaparral environment tested with structural equation modeling.

The xylem in plants is specialized to transport water, mechanically support the plant body, and store water and carbohydrates. Balancing these functions leads to trade-offs that are linked to xylem structure. We proposed a multivariate hypothesis regarding the main xylem functions and tested it using structural equation modeling. We sampled 29 native shrub species from field sites in semiarid Southern California. We quantified xylem water transport (embolism resistance and transport efficiency), mechanical strength, storage of water (capacitance) and starch, minimum hydrostatic pressures (Pmin), and proportions of fibers, vessels, and parenchyma, which were treated as a latent variable representing "cellular trade-offs." We found that xylem functions (transport, mechanical support, water storage, and starch storage) were independent, a result driven by PminPmin was strongly and directly or indirectly associated with all xylem functions as a hub trait. More negative Pmin was associated with increased embolism resistance and tissue strength and reduced capacitance and starch storage. We found strong support for a trade-off between embolism resistance and transport efficiency. Tissue strength was not directly associated with embolism resistance or transport efficiency, and any associations were indirect involving Pmin With Pmin removed from the model, cellular trade-offs were central and related to all other traits. We conclude that xylem traits are broadly governed by functional trade-offs and that the Pmin experienced by plants in the field exerts a strong influence over these relationships. Angiosperm xylem contains different cell types that contribute to different functions and that underpin trade-offs.

Comparative genomics reveals that metabolism underlies evolution of entomopathogenicity in bee-loving Ascosphaera spp. fungi.

Ascosphaera (Eurotiomycetes: Onygenales) is a diverse genus of fungi that is exclusively found in association with bee nests and comprises both saprophytic and entomopathogenic species. To date, most genomic analyses have been focused on the honeybee pathogen A. apis, and we lack a genomic understanding of how pathogenesis evolved more broadly in the genus. To address this gap we sequenced the genomes of the leaf-cutting bee pathogen A. aggregata as well as three commensal species: A. pollenicola, A. atra and A. acerosa. De novo annotation and comparison of the assembled genomes was carried out, including the previously published genome of A. apis. To identify candidate virulence genes in the pathogenic species, we performed secondary metabolite-oriented analyses and clustering of biosynthetic gene clusters (BGCs). Additionally, we captured single copy orthologs to infer their phylogeny and created codon-aware alignments to determine orthologs under selective pressure in our pathogenic species. Our results show several shared BGCs between A. apis, A. aggregata and A. pollenicola, with antifungal resistance related genes present in the bee pathogens and commensals. Genes involved in metabolism and protein processing exhibit signatures of enrichment and positive selection under a fitted branch-site model. Additional known virulence genes in A. pollenicola, A. acerosa and A. atra are identified, supporting previous hypotheses that these commensals may be opportunistic pathogens. Finally, we discuss the importance of such genes in other fungal pathogens, suggesting a common route to evolution of pathogenicity in Ascosphaera.

COVID-19: In the Eyes of a Filipino Child.

Objectives: explore thoughts and feelings of children on COVID-19, find out how they cope, and what they did during lockdown. It was total lockdown in Luzon, Philippines, April 2020 - when survey was conducted; pre-tested open-end questionnaire was administered to children who answered either by paper and pen, or through social media, with parents' cooperation. Participants: 200 boys and girls, 6-12 years old, public and private schools in NCR-Luzon. Results: Participants heard COVID-19, pandemic and lock down from media and family; described as deadly, dangerous, contagious, world-wide, death-causing virus; about 90% expressed sadness, fear, boredom, anger, disappointments and difficult time; employed self-enhanced coping mechanisms, and engaged in hobbies and interests to assuage thoughts and feelings; family appeared as saving grace.Recommendations: develop strategies to assist children during critical events; studies - find out effects of pandemic on participants' health; visit participants after two years to find out reminiscence of pandemic experience.

Performance of a deep neural network in teledermatology: a single-centre prospective diagnostic study.

BACKGROUND: The use of artificial intelligence (AI) algorithms for the diagnosis of skin diseases has shown promise in experimental settings but has not been yet tested in real-life conditions. OBJECTIVE: To assess the diagnostic performance and potential clinical utility of a 174-multiclass AI algorithm in a real-life telemedicine setting. METHODS: Prospective, diagnostic accuracy study including consecutive patients who submitted images for teledermatology evaluation. The treating dermatologist chose a single image to upload to a web application during teleconsultation. A follow-up reader study including nine healthcare providers (3 dermatologists, 3 dermatology residents and 3 general practitioners) was performed. RESULTS: A total of 340 cases from 281 patients met study inclusion criteria. The mean (SD) age of patients was 33.7 (17.5) years; 63% (n = 177) were female. Exposure to the AI algorithm results was considered useful in 11.8% of visits (n = 40) and the teledermatologist correctly modified the real-time diagnosis in 0.6% (n = 2) of cases. The overall top-1 accuracy of the algorithm (41.2%) was lower than that of the dermatologists (60.1%), residents (57.8%) and general practitioners (49.3%) (all comparisons P < 0.05, in the reader study). When the analysis was limited to the diagnoses on which the algorithm had been explicitly trained, the balanced top-1 accuracy of the algorithm (47.6%) was comparable to the dermatologists (49.7%) and residents (47.7%) but superior to the general practitioners (39.7%; P = 0.049). Algorithm performance was associated with patient skin type and image quality. CONCLUSIONS: A 174-disease class AI algorithm appears to be a promising tool in the triage and evaluation of lesions with patient-taken photographs via telemedicine.

Effect of an intensive lifestyle intervention on the prevalence of metabolic syndrome and its components among overweight and obese adults.

BACKGROUND: Despite the fact that up to a third of the global population has metabolic syndrome (MetS), it has been overlooked in clinical settings. This study assesses the impact of a physician-supervised nonsurgical weight management program on the prevalence of MetS and its key indicators. METHODS: Four-hundred seventy-nine overweight and obese participants aged 19 years or older were included in a prospective longitudinal study. Changes in MetS and its key indicators were assessed using the binomial exact, chi-square and Wilcoxon signed-rank tests in an intent-to-treat study population. Differences in age strata were assessed using a generalized linear model. RESULTS: Fifty-two percent of participants (n = 249) had MetS at baseline. Prevalence of MetS decreased steadily with significant changes from baseline observed at weeks 13 (31.8%, P < 0.0001), 26 (28.7%, P < 0.0012) and 39 (21.6%, P < 0.0002); changes from baseline were observed at week 52 as statistically significant (16.7%, P < 0.0012). Improvements in anthropometrics and levels of key indicators of MetS were observed throughout the study. CONCLUSION: These findings confirm that weight loss is inversely associated with prevalence of MetS and its key indicators among overweight and obese individuals. Future studies may benefit from a larger sample size and better retention (ClinicalTrials.gov ID: NCT03588117).

Optimization of Ni (II) biosorption from aqueous solution on modified lemon peel.

The valorization of agricultural waste peels as a low-cost biosorbent is a promising approach to water treatment. In this work, the improvement of the adsorption capacity of lemon peel to remove Ni (II) from aqueous effluents was explored using several chemical modifiers: HNO3, HCl, H3PO4, CaCl2, NH3 and NaOH. The surface pretreatment using NaOH was selected as the best option because of the improvement of the maximum adsorption capacity. The maximum adsorption capacity was of 36.74 mg g-1 according to the Langmuir model at optimum conditions (pH = 5, S/L = 5 g L-1, 25 °C). The pseudo-first order model of biosorption kinetics provides the best fit for experimental data. From thermodynamic studies, it was concluded that Ni (II) biosorption by modified lemon peel was endothermic and spontaneous. After five consecutives adsorption-desorption cycles using 0.1 M of HNO3 and H2SO4, a recovery of 90% of Ni (II) was obtained. Regarding characterization of the biosorbent, the surface morphology was studied by Scanning Electron Microscopy while the functional groups responsible for Ni (II) adsorption were evaluated by Fourier transform infrared spectroscopy.

Progesterone in vitro increases growth, motility and progesterone receptor expression in third stage larvae of Toxocara canis.

The in vitro effect of progesterone in T. canis larvae on their enlargement and motility were evaluated, together to the possible presence of progesterone receptors (PRs). T. canis larvae were cultured in RPMI-1640 with different concentrations of progesterone (0, 20, 40, 80, 400 and 800 ng/mL). Enlargement and increases in motility were dependent on the concentration only from 0 to 80 ng/mL (p < 0.05). The mean percentage of PR + cells in newly obtained larvae as measured by flow cytometry was 8.16 ± 0.4. The number of PR + cells increased depending on concentration from 0 to 80 ng/mL (p < 0.001). Cells obtained from larvae stimulated at any of the studied hormone concentrations showed greater mean fluorescence intensity when compared to non-stimulated cells. Additionally, the expression and location of PR + cells were determined in the larvae. The sequence of an amplicon (420-bp) obtained by PCR from T. canis larvae showed 100% homology with a gene fragment that codes for the PR of the dog. PR + cells were immunolocated using confocal microscopy in the intestinal region of the larvae that had been recently obtained. The results of this study show that T. canis larvae can recognize and respond to the presence of progesterone through a molecule possibly able to bind it. Since we previously observed a similar response to prolactin, we suggest that both hormones could participate sequentially in the reactivation of T. canis larvae in pregnant bitches.

RAD51 foci as a functional biomarker of homologous recombination repair and PARP inhibitor resistance in germline BRCA-mutated breast cancer.

Background: BRCA1 and BRCA2 (BRCA1/2)-deficient tumors display impaired homologous recombination repair (HRR) and enhanced sensitivity to DNA damaging agents or to poly(ADP-ribose) polymerase (PARP) inhibitors (PARPi). Their efficacy in germline BRCA1/2 (gBRCA1/2)-mutated metastatic breast cancers has been recently confirmed in clinical trials. Numerous mechanisms of PARPi resistance have been described, whose clinical relevance in gBRCA-mutated breast cancer is unknown. This highlights the need to identify functional biomarkers to better predict PARPi sensitivity. Patients and methods: We investigated the in vivo mechanisms of PARPi resistance in gBRCA1 patient-derived tumor xenografts (PDXs) exhibiting differential response to PARPi. Analysis included exome sequencing and immunostaining of DNA damage response proteins to functionally evaluate HRR. Findings were validated in a retrospective sample set from gBRCA1/2-cancer patients treated with PARPi. Results: RAD51 nuclear foci, a surrogate marker of HRR functionality, were the only common feature in PDX and patient samples with primary or acquired PARPi resistance. Consistently, low RAD51 was associated with objective response to PARPi. Evaluation of the RAD51 biomarker in untreated tumors was feasible due to endogenous DNA damage. In PARPi-resistant gBRCA1 PDXs, genetic analysis found no in-frame secondary mutations, but BRCA1 hypomorphic proteins in 60% of the models, TP53BP1-loss in 20% and RAD51-amplification in one sample, none mutually exclusive. Conversely, one of three PARPi-resistant gBRCA2 tumors displayed BRCA2 restoration by exome sequencing. In PDXs, PARPi resistance could be reverted upon combination of a PARPi with an ataxia-telangiectasia mutated (ATM) inhibitor. Conclusion: Detection of RAD51 foci in gBRCA tumors correlates with PARPi resistance regardless of the underlying mechanism restoring HRR function. This is a promising biomarker to be used in the clinic to better select patients for PARPi therapy. Our study also supports the clinical development of PARPi combinations such as those with ATM inhibitors.

Desiccating stress-induced disruption of ocular surface immune tolerance drives dry eye disease.

Dry eye is an allegedly autoimmune disorder for which the initiating mechanisms and the targeted antigens in the ocular surface are not known, yet there is extensive evidence that a localized T helper type 1 (Th1)/Th17 effector T cell response is responsible for its pathogenesis. In this work, we explore the reconciling hypothesis that desiccating stress, which is usually considered an exacerbating factor, could actually be sufficient to skew the ocular surface's mucosal response to any antigen and therefore drive the disease. Using a mouse model of dry eye, we found that desiccating stress causes a nuclear factor kappa B (NF-κB)- and time-dependent disruption of the ocular surface's immune tolerance to exogenous ovalbumin. This pathogenic event is mediated by increased Th1 and Th17 T cells and reduced regulatory T cells in the draining lymph nodes. Conversely, topical NF-κB inhibitors reduced corneal epithelial damage and interleukin (IL)-1ß and IL-6 levels in the ocular surface of mice under desiccating stress. The observed effect was mediated by an augmented regulatory T cell response, a finding that highlights the role of mucosal tolerance disruption in dry eye pathogenesis. Remarkably, the NF-κB pathway is also involved in mucosal tolerance disruption in other ocular surface disorders. Together, these results suggest that targeting of mucosal NF-κB activation could have therapeutic potential in dry eye.

High-throughput sequencing of cerebrospinal fluid for diagnosis of chronic Propionibacterium acnes meningitis in an allogeneic stem cell transplant recipient.

BACKGROUND: A 40-year-old man with chronic myelogenous leukemia presented multiple times over a period of 3 years with episodes of confusion, wide-based gait and falls because of recurrent hydrocephalus despite repeated therapeutic lumbar punctures. These problems occurred in the context of persistent cerebrospinal fluid (CSF) pleocytosis and leptomeningeal enhancement. Extensive diagnostic workups and therapeutic trials had failed to identify a clinically plausible cause or produce any significant improvement in the CSF and neuroimaging abnormalities. METHODS: We used high-throughput metagenomic shotgun sequencing to identify microbes in 2 CSF samples collected from the patient during his illness. These results were compared to sequence data from 1 CSF sample collected during treatment and 5 control CSF samples from other patients. RESULTS: We found sequences representing 53% and 67% of the Propionibacterium acnes genome in 2 CSF samples collected from the patient during his illness. Directed antimicrobial therapy was administered for 6 weeks with resolution of CSF and neuroimaging abnormalities. Sequencing of a sample obtained during treatment demonstrated that the P. acnes levels were decreased to background levels. After insertion of a ventriculo-peritoneal shunt, the patient returned to baseline status. CONCLUSIONS: High-throughput metagenomic shotgun sequencing revealed P. acnes as the cause of chronic meningitis that had eluded conventional attempts at diagnosis. Treatment directed at this organism resulted in cure of the infection and clinical improvement.

Effect of exposure to adverse climatic conditions on production in Manchega dairy sheep.

The present study aimed to examine the effects of exposure to adverse weather conditions on milk production to assess the thermotolerance capability of the Manchega breed, a dairy sheep reared in the Mediterranean area, and the extent of decline in production outside the thermal comfort zone. To achieve this purpose, we merged data from the official milk recording of the breed with weather information and used to describe the cold and heat stress response for production traits. Production data consisted of 1,094,804 test-day records from the first 3 lactations of 177,605 ewes gathered between years 2000 to 2010. For each production trait and climate variable, the thermal load production response was characterized by the estimation of cold and heat stress thresholds that define a thermoneutral zone and the slopes of production decay outside this thermoneutral zone. Overall, we observed a comfort region between 10 and 22°C for daily average temperature, 18 and 30°C for daily maximum temperature, and from 9 to 18 units for a temperature-humidity index (THI) for all traits. Decline in production due to cold stress effects was of a greater magnitude than heat stress effects, especially for milk yield. Production losses ranged between 7 and 16 and from 0.2 to 0.6g/d per °C (or THI unit) for milk and for fat and protein yields, respectively. For heat stress, the observed decline in production was of 1 to 5 and 0.1 to 0.3g/d per °C (or THI unit) above the threshold for milk yield and for fat and protein yields, respectively. Highly productive animals showed a narrower comfort zone and higher slopes of decay. The study of lagged effects of thermal load showed how consequences of cold and heat stress are already visible in the first hours after exposure. Thus, production losses were due mainly to climate conditions on the day of control and the day before, with conditions on the previous days having a smaller effect. Annual economic losses due to thermal (cold and heat) stress ranged from 0.1 up to 4% of total profit depending on which climate variable was considered. Although of small magnitude, the effect of adverse climatic conditions on total annual farm profit is not negligible; thus, we consider the implementation of strategies aimed at reducing these losses to be important. These strategies could be target improving the mitigation strategies as well as obtaining more thermotolerant animals through selection.

Spontaneous gallbladder perforation in a child secondary to a gallbladder cyst: a rare presentation and review of literature.

Spontaneous gallbladder perforation is rare in children. The etiology of gallbladder perforation varies greatly and is often unknown. Identified causes include infection, congenital, stones or choledochal cysts. Presently there are only five reported cases of spontaneous gallbladder perforation in children in the English literature. As such, the optimal method of diagnosis and management remains controversial. We report the case of a 2-year-old girl who presented with peritonitis secondary to spontaneous gallbladder perforation.

Anticancer mechanisms of cannabinoids.

In addition to the well-known palliative effects of cannabinoids on some cancer-associated symptoms, a large body of evidence shows that these molecules can decrease tumour growth in animal models of cancer. They do so by modulating key cell signalling pathways involved in the control of cancer cell proliferation and survival. In addition, cannabinoids inhibit angiogenesis and decrease metastasis in various tumour types in laboratory animals. In this review, we discuss the current understanding of cannabinoids as antitumour agents, focusing on recent discoveries about their molecular mechanisms of action, including resistance mechanisms and opportunities for their use in combination therapy. Those observations have already contributed to the foundation for the development of the first clinical studies that will analyze the safety and potential clinical benefit of cannabinoids as anticancer agents.

Electrochemical magnetoimmunosensor for the ultrasensitive determination of interleukin-6 in saliva and urine using poly-HRP streptavidin conjugates as labels for signal amplification.

A novel magnetoimmunosensor design for interleukin-6 (IL-6) which involved the covalent immobilization of anti-IL-6 antibodies onto carboxyl-functionalized magnetic microparticles and a sandwich-type immunoassay with signal amplification using poly-HRP-streptavidin conjugates is reported. All the variables concerning the preparation and the electroanalytical performance of the immunosensor were optimized. The use of poly-HRP-strept conjugates as enzymatic labels instead of conventional HRP-strept allowed enhanced signal-to-blank current ratios to be obtained. A linear calibration plot between the measured steady-state current and the log of IL-6 concentration was achieved in the 1.75 to 500 pg/mL range, which was not feasible when using HRP-strep as label. A limit of detection of 0.39 pg/mL IL-6 was obtained. The anti-IL-6-MB conjugates exhibited an excellent storage stability providing amperometric responses with no significant loss during at least 36 days. The magnetoimmunosensor showed also an excellent selectivity against potentially interfering substances. The immunosensor was used to determine IL-6 in urine samples spiked at three different concentration levels with clinical relevance. Moreover, IL-6 was measured in three different saliva samples corresponding to a periodontitis patient, a smoker volunteer, and a non-smoker volunteer. The obtained results were statistically in agreement with those provided by a commercial ELISA kit.

Understanding sperm heterogeneity: biological and practical implications.

Sperm are the most diverse cell type known. This diversity is thought to reflect adaptation to conditions under which sperm function as a way to ensure the survival of sperm in fertilization environments and to maximize fertilizing capacity thereof. The existence of morphological diversity among species is widely assumed, although this diversity seems less clear as we go deeper (between males, between ejaculates from the same male and even within the same ejaculate), with different theories addressing this heterogeneity. Moreover, the development of assisted reproductive techniques (ART) has led to changes in the physiological conditions in which sperm fertilize, which could lead, ultimately, to a selection towards more favourable sperm design. Regardless of the origin of this diversity, when studying the relationship between shape and function of sperm, it is advisable to assess the degree of heterogeneity of sperm and takes into account to be more likely to identify those morphological characteristics determining the fertile ability of sperm. Otherwise, these relationships could be hidden as a result of considering an average shape not representative of morphological characteristics of sperm. In addition, the knowledge of this morphological diversity in terms of changes arising from modifications in the sperm environment and mechanisms that generate these changes could be useful for understanding the reproductive capacity of males but also in enhancing their fertile ability.

Characterization of pea (Pisum sativum) seed protein fractions.

BACKGROUND: Legume seed proteins have to be chemically characterized in order to properly link their nutritional effects with their chemical structure. RESULTS: Vicilin and albumin fractions devoid of cross-contamination, as assessed by mass peptide fingerprinting analysis, were obtained from defatted pea (Pisum sativum cv. Bilbo) meal. The extracted protein fractions contained 56.7-67.7 g non-starch polysaccharides kg⁻¹. The vicilin fraction was higher than legumins in arginine, isoleucine, leucine, phenylalanine and lysine. The most abundant amino acids in the albumin fraction were aspartic acid, glutamic acid, lysine and arginine, and the amounts of methionine were more than double than those in legumins and vicilins. The pea albumin fraction showed a clear enrichment of protease inhibitory activity when compared with the seed meal. In vitro digestibility values for pea proteins were 0.63 ± 0.04, 0.88 ± 0.04 and 0.41 ± 0.23 for legumins, vicilins and albumins respectively. CONCLUSION: Vicilin and albumin fractions devoid of cross-contamination with other proteins were obtained from pea seed meal. The vicilin fraction also contained low amounts of soluble non-starch polysaccharides and was enriched in isoleucine, leucine, phenylalanine and lysine. In vitro digestibility values for pea proteins were similar or even numerically higher than those for control proteins.

Divergent Gut Microbiota: Archaeal and Bacterial Signatures Unveil Unique Patterns in Colombian Cyclists Compared to Weightlifters and Non-Athletes.

Engagement in physical activity, across various sports, promotes a diverse microbiota in active individuals. This study examines the gut microbiota of Colombian athletes, specifically weightlifters (n = 16) and road cyclists (n = 13), compared to non-athletes (n = 15). Using Kruskal-Wallis tests, the physical activity level of a group of non-athletic individuals and the sports experience of a group of professional athletes is analyzed. The median age of participants is 24 years, comprising 25 men and 19 women. The microbiota is collected using fecal samples. Participants provided these samples during their pre-competitive stage, specifically during the concentration phase occurring two weeks prior to national competitions. This timing is chosen to capture the microbial composition during a period of heightened physical preparation. Questionnaire responses and microbial composition assessments identify disparities among groups. Microbial composition analysis explores core microbiome, abundance, and taxonomy using Pavian, MicrobiomeAnalyst 2.0, and GraPhlAn. ANCOM-BC2 reveals differentially abundant species. Road cyclists exhibit decreased Bacteria and increased Archaea abundance. Phylum-level variations included Planctomycetes, Acidobacteria, and Proteobacteria, while Bacteroidetes prevailed. Key families influencing gut microbiota are Bacteroidaceae, Muribaculaceae, and Selenomonadaceae. Weightlifters exhibit unique viral and archaeal community connections, while cyclists showed specialized microbial interplay influenced by endurance exercise. Correlation network analysis emphasizes distinctive microbial interactions within athlete groups, shedding light on the impact of physical activities on gut microbiota and athlete health.

Improved cryopreservation protocol for Blanca-Celtibérica buck semen collected by electroejaculation.

The collection of sperm samples by electroejaculation (EE) leads to an increase of the production of seminal plasma which could modify the tolerance of spermatozoa to the cryopreservation procedure. This study aims to compare a standard sperm cryopreservation protocol for samples collected by artificial vagina (AV) with the same protocol and modifications to this for samples obtained by EE. Semen from six males of Blanca-Celtibérica goat breed was collected by AV (control) and EE, and three experiments were conducted. In Experiment 1, it was examined the effects of egg yolk concentration contained in freezing extender (0%, 1.5%, 10% and 20% of egg yolk); in Experiment 2, it was evaluated the cooling rate from 30 to 5 °C (fast: 10 min and slow: 90 min) and the temperature of glycerol addition (30 and 5 °C); and in Experiment 3, it was examined the time of equilibration at 5 °C (0, 1, 2 or 3h). A heterologous in vitro fertilization test was carried out in order to compare the fertility of control samples with that resulting from the EE protocol which showed the highest sperm quality. Results showed greater sperm motility parameters after thawing for control samples cryopreserved in standard conditions in the three experiments. For samples collected by EE, extender with 20% egg yolk, a slow cooling rate and a longer equilibration time (3h) provided higher sperm quality, and no differences were observed between temperatures of glycerol addition. Samples collected by EE and cryopreserved with the protocol which yielded the best sperm quality after thawing showed higher fertility compared to AV.