Sperm Cryodamage in Ruminants: Understanding the Molecular Changes Induced by the Cryopreservation Process to Optimize Sperm Quality.

Sperm cryopreservation represents a powerful tool for livestock breeding. Several efforts have been made to improve the efficiency of sperm cryopreservation in different ruminant species. However, a significant amount of sperm still suffers considerable cryodamage, which may affect sperm quality and fertility. Recently, the use of different "omics" technologies in sperm cryobiology, especially proteomics studies, has led to a better understanding of the molecular modifications induced by sperm cryopreservation, facilitating the identification of different freezability biomarkers and certain proteins that can be added before cryopreservation to enhance sperm cryosurvival. This review provides an updated overview of the molecular mechanisms involved in sperm cryodamage, which are in part responsible for the structural, functional and fertility changes observed in frozen-thawed ruminant sperm. Moreover, the molecular basis of those factors that can affect the sperm freezing resilience of different ruminant species is also discussed as well as the molecular aspects of those novel strategies that have been developed to reduce sperm cryodamage, including new cryoprotectants, antioxidants, proteins, nanoparticles and vitrification.

Quality Standards for Allergen Immunotherapy Clinics in Spain: Consensus Document.

BACKGROUND AND OBJECTIVE: Allergen immunotherapy clinics (AITCs) in Spain differ widely in terms of structure, organization, resources, and portfolio of services. Therefore, it is essential to unify treatment criteria and define quality standards for the most complex AITCs. Objective: To establish a series of recommendations that make it possible to guarantee quality and safety in the administration of immunotherapy and define quality standards for the most complex AITCs. METHODS: This project began with an online survey of 65 allergy departments/units throughout Spain in 2013. Next, a 2-phase consensus process was carried out. In the first phase, 10 experts defined and agreed on the standards using the RAND/UCLA Appropriateness method; in the second, the agreements were validated by means of a 2-round Delphi consultation with 84 experts. RESULTS: Consensus was reached on minimum safety and quality criteria in the administration of allergen immunotherapy, and 2 levels of highly complex AITCs were defined: accredited AITCs and accredited AITCs with excellence. Consensus was also reached on quality standards and accreditation criteria for both levels. CONCLUSIONS: This project is pioneering in terms of its purpose (the definition of quality standards for AITCs) and of the use of structured participation techniques (combination of the RAND/UCLA and Delphi methods). It enabled the design of minimum standards for quality and safety in administering AIT, as well as quality criteria for accreditation of AITCs supported by a broad panel of experts from the Spanish Society of Allergology and Clinical Immunology.

Freezing-Thawing Procedures Remodel the Proteome of Ram Sperm before and after In Vitro Capacitation.

Mammalian sperm must undergo a set of structural and functional changes collectively termed as capacitation to ensure a successful oocyte fertilization. However, capacitation can be compromised by cryopreservation procedures, which alter the proteome and longevity of sperm. To date, how the protein changes induced by cryopreservation could affect the acquisition of sperm fertilizing potential remains unexplored. The present study investigated the protein profile of ram sperm during in vitro capacitation before and after cryopreservation to elucidate the impact of cryopreservation on sperm capacitation at a molecular level. Fresh and cryopreserved ram sperm were incubated under capacitating (CAP) and non-capacitating (NC) conditions for 240 min. The sperm proteome of these four treatments was analyzed and compared at different incubation times using reverse phase liquid chromatography coupled to mass spectrometry (RP-LC-MS/MS). The comparison between fresh and cryopreserved sperm suggested that cryopreservation facilitated an apoptosis-stress response and redox process, while the comparison between sperm incubated in CAP and NC conditions showed that capacitation increased those biological processes associated with signaling, metabolism, motility, and reproductive processes. In addition, 14 proteins related to mitochondrial activity, sperm motility, oocyte recognition, signaling, spermatogenesis, and the apoptosis-stress response underwent significant changes in abundance over time when fresh and cryopreserved sperm incubated in CAP and NC conditions were compared. Our results indicate that disturbances in a ram sperm proteome after cryopreservation may alter the quality of sperm and its specific machinery to sustain capacitation under in vitro conditions.

Objective evaluation of ram and buck sperm motility by using a novel sperm tracker software.

This work offers researchers the first version of an open-source sperm tracker software (Sperm Motility Tracker, V1.0) containing a novel suit of algorithms to analyze sperm motility using ram and buck sperm as models. The computer-assisted semen analysis is used in several publications with increasing trend worldwide in the last years, showing the importance of objective methodologies to evaluate semen quality. However, commercial systems are costly and versatility is constrained. In the proposed method, segmentation is applied and the tracking stage is performed by using individual Kalman filters and a simplified occlusion handling method. The tracking performance in terms of precision (number of true tracks), the percentage of fragmented paths and percentage of correctly detected particles were manually validated by three experts and compared with the performance of a commercial motility analyzer (Microptic's SCA). The precision obtained with our sperm motility tracker was higher than the one obtained with a commercial software at the current acquisition frame rate of 25 fps (P < 0.0001), concomitantly with a similar percentage of fragmentized tracks (P = 0.0709) at sperm concentrations ranging 25-37 × 106 cells/mL. Moreover, our tracker was able to detect trajectories that were unseen by SCA. Kinetic values obtained by using both methods were contrasted. The higher values found were explained based on the better performance of our sperm tracker to report speed parameters for very fast motile sperm. To standardize results, acquisition conditions are suggested. This open-source sperm tracker software has a good plasticity allowing researchers to upgrade according requirements and to apply the tool for sperm from a variety of species.

An In Vitro Evaluation of Biochemical Processes Involved in Lead-Induced Changes on Ram Spermatozoa.

Lead (Pb(2+) ) is a toxic heavy metal which interferes with several physiological processes regulated by Ca(2+) , including those characterized by changes of the membrane stability and the motility of spermatozoa necessary for the fertilization of the oocyte. In this study, ejaculated sperm from six rams (Ovis aries) have been incubated in vitro with or without 50 ng Pb(2+) /ml during 30 min and in the presence or absence of three different potential modulators of the effects of Pb(2+) on changes in the sperm membrane before fertilization: charybdotoxin, quinacrine and staurosporine. Sperm samples incubated with Pb(2+) have shown significant reductions in acrosome integrity and sperm viability and an increase in progressive movement. None of the studied potential modulators had a protective effect against Pb(2+) action. On the contrary, Pb(2+) -incubated sperm in the presence of staurosporine had lower acrosome integrity, and lower sperm viability was observed when spermatozoa were incubated with Pb(2+) + charybdotoxin. Quinacrine was the only tested substance capable of increasing the concentration of Pb(2+) in spermatozoa; thus, the enhancement of Pb(2+) effects produced by staurosporine and charybdotoxin was not produced by an increased uptake of Pb(2+) by spermatozoa. However, the increase of intracellular Pb(2+) in those spermatozoa incubated with quinacrine did not result in an adverse effect on sperm motility or viability although the acrosome integrity was negatively affected.

Selection of red deer spermatozoa with different cryoresistance using density gradients.

The objective of sperm selection media is selecting the best spermatozoa and to remove seminal plasma and diluent for using them in assisted reproductive techniques. It is known that individuals show different cryoresistance in response to the same freezing procedure. Our hypothesis was that the efficacy of selection media could be dissimilar for samples with different sperm quality after thawing. Epididymal sperm samples from mature Iberian red deer were collected and frozen. Males were classified as with high post-thaw sperm quality when sperm motility (SM) ≥ 70%, or as with low post-thaw sperm quality when SM ≤ 69%. Samples were centrifuged using the following density gradients (DG): Percoll® , Puresperm® and Bovipure™ , and several functional sperm parameters were assessed after sperm selecting and washing. Males classified with high sperm quality had higher post-thawing values (p > .05) for all parameters evaluated, except for linearity index, than those categorized as low sperm quality. After selection, some sperm characteristics improved (viability, apoptosis and mitochondrial activity) for both groups, showing the males with high sperm quality higher values in all sperm parameters except for kinematic traits and DNA fragmentation index (%DFI), regardless of DG. Bovipure™ yield lower values of sperm motility, viability, apoptosis and mitochondrial activity in relation to Percoll® and Puresperm® considering both quality groups. There was an interaction between the type of DG and sperm quality group for sperm viability (p = .040) and apoptosis (p = .003). Thus, Percoll® selected less live and more apoptotic spermatozoa than Puresperm® and Bovipure™ for males with low sperm quality. In conclusion, the DG are more efficient selecting spermatozoa from samples with high sperm quality, acting differently depending on initial sperm quality.

Oestrous sheep serum balances ROS levels to supply in vitro capacitation of ram spermatozoa.

Reactive oxygen species (ROS) are fundamental for intracellular signalling. In spermatozoa, they are involved both to apoptosis and to capacitation, and changes in ROS levels can alter the balance between these two processes. Oestrous sheep serum (OSS) is considered an efficient agent for in vitro capacitation of ram spermatozoa. We have explored the effects of OSS on ram sperm physiology, especially on ROS production, during in vitro capacitation. Semen samples from 15 rams were cryopreserved. After thawing, samples were submitted to four treatments: control (CTL), 10% OSS supplementation for in vitro sperm capacitation, caspase inhibitor (INH, Z-VAD-FMK 100 µM) and OSS (10%) plus caspase inhibitor (I + E). Sperm samples were incubated for 30 min at 38.5°C and 5% CO2 and evaluated motility and kinetic parameters by computer-assisted semen analysis (CASA) and viability (propidium iodide), apoptotic-like membrane changes (YO-PRO-1), acrosomal status (PNA-FITC), intracellular calcium (FLUO-3), membrane fluidity (M540) and ROS production (CM-H2 DCFDA) by flow cytometry. OSS induced changes in kinetic parameters compatible with capacitation, with a decrease in the percentage of progressive motility and linearity, and an increase in the amplitude of the lateral displacement of the sperm head (p < .05). Moreover, OSS increased the proportion of M540+ viable spermatozoa, YO-PRO-1+ and acrosome-reacted spermatozoa (p < .05). After incubation, OSS and I+E achieved lower ROS levels (p < .05). Ca(2+) levels did not change with the incubation, but were slightly higher (p < .05) when both OSS and the inhibitor were present. We suggest that OSS may modulate ROS levels, allowing intracellular signalling for capacitation to occur while preventing higher levels that could trigger apoptosis.

The Effect of Oxidative Stress on Thawed Bulk-Sorted Red Deer Sperm.

The aims of this study were to assess the effects of the sex-sorting process on post-thaw sperm quality as well as on induced oxidative stress damage (H2 O2 0 mm = H000; H2 O2 50 mm = H050; H2 O2 100 mm = H100) and the protective action of reduced glutathione (GSH) and Trolox, when comparing sorted (BSS) and non-sorted (NS) red deer spermatozoa incubated at 37°C. Sperm samples from three stags were collected by electroejaculation and frozen. Immediately after thawing, sperm motility was higher (p < 0.05) for NS (59% ± 3.3) than BSS (36.9% ± 5.8) sperm. Furthermore, the percentage of apoptotic sperm was higher (p < 0.05) for BSS (21.6% ± 5.0) than NS sperm (14.6% ± 1.2). The presence of H2 O2 increased DNA damage in NS (H000 = 4.1% ± 0.9; H050 = 9.3% ± 0.7; and H100 = 10.9% ± 2.3), but not in BSS sperm. However, in the presence of oxidant, GSH addition improved (p < 0.05) sperm motility in both groups of sperm samples as compared to their controls (NS: 44.5 ± 4.8 vs 21.1 ± 3.9 and BSS: 33.3 ± 8.1 vs 8.9 ± 1.8). These results demonstrate that the sperm-sorting process induces sublethal effects, albeit selecting a sperm population with a chromatin more resistant to oxidative stress than that in non-sorted sperm. Moreover, addition of GSH at 1 mm may be a good choice for maintaining the quality of stressed sperm samples, unlike Trolox, which inhibited sperm motility.

Free-radical production after post-thaw incubation of ram spermatozoa is related to decreased in vivo fertility.

The aim of the present study was to evaluate the effect of sperm reactive oxygen species (ROS) production and DNA changes on male fertility. For that purpose, six rams with significantly different pregnancy rates were used; these were classified as having high fertility, i.e. 59.4% average pregnancy rate, or low fertility, i.e. 23.1% average pregnancy rate. Sperm quality was assessed after a two-step process of sample thawing followed by an incubation of 2h, either in the freezing extender (37°C) or after dilution in synthetic oviductal fluid (SOF; 38°C, 5%CO2). Sperm viability (YO-PRO-1), ROS production (5-(and-6)-chloromethyl-2',7'-dichlorodihydrofluorescein acetyl ester (CM-H2DCFDA)) and undamaged chromatin (sperm chromatin structure assay, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling, chromomycin A3) were evaluated by flow cytometry. Although no significant differences in sperm viability were observed, our results showed increased ROS production during incubation in the freezing extender as well as in SOF medium. Comparison between fertility groups showed significant differences in ROS production after 2h of incubation for the two treatments. Regarding DNA integrity, our results showed no significant differences either between treatments and incubation times or fertility groups. Linear regression analysis showed that ROS production determined by CM-H2DCFDA was a good indicator parameter for in vivo male fertility of SOF-incubated samples, yielding a fair correlation between both parameters (r=-0.92). These results indicate that detection of ROS production by CM-H2DCFDA and flow cytometry after 2h of incubation in SOF could be a useful procedure for predicting fertility of ram spermatozoa.

The effects of surgical checklists on morbidity and mortality: a pre- and post-intervention study.

BACKGROUND: Surgical checklists (SCs) have been developed to enhance teamwork and facilitate handovers, thereby improving the safety of surgical patients in health care organisations. The aim of this study was to determine whether the implementation of a 39-item SC reduced mortality and surgical adverse events (AEs) in patients undergoing inpatient surgery. METHODS: A retrospective pre- and post-intervention study of two cohorts of surgical patients was conducted (n = 1602) in a tertiary teaching hospital. The patients' homogeneity was confirmed by studying 40 comorbidities, 13 analytical determinations and 14 patient- and intervention-related variables. A 39-item SC adapted from one by the World Health Organization was used. The primary endpoint was the occurrence of any AE, including death, within 30 days of the operation. Twenty-three types of AEs were analysed. RESULTS: Following implementation of the checklist, the rate of AEs per 100 patients decreased from 31.5% to 26.5% (P = 0.39), the rate of infectious AEs decreased from 13.9 to 9.6 (P = 0.037) and non-infectious AEs decreased from 17.5 to 16.8 (P = 0.82). For non-elective patients, total AEs decreased from 60.4 to 37.0 (P = 0.017). The proportion of patients with one or more AE decreased from 18.1% to 16.2% (P = 0.35), and the death rate at 30 days decreased from 1.5% to 0.9% (P = 0.35). CONCLUSION: The overall AE rate did not decrease significantly between the two periods. However, the rate of infectious AEs and overall AEs in patients with non-elective admissions had statistically significant reductions. Further research is needed to determine how and in which patients SC introduction can work successfully.

Effects of vitrification on ram spermatozoa using free-egg yolk extenders.

The present study aimed to examine the behavior of ram spermatozoa subjected to a vitrification process in free-egg yolk diluents in relation with conventional diluents and cryopreservation protocol used in this species. Previously it was investigated the toxicity of cryoprotectants, sucrose and glycerol, based on different concentrations (sucrose at 0.03 M, 0.05 M, 0.15 M and 0.25 M; and glycerol at 3%, 7%, 14% and 18%) compared to a commercial extender (Biladyl® with 20% egg yolk and 7% glyerol). Cryoprotectants which reported less toxicity were chosen to perform the vitrification and results were compared with the conventional cryopreservation. Semen from three rams was collected by electroejaculation. The sperm evaluation was carried out at 0, 2 and 4h through the incubation time at 37°C for the experiment of toxicity and, at thawing when cryopreservation was performed. The sperm quality throughout the incubation time always resulted lower (P⩽0.05) for the free-egg yolk diluents in relation to Biladyl® (control), obtaining the lowest values of sperm quality with the highest concentrations of sucrose and glycerol. The vitrification was carried out with combinations of sucrose and glycerol (sucrose at 0.03 and 0.05 M with 3% and 7% of glycerol, respectively) and with Biladyl® (at different sperm concentrations). The vitrification decreased drastically (P⩽0.05) the sperm quality when combinations of sucrose and glycerol were used. Nevertheless, the sperm samples vitrified with Biladyl® at the lowest sperm concentration showed acceptable values of viability, acrosome integrity and DFI, although the sperm motility was strongly decreased. In conclusion, the use of vitrification with diluents based on combinations of sucrose and glycerol did not work for semen cryopreservation of ram. Promising results were obtained when diluents with egg yolk were used in the vitrification procedure, although more studies are necessary to improve this technique and the use of diluents without egg yolk.

Two biomarker-directed randomized trials in European and Chinese patients with nonsmall-cell lung cancer: the BRCA1-RAP80 Expression Customization (BREC) studies.

BACKGROUND: In a Spanish Lung Cancer Group (SLCG) phase II trial, the combination of BRCA1 and receptor-associated protein 80 (RAP80) expression was significantly associated with outcome in Caucasian patients with nonsmall-cell lung cancer (NSCLC). The SLCG therefore undertook an industry-independent collaborative randomized phase III trial comparing nonselected cisplatin-based chemotherapy with therapy customized according to BRCA1/RAP80 expression. An analogous randomized phase II trial was carried out in China under the auspices of the SLCG to evaluate the effect of BRCA1/RAP80 expression in Asian patients. PATIENTS AND METHODS: Eligibility criteria included stage IIIB-IV NSCLC and sufficient tumor specimen for molecular analysis. Randomization to the control or experimental arm was 1 : 1 in the SLCG trial and 1 : 3 in the Chinese trial. In both trials, patients in the control arm received docetaxel/cisplatin; in the experimental arm, patients with low RAP80 expression received gemcitabine/cisplatin, those with intermediate/high RAP80 expression and low/intermediate BRCA1 expression received docetaxel/cisplatin, and those with intermediate/high RAP80 expression and high BRCA1 expression received docetaxel alone. The primary end point was progression-free survival (PFS). RESULTS: Two hundred and seventy-nine patients in the SLCG trial and 124 in the Chinese trial were assessable for PFS. PFS in the control and experimental arms in the SLCG trial was 5.49 and 4.38 months, respectively [log rank P = 0.07; hazard ratio (HR) 1.28; P = 0.03]. In the Chinese trial, PFS was 4.74 and 3.78 months, respectively (log rank P = 0.82; HR 0.95; P = 0.82). CONCLUSION: Accrual was prematurely closed on the SLCG trial due to the absence of clinical benefit in the experimental over the control arm. However, the BREC studies provide proof of concept that an international, nonindustry, biomarker-directed trial is feasible. Thanks to the groundwork laid by these studies, we expect that ongoing further research on alternative biomarkers to elucidate DNA repair mechanisms will help define novel therapeutic approaches. TRIAL REGISTRATION: NCT00617656/GECP-BREC and ChiCTR-TRC-12001860/BREC-CHINA.

Dynamics of sperm subpopulations based on motility and plasma membrane status in thawed ram spermatozoa incubated under conditions that support in vitro capacitation and fertilisation.

The present study evaluated modifications occurring in thawed ram spermatozoa during incubation in different media that supported in vitro capacitation and fertilisation, and examines how these changes relate to IVF. Thawed sperm samples were incubated under capacitating (Cap) and non-capacitating (non-Cap) conditions for 0, 1 and 2h and used in an IVF test. During incubation, changes related to membrane status and the motility pattern of spermatozoa were assessed, the latter being used to characterise sperm subpopulations. A significantly greater increase (P≤0.05) in the percentage of spermatozoa with higher membrane fluidity was observed in samples incubated with Cap medium from the beginning of incubation. In addition, changes over time in the distribution of the motile subpopulation were particularly evident when spermatozoa were incubated with Cap medium, with a noted increase in spermatozoa classified as 'hyperactivated like', with major changes occurring after 1h incubation. Both characteristics (i.e. membrane fluidity and the percentage of the hyperactivated-like subpopulation) were significantly related with in vitro fertility, and only sperm samples incubated with the Cap medium were capable of fertilising oocytes. These results support the idea that changes in sperm membrane fluidity and motility pattern (i.e. an increase in hyperactivated spermatozoa) are needed for fertilisation to take place.

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.

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.

Identification of optimal concentrations and incubation times for the study of in vitro effects of Pb in ram spermatozoa.

In vitro effects of lead (Pb) on ram (Ovis aries) spermatozoa were studied to establish a threshold level that affects sperm function. Spermatozoa were incubated between 15 and 180 min with Pb concentrations ranging from 0 to 5,000 ng/mL. Sperm motility, acrosome integrity, membrane functionality and sperm viability were all negatively affected by Pb and incubation time. Acrosome integrity was linearly affected by Pb levels at an incubation time of 30 min, and 50 ng/mL was the lowest Pb level producing such effect. These experimental conditions can be appropriate for in vitro studies of the mechanisms of action of Pb on spermatozoa.

Diagnosis and treatment of allergic rhinitis in children: results of the PETRA study.

BACKGROUND: Good control of allergic rhinitis (AR) in children is desirable because it is associated with diseases such as asthma. The aim of this analysis of the PETRA study was to characterise its diagnosis and treatment in Spanish children. METHODS: Data were analysed for paediatric patients (age 5-17 years, inclusive) included in the PETRA study, which included consecutive patients with allergic rhinitis attending respiratory specialists throughout Spain. Demographic information, disease characteristics (duration, severity according to the Allergic Rhinitis and its Impact on Asthma [ARIA] classification), diagnostic procedures, treatments and physicians' attitudes to treatment were recorded. RESULTS: Of the original sample of 1043 patients, 260 children were included (mean age, 11.7 years; 56.2% boys; 61.9% allergic to house dust mites (HDM) and 38.1% allergic to grass pollen). By ARIA classification, 180/260 (69.4%) had persistent AR and 176/280 (63%) had moderate disease. Asthma was reported in 89/161 (55%) with HDM allergy and 44/99 (45%) with grass pollen allergy. Symptomatic treatment was prescribed in 98.5%, although disease control had been no better than poor in 57.3%. Allergen specific immunotherapy was administered to 56.9%, and was used more often for HDM AR. When asked why specific immunotherapy was not prescribed, two-thirds of the investigators preferred a wait-and-see approach, prescribing immunotherapy if symptoms worsened or asthma developed. CONCLUSIONS: Paediatric patients treated by specialists for allergic rhinitis have moderate or severe disease. Symptomatic treatment was extensively prescribed but often did not achieve good disease control. Many specialists preferred a wait-and-see approach before prescribing immunotherapy.

Effect of several antioxidants on thawed ram spermatozoa submitted to 37°C up to four hours.

Thawed ram spermatozoa were incubated at 37°C in the presence of dehydroascorbic acid (DHA), TEMPOL (TPL), N-acetyl-cysteine (NAC) and rutin (RUT), at 0.1 and 1 mm, in order to test their effects on sperm physiology. Cryopreserved spermatozoa from four rams were thawed, pooled, washed and incubated in TALP-Hepes with 1 mm or 0.1 mm of each antioxidant, performing a replicate with induced oxidative stress (Fe(2+) /ascorbate). Motility (CASA), viability and mitochondrial membrane potential (flow cytometry) were analysed at 2 and 4 h. Lipoperoxidation (MDA production), intracellular reactive oxygen species (ROS) and DNA status (TUNEL) were analysed at 4 h. Antioxidants, except DHA 0.1 mm, decreased motility and kinematic parameters, but had little effect on viability or mitochondrial activity. Except 1 mm DHA, the antioxidants reduced ROS at 4 h. Moreover, NAC 1 mm, rutin and TEMPOL reduced ROS and DNA damage in the presence of oxidative stress. N-acetyl-cysteine, rutin 1 mm and TEMPOL reduced lipoperoxidation in the presence of oxidative stress. However, DHA did not affect lipoperoxidation. At 1 mm, DHA increased DNA damage in the absence of oxidative stress. Dehydroascorbic acid effects could arise from spermatozoa having a low capacity for reducing it to ascorbic acid, and it may be tested in the presence of other antioxidants or reducing power. Future research should focus in testing whether the inhibition of motility observed for NAC, rutin and TEMPOL is reversible. These antioxidants might be useful at lower temperatures (refrigerated storage or cryopreservation) when their protective effects could be advantageous.

In vitro caries-preventive effect of fluoridated orthodontic resins against cariogenic challenge stimulation.

AIM: The aim of the present study was to evaluate the in vitro caries preventive effect of fluoridated orthodontic resins under pH cycling with two types of acid demineralizing saliva. MATERIALS AND METHODS: Brackets were bonded to 120 extracted human premolars, using Rely-a-bond (n = 40), Tru- Bond (n = 40) and Ortho-one (n = 40) orthodontic bonding agents. Each group of resin was divided into 2 subgroups (n = 20): immersion in remineralizing artificial saliva for 14 days and acid saliva with pH 4.3. After 14 days of pH cycling the caries preventive effect on the development of white spot lesion was evaluated considering the presence of inhibition zones to white spot lesions using two scores: 0 = absence and 1 = presence. Kruskal-Wallis ANOVA and Mann-Whitney U tests were used. RESULTS: Formation of white spot lesions was observed only under pH cycling using acid saliva with pH 4.3; with Rely-a-bond and Tru-Bond being significantly more effective in preventing the appearance of white spot lesions effect than Ortho-one. CONCLUSION: The acidity of the demineralizing solution influenced the formation of white spot lesions around orthodontic brackets under highly cariogenic conditions. Rely-a-bond and Tru-bond presented higher caries-preventive effect than Orthoone. CLINICAL SIGNIFICANCE: The development of fluoride-containing materials cannot be regarded as a permanent means to control dental caries lesions, but a complement along with other preventive methods.

Oral desensitization as a useful treatment in 2-year-old children with cow's milk allergy.

BACKGROUND: Limited published evidence shows oral desensitization to be a potential intervention option for cow's milk protein (CMPs) allergy. OBJECTIVE: The aim of this study was to evaluate the safety and efficacy of oral desensitization in 2-year-old children with cow's milk allergy, as a treatment alternative to elimination diet. METHODS: A total of 60 children aged 24-36 months with IgE-mediated allergy to CMPs were included in this multi-center study and were randomized into two groups. Thirty children (group A: treatment group) began oral desensitization immediately, whereas the remaining 30 (group B: control group) were kept on a milk-free diet and followed-up for 1 year. RESULTS: After 1-year follow-up period, 90% of the children in group A had become completely tolerant vs. 23% of the children in group B. In group A, cow's milk skin reactivity and serum-specific IgE to milk and casein decreased significantly from the initial assessment, whereas group B showed no significant change after 1 year of follow-up. Twenty-four patients (80%) developed some reaction during the treatment period: 14 children developed moderate reaction (47%) and 10 mild reaction (33%). The most common manifestations were urticaria-angioedema, followed by cough. CONCLUSIONS AND CLINICAL RELEVANCE: In this study, oral desensitization was found to be effective in a significant percentage of 2-year-old children with cow's milk allergy. Oral desensitization appears to be efficacious as an alternative to elimination diet in the treatment of 2-year-old children with cow's milk allergy. The side-effect profile appears acceptable but requires further study.