Empeoramiento clínico paradójico en una crisis asmática moderada-grave / Paradoxical clinical worsening in a moderate-severe asthmatic attack

La reagudización o crisis asmática es uno de los motivos de consulta más frecuentes en las consultas de Atención Primaria y en los servicios de urgencias pediátricas. Se trata de una patología con un algoritmo de actuación y de tratamiento según la gravedad bien establecido, con fármacos con un buen perfil de seguridad para la edad pediátrica. Se presenta un caso con mala respuesta inicial que ilustra un efecto paradójico del salbutamol (AU)

Asthma exacerbations are among the most frequent reasons for paediatric primary care and emergency care visits. Asthma is a disease with a well-established management and treatment algorithm based on severity, and drugs with a good safety profile for the paediatric population are available for its treatment. We present a case with a poor inital response illustrating a paradoxical reaction to salbutamol. (AU)

Protamine composition of koala and wombat spermatozoa provides new insights into DNA stability following cryopreservation.

To investigate differences in the post-thaw DNA stability of koala and wombat spermatozoa, protamine amino acid sequences were compared and it was found that there were three more arginine residues for the wombat. Koala and wombat spermatozoa, cryopreserved using identical protocols, were examined for changes in sperm DNA fragmentation (SDF) dynamics over 24h of post-thaw incubation. Following validation of a wombat sperm chromatin dispersion test, wombat DNA showed a rate of SDF that was 6-fold higher than for koala spermatozoa (P=0.038). Finally, we examined whether expected differences in chromatin compactness, associated with protamine sequence, had an effect on restriction site accessibility of sperm DNA. Thawed spermatozoa were exposed to Alu I and EcoR1 endonuclease restriction enzymes and the SDF dynamics were observed. Koala spermatozoa exposed to Alu I showed a greater rate of SDF (P=0.01), whereas wombat spermatozoa exposed to EcoR1 showed a greater rate of SDF (P=0.032). We conclude that restriction sites in these species are differentially present or exposed and potentially account for differences in SDF dynamics. Although differences in the arginine composition of protamine may explain relative differences in SDF following cryopreservation, they do not support the hypothesis that increased arginine composition increases DNA stability; rather, increased arginine composition in the wombat may reduce post-thaw chromatin swelling.

Sperm DNA fragmentation in donors and normozoospermic patients attending for a first spermiogram: Static and dynamic assessment.

Static assessment of sperm DNA Fragmentation (SDF at the time of ejaculation or sperm thawing when cryopreserved) and the dynamic assessment of SDF (SDF assessed after T2 hr, T6 hr and T24 hr of sperm thawing) were used to establish cut-off values associated with sperm donors when compared with closely related normozoospermic patients. Cryopreserved samples from donors revealed SDF levels two times lower in comparison with the patients. Donor sperm DNA exhibited a 2.5 times higher longevity when compared with the patients. Static values of SDF after thawing of approximately 11% identify the donors with a 71% of sensitivity and 84% specificity. With respect to the dynamic assessment, SDF increases of 2.3 per hr during the first 2 hr of incubation identify the donors with 70% of sensitivity and 66% of specificity. Creating the Rate of Combined Damage (RCD) defined as the product of SDF-T0 by the increase in the damage registered during the first 2 hr of incubation (r-SDF-T0-2 ), an index of RCD = 22.2 units has an identification capacity of donors with a 78% sensitivity and 77% specificity. Such cut-off values could be used to characterise donors with high chromatin resistance to damage when meeting the above-established criteria.

Cryopreservation of saltwater crocodile (Crocodylus porosus) spermatozoa.

The aim of the present study was to develop a protocol for the successful cryopreservation of Saltwater crocodile spermatozoa. Sperm cells were frozen above liquid nitrogen vapour in phosphate-buffered saline (PBS) containing either 0.3M trehalose, 0.3M raffinose or 0.3M sucrose and compared with glycerol (0.3-2.7M). Although the highest levels of mean post-thaw motility were observed following cryopreservation in 0.3M trehalose (7.6%) and 0.3M sucrose (7.3%), plasma membrane integrity (PI) was best following cryopreservation in 2.7M glycerol (52.5%). A pilot study then assessed the cytotoxicity of glycerol and sucrose prior to cryopreservation and revealed no loss of survival when spermatozoa were diluted in 0.68M glycerol or 0.2-0.3M sucrose once cryoprotectants were washed out with PBS or Biggers, Whitten and Whittingham medium containing sperm capacitation agents (BWWCAP). A final study refined the combined use of permeating (0.68 or 1.35M glycerol) and non-permeating (0.2 or 0.3M sucrose) cryoprotectants. Spermatozoa were cryopreserved in liquid nitrogen vapour at rates of approximately -21°Cmin-1 (fast freeze) or -6.0°Cmin-1 (slow freeze). Post-thaw survival was highest with a combination of 0.2M sucrose and 0.68M glycerol and when these cryoprotectants were washed out with BWWCAP, regardless of whether spermatozoa were frozen using a fast (motility 14.2±4.7%; PI 20.7±2.0%) or slow (motility 12.0±2.7%; PI 22±4%) cryopreservation rate.

The assessment of sperm DNA fragmentation in the saltwater crocodile (Crocodylus porosus).

Herein we report a method of assessing DNA fragmentation in the saltwater crocodile using the sperm chromatin dispersion test (SCDt) after including frozen-thawed spermatozoa in a microgel (Halomax; Halotech DNA, Madrid, Spain). Following controlled protein depletion, which included a reducing agent, sperm nuclei with fragmented DNA showed a homogeneous and larger halo of chromatin dispersion with a corresponding reduced nucleoid core compared with sperm with non-fragmented DNA. The presence of DNA damage was confirmed directly by incorporation of modified nucleotides using in situ nick translation (ISNT) and indirectly by studying the correlation of the SCDt with the results of DNA damage visualisation using a two-tailed comet assay (r=0.90; P=0.037). Results of the SCDt immediately following thawing and after 5h incubation at 37°C in order to induce a range of DNA damage revealed individual crocodile differences in both the baseline level of DNA damage and DNA longevity.

Effects of freezing and activation on membrane quality and DNA damage in Xenopus tropicalis and Xenopus laevis spermatozoa.

There is growing concern over the effect of sperm cryopreservation on DNA integrity and the subsequent development of offspring generated from this cryopreserved material. In the present study, membrane integrity and DNA stability of Xenopus laevis and Xenopus tropicalis spermatozoa were evaluated in response to cryopreservation with or without activation, a process that happens upon exposure to water to spermatozoa of some aquatic species. A dye exclusion assay revealed that sperm plasma membrane integrity in both species decreased after freezing, more so for X. laevis than X. tropicalis spermatozoa. The sperm chromatin dispersion (SCD) test showed that for both X. tropicalis and X. laevis, activated frozen spermatozoa produced the highest levels of DNA fragmentation compared with all fresh samples and frozen non-activated samples (P<0.05). Understanding the nature of DNA and membrane damage that occurs in cryopreserved spermatozoa from Xenopus species represents the first step in exploiting these powerful model organisms to understand the developmental consequences of fertilising with cryopreservation-damaged spermatozoa.

Administración accidental de aceite puro esencial de arbol de té (Melaleuca alternifolia) en lactantes / Accidental pure essential tea tree oil (Melaleuca alternifolia) administration in two infants

El aceite de árbol de té es una sustancia que se obtiene mediante la destilación de las hojas y ramas frescas del árbol Melaleuca alternifolia. En los últimos años se ha popularizado su uso sobre todo como agente antiinfeccioso tópico en una gran variedad de enfermedades. Se trata de una sustancia natural que tiene un potencial efecto tóxico demostrado sobre todo si se ingiere. Presentamos dos casos de pacientes atendidos en nuestro Servicio de Urgencias tras administración e ingestión accidental de aceite esencial de árbol de té, en ambos casos los padres confundieron el envase con el de la vitamina D. La atención de estos pacientes nos llevó a realizar una búsqueda bibliográfica de casos publicados de intoxicación por ingesta de aceite de árbol de té y a poner de manifiesto la ausencia de advertencias de seguridad en los envases de aceite de árbol de té que actualmente se comercializan en nuestro país (AU)

Tea tree oil is an essential oil obtained by steam distillation of the leaves and terminal branches of Melaleuca alternifolia. In recent years, it has become popular as an antimicrobial agent against a large number of diseases. It is a natural substance that has a potential toxic effect especially if ingested. We report two cases of infants who came to our Emergency Department because they were accidentally given tea tree oil. In both cases parents mistook the tea tree oil bottle with the D vitamin bottle. The care of these patients led us to perform a literature search of published cases of tea tree oil poisoning and highlight the absence of safety warnings on tea tree oil packages currently marketed in our country (AU)

Análisis de atención inicial y manejo del traumatismo craneoencefálico estable en Urgencias de Pediatría. Estudio comparativo / Analysis of initial care and management of stable cranioencephalic traumatism in Pediatric Emergency Department. A comparative study

El traumatismo craneoencefálico (TCE) constituye un motivo de consulta frecuente en Urgencias de Pediatría; la mayoría de tipo leve. Tras su valoración con el triángulo de evaluación pediátrica y clasificación del nivel de urgencia, se debe aplicar un protocolo de actuación que establezca su adecuado manejo y solicitud de prueba de imagen. El objetivo del estudio es analizar las características de dos grupos de pacientes valorados con 4 años de diferencia, establecer su valoración inicial, pruebas de imagen y necesidad de observación hospitalaria. Resultados. Total 169 episodios (prevalencia 2,6%). Año 2010: 92 episodios, 90 pacientes. Año 2014: 77 episodios, 76 pacientes. En ambos años predomina el sexo masculino, el motivo de consulta más prevalente es el TCE aislado, y cuando asocia clínica predominan los vómitos, y en la exploración la existencia de herida externa. El registro de constantes de frecuencia cardiaca y presión arterial se ha incrementado significativamente. Se ha reducido el porcentaje de radiografías de cráneo simple realizadas de forma global en 1,4% y en la franja de edad entre 1 y 2 años en un 7,1%. El porcentaje de tomografía axial computarizada craneal (TAC) fue similar en ambos años. Precisó observación un 13% en 2010 y 9,1% en 2014. Discusión. Es imprescindible conseguir un equilibrio en la valoración urgente del TCE estable que permita reducir la radiación aplicada al paciente en forma de radiología convencional, mantener la adecuación de la indicación de la TAC craneal ajustada y su estancia en forma de observación hospitalaria

Cranioencephalic traumatism (CET) is a cause of frequent medical visits in the Pediatric Emergency Department, most of them being mild. After evaluation with the pediatric assessment triangle and classification on an emergency level, an action protocol should be applied that establishes its adequate management and request for imaging test. The purpose of the study is to analyze the characteristics of two groups of patients evaluated with a different of 4 years, to establish their initial evaluation, imaging tests and need for hospital observation. Results. Total 169 episodes (prevalence 2.6%). Year 2010: 92 episodes, 90 patients. Year 2014: 77 episodes, 76 patients. In both years, there was a predominance of male gender, the most prevalent reason for the visit was isolated CET, and when symptoms were associated, vomiting clinically predominated, while in the physical exam, the external wound predominated. The recording of the vital signs of heart rate and blood pressure has significantly increased. The percentage of simple brain x-rays performed globally has reduced by 1.4% and the age range between 1 and 2 years by 7.1%. The percentage of cranial computed tomography was similar in both years. A total of 13% required observation in 2010 and 9.1% in 2014. Discussion. It is essential to achieve a balance in urgent assessment of stable CET that would make it possible to reduce the radiation applied to the patient in form of conventional radiology, to maintain adaptation of the indication of the cranial CT scan indication and the patients stay in form of hospital observation

Spermatozoa of Sminthopsis murina (Mammalia: Metatheria) exhibit an unusually high degree of chromatin stability in the absence of disulphide bonding in protamine 1.

Although all but a single genus (Planigale) of the metatheria so far examined contain no cysteine residues in protamine 1, we report a remarkable level of chromatin stability in the spermatozoa of the common dunnart, Sminthopsis murina. S. murina cauda epididymal spermatozoa and somatic epithelial cells were exposed to a combination of graded treatments to lyse sperm protein and induce sperm DNA damage via standard freeze-thaw protocols and post-thaw incubation at 37°C for 48h, exposure to sodium nitroprusside (SNP) and the enzyme AluI restriction endonuclease. Sperm DNA fragmentation was assessed using the comet assay and sperm chromatin dispersal test. Although S. murina somatic cells showed DNA fragmentation following protein lysis and after treatment with all the protocols specifically designed to induce chromatin damage, sperm DNA fragmentation was only observed following moderate to severe proteolytic exposure and treatment with the restriction endonuclease; there was also an increase in the baseline halo of spermatozoa treated with an aggressive reducing agent, but no corresponding evidence of fragmented DNA, suggesting that cysteine residues may be functioning to conform tertiary and/or quaternary chromatin structure. Given that the protamine 1 of S. murina contains no cysteine, we suggest that the source of these residues is possibly the histone fraction of the chromatin and that the high level of stability is potentially related to prolonged sperm survival in the female's reproductive tract.

Effect of cryopreservation on the sperm DNA fragmentation dynamics of the bottlenose dolphin (Tursiops truncatus).

Sperm DNA fragmentation is one of the major causes of infertility; the sperm chromatin dispersion test (SCDt) evaluates this parameter and offers the advantage of species-specific validated protocol and ease of use under field conditions. The main purpose of this study was to evaluate sperm DNA fragmentation dynamics in both fresh and post-thaw bottlenose dolphin sperm using the SCDt following different cryopreservation protocols to gain new information about the post-thaw differential sperm DNA longevity in this species. Fresh and cryopreserved semen samples from five bottlenose dolphins were examined for sperm DNA fragmentation dynamics using the SCDt (Halomax(®)). Sperm DNA fragmentation was assessed immediately at collection and following cryopreservation (T0) and then after 0.5, 1, 4, 8, 24, 48 and 72 h incubation at 37°C. Serially collected ejaculates from four dolphins were frozen using different cryopreservation protocols in a TES-TRIS-fructose buffer (TTF), an egg-yolk-free vegetable lipid LP1 buffer (LP1) and human sperm preservation medium (HSPM). Fresh ejaculated spermatozoa initially showed low levels of DNA fragmentation for up to 48 h. Lower Sperm DNA fragmentation (SDF) was found in the second fresh ejaculate compared to the first when more than one sample was collected on the same day (p < 0.05); this difference was not apparent in any other seminal characteristic. While there was no difference observed in SDF between fresh and frozen-thawed sperm using the different cryopreservation protocols immediately after thawing (T0), frozen-thawed spermatozoa incubated at 37°C showed an increase in the rate of SDF after 24 h. Sperm frozen in the LP1(℗) buffer had higher levels (p < 0.05) of DNA fragmentation after 24- and 48-h incubation than those frozen in TTF or HSPM. No correlation was found between any seminal characteristic and DNA fragmentation in either fresh and/or frozen-thawed samples.

Validation of the sperm chromatin dispersion (SCD) test in the amphibian Xenopus laevis using in situ nick translation and comet assay.

The integrity of sperm DNA is becoming increasingly recognised as an important parameter of semen quality, but there are no published reports of this procedure for any amphibian. The primary aim of this study was to apply a modified sperm chromatin dispersion (SCD) test (Halomax) to an amphibian sperm model (African clawed frog; Xenopus laevis) and to validate the assay against in situ nick translation (ISNT) and the double-comet assay procedure. Inactivated spermatozoa were collected from fresh testes (n=3). Sperm DNA fragmentation (SDF) for each sperm sample was conducted immediately following activation (T0) and again after 1h (T1) and 24h (T24) of incubation at room temperature in order to produce a range of spermatozoa with differing levels of DNA damage. The SCD procedure resulted in the production of three nuclear morphotypes; amphibian sperm morphotype 1 (ASM-1) and ASM-2 showed no evidence of DNA damage, whereas ASM-3 spermatozoa were highly fragmented with large halos of dispersed DNA fragments and a reduced nuclear core. ISNT confirmed that ASM-3 nuclei contained damaged DNA. There was a significant correlation (r=0.9613) between the levels of ASM-3 detected by the SCD test and SDF revealed by the double-comet assay.

Localisation and quantification of alkali-labile sites in human spermatozoa by DNA breakage detection-fluorescence in situ hybridisation.

The localisation and quantification of constitutive alkali-labile sites (ALSs) were investigated using a protocol of DNA breakage detection plus fluorescence in situ hybridisation (DBD-FISH) and alkaline single-cell gel electrophoresis (SCGE or comet assay), in spermatozoa of infertile and fertile men. Semen samples from 10 normozoospermic patients undergoing infertility treatment and 10 fertile men were included in this study. ALSs were localised and quantified by DBD-FISH. The region most sensitive to alkali treatment in human spermatozoa was located in the basal region of the head. ALSs were more frequent in spermatozoa of infertile men than in those of fertile men. These results were confirmed by SCGE comet assays. In conclusion, the most intense localisation of hybridisation signals in human spermatozoa, representing the highest density of constitutive ALSs, was not randomly distributed and was predominantly located in the base of the head. Moreover, infertile men presented with an increase in ALS frequency. Further studies are necessary to determine the association between ALS, sperm chromatin organisation and infertility.

Validation of a field based chromatin dispersion assay to assess sperm DNA fragmentation in the bottlenose dolphin (Tursiops truncatus).

Over the last two decades, there have been significant advances in the use of assisted reproductive technology for genetic and reproductive management of captive dolphin populations, including evaluation of sperm DNA quality. This study validated a customized sperm chromatin dispersion test (SCDt) for the bottlenose dolphin (Tursiops truncatus) as a means of assessing sperm DNA damage both in the field and in the laboratory. After performing the SCDt, two different sperm morphotypes were identified: (i) sperm with fragmented DNA showed large haloes of dispersed DNA fragments emerging from a compact sperm nucleoid core and (ii) sperm containing non-fragmented DNA displayed small compact haloes surrounded by a dense core of non-dispersed DNA and protein complex. Estimates of sperm DNA fragmentation by means of SCDt were directly comparable to results obtained following a two-tailed comet assay and showed a significant degree of correlation (r = 0.961; p < 0.001). This investigation also revealed that the SCDt, with minor modifications to the standard protocol, can be successfully conducted in the field using a LED florescence microscopy obtaining a high correlation (r = 0.993; p = 0.01) between the data obtained in the laboratory and in the field.

Characterisation of a subpopulation of sperm with massive nuclear damage, as recognised with the sperm chromatin dispersion test.

Assessment of human sperm DNA fragmentation by the sperm chromatin dispersion (SCD) test is based on the detection of haloes of spreading DNA loops after sequential DNA denaturing and protamine removal. After the SCD test, sperm without DNA fragmentation show chromatin haloes emerging from the central nuclear core, while sperm containing fragmented DNA present small or no haloes. The nuclear degraded sperm are recognised as a differentiated category within the sperm with fragmented DNA, whose cores appear irregularly and/or faintly stained. This subpopulation is more prevalent in patients with varicocele. Protein staining with 2.7-dibrom-4-hydroxy-mercury-fluorescein demonstrated that degraded sperm intensely lose nuclear core proteins after the SCD processing. Moreover, degraded sperm are 65% more faintly labelled for DNA breaks after in situ nick translation (ISNT) on average, due to extensive DNA loss. A two-dimensional comet assay under sequential neutral and alkaline conditions demonstrated that degraded sperm contain both massive double- and single-strand DNA breaks. The degraded sperm appear as a subpopulation with stronger nuclear damage, affecting both DNA and protein fractions, possibly due to intense intratesticular oxidative stress, what could explain its higher proportion in patients with varicocele.

Incipient post-zygotic barrier in a model system of ecological speciation with gene flow.

The role of post-zygotic isolation in nonallopatric ecological speciation is still mostly unknown and information on the nature and strength of these barriers in well-known speciation models is essential for a deeper understanding of such processes. The Galician ecotypes of the marine snail Littorina saxatilis represent one of the best studied cases of nonallopatric ecological speciation. Here, we test the existence of incipient post-zygotic isolation by comparing the fertility of male hybrids with that of both pure forms [ridged and banded (RB) and smooth and unbanded (SU) ecotypes]. We analysed the degree of sperm DNA fragmentation (SDF) of individuals morphologically classified as RB, SU and hybrids, sampled from two locations. SDF analyses were chosen to study sperm quality because, in other animal species, SDF rates correlate with important parameters for speciation research, such as fertilization and abortion rates and viability of adult progeny. In the present work, hybrids showed significantly higher SDF rates than RB and SU males in one location and significantly higher variances in both locations. These results suggest the existence of an incipient post-zygotic barrier, the strength of which may vary across the Galician shore, and highlight the potential of SDF analyses for speciation research.

Comparison of membrane-permeant fluorescent probes for sperm viability assessment in the ram.

This study was designed to compare the effectiveness and usability of four permeant fluorochromes (CFDA; SYBR-14; Hoechst-33342; and acridine orange), combined with propidium iodide to assess sperm membrane integrity. Three different experiments were conducted. The first trial was designed to study the optimal dye concentration and minimum incubation time required to achieve optimum fluorescence intensities and contrast for each fluorochrome combination using ram fresh semen samples. Both SYBR-14 and acridine orange allowed a direct assessment of sperm membrane integrity, without the need of incubating samples, whereas a minimum of 4 and 6 min of incubation at 37°C was necessary to achieve optimum fluorescence intensities in the CFDA and Hoechst groups, respectively. In the second trial, fresh semen samples were mixed with different volumes of membrane-affected sperm (semen treated with three cycles of freezing to -20°C and thawing at room temperature) to produce semen samples with known proportions of damaged spermatozoa. The results were compared with the theoretical values predicted on the basis of the estimations made on fresh and frozen samples. The proportions of damaged sperm in each sample determined using the four fluorochrome combinations agreed with the predicted theoretical values, with the acridine orange/propidium iodide providing the best adjustment. The third experiment was performed to compare the results of sperm membrane integrity using the four fluorochrome combinations. The proportions of plasmalemma-intact sperm determined by acridine orange and SYBR-14 were greater (p < 0.0001) than the proportions of intact sperm determined by CFDA and Hoechst stains. It was concluded that the most efficient combinations to be used in ram sperm were AO/PI and SYBR/PI because it allowed a direct assessment of sperm viability without the need to incubate samples and obtaining reliable results.

Osmotic stress and cryoinjury of koala sperm: an integrative study of the plasma membrane, chromatin stability and mitochondrial function.

This study investigated whether cryopreservation-induced injury to koala spermatozoa could be explained using an experimental model that mimics the structural and physiological effects of osmotic flux. DNA labelling after in situ nick translation of thawed cryopreserved spermatozoa revealed a positive correlation (r=0.573; P<0.001; n=50) between the area of relaxed chromatin in the nucleus and the degree of nucleotide labelling. While the chromatin of some spermatozoa increased more than eight times its normal size, not all sperm nuclei with relaxed chromatin showed evidence of nucleotide incorporation. Preferential staining associated with sperm DNA fragmentation (SDF) was typically located in the peri-acrosomal and peripheral regions of the sperm head and at the base of the spermatozoa where it appear to be 'hot spots' of DNA damage following cryopreservation. Results of the comparative effects of anisotonic media and cryopreservation on the integrity of koala spermatozoa revealed that injury induced by exposure to osmotic flux, essentially imitated the results found following cryopreservation. Plasma membrane integrity, chromatin relaxation and SDF appeared particularly susceptible to extreme hypotonic environments. Mitochondrial membrane potential (MMP), while susceptible to extreme hypo- and hypertonic environments, showed an ability to rebound from hypertonic stress when returned to isotonic conditions. Koala spermatozoa exposed to 64 mOsm/kg media showed an equivalent, or more severe, degree of structural and physiological injury to that of frozen-thawed spermatozoa, supporting the hypothesis that cryoinjury is principally associated with a hypo-osmotic effect. A direct comparison of SDF of thawed cryopreserved spermatozoa and those exposed to a 64 mOsm/kg excursion showed a significant correlation (r=0.878; P<0.05; n=5); however, no correlation was found when the percentage of sperm with relaxed chromatin was compared. While a cryo-induced osmotic injury model appears to explain post-thaw changes in koala SDF, the mechanisms resulting in relaxed chromatin require further study. A lack of correlation between the percentage of sperm with relaxed chromatin and SDF suggests that the timing of these pathologies are asynchronous. We propose an integrative model of cryo-induced osmotic injury that involves a combination of structural damage (rupture of membrane) and oxidative stress that first leads to the reduction of MMP and the relaxation of chromatin, which is then ultimately followed by an increase in DNA fragmentation.

Varicocele, leucocitospermia y su impacto en la fragmentación del ADN espermático / Varicocele, leukocytospermia and its impact on the spermatic DNA fragmentation

Introducción: El estrés oxidativo en una de las causas que puede explicar la presencia de unos niveles elevados de daño en la molécula de ADN. En 2 cuadros clínicos que afectan a la línea germinal del varón, la leucocitospermia y el varicocele, se reconoce la incidencia de niveles elevados de estrés oxidativo. Objetivos: En el presente trabajo se compararon 2 cuadros clínicos, varicocele y leucocitospermia, con objeto de comprobar si existe una intensidad diferencial de la fragmentación del ADN espermático. Material y métodos: Se incluyeron como controles externos donantes de espermatozoides con fertilidad probada y pacientes con factor masculino no determinado. A diferencia de otros estudios de fragmentación del ADN espermático, en este caso, se consideraron tanto los niveles de fragmentación absolutos (SDF), como la proporción de espermatozoides degradados en el total de fragmentados (índice de degradación [ID]) de acuerdo con la información obtenida tras la aplicación del test Halosperm. Resultados: Se demostró un aumento muy significativo de la fragmentación del ADN espermático en las muestras seminales de pacientes con varicocele y con leucocitospermia. Los pacientes con varicocele mostraron un ID 2 veces mayor que el observado en pacientes con factor masculino no determinado o en pacientes con leucocitospermia, y 6 veces mayor que en los donantes. Discusión: La presencia de unos niveles de estrés oxidativo elevados podría ser una explicación asumible para justificar los altos niveles de daño observado en los espermatozoides de los pacientes tanto con varicocele como con leucocitospermia, pero probablemente estos niveles sean más elevados en el caso del varicocele ya que los niveles de degradación del ADN espermático son superiores a los observados en cuadros de leucocitospermia... (AU)

Introduction: High levels of oxidative stress can explain the presence of high levels of damage in the DNA molecule. The impact of high levels of oxidative stress in 2 clinical circumstances affecting the male germ line has been well established: leukocytospermia and varicocele. Objective: To assess sperm DNA fragmentation in patients diagnosed with leukocytospermic and varicocele. Material and methods: Leukocytospermic and varicocele patients and external controls (donors with proven fertility and patients with undetermined male factor). Unlike in other studies of sperm DNA fragmentation, in this study both the proportion of damaged sperm after using the sperm chromatin dispersion test (Halosperm), and the proportion of degraded sperm in total fragmented (degradation index [DI]) were taken into consideration. Results: A highly significant increase in sperm DNA fragmentation has been observed in semen samples of patients with varicocele and leukocytospermia. Varicocele patients showed a DI twice as high as that observed in patients with undetermined male factor or in patients with leukocytospermia, and 6 times as high as that observed in the donors. Discussion: The presence of high levels of oxidative stress could be an acceptable explanation for the high levels of damage observed in the spermatozoa of varicocele patients or with leukocytospermia; the level of sperm degradation is higher in the case of varicocele than those observed in leukocytospermia. Conclusions: SDF levels in patients with leukocytospermia and varicocele are significantly higher than those observed in donors or men with undetermined male factor. The DI in sperm samples from patients with varicocele is the highest of all the samples studied in this analysis. The routine determination of the DI may have a practical value, by guiding the patient towards the potential diagnosis of varicocele, even when this is subclinical (AU)

Is sperm DNA fragmentation a good marker for field AI bull fertility?

This paper aimed at investigating the potential use of sperm DNA fragmentation (SDF) to improve the routine screening of infertility of Holstein bulls. Cryopreserved sperm samples from 201 Holstein bulls provided by an AI center were used in the analyses of SDF at 0 (SDF_0) and 6 (SDF_6) h of incubation at 37°C. A refinement of the sperm chromatin dispersion test implemented in the Sperm-Halomax kit was employed to measure SDF. Records on routinely collected semen traits (volume, concentration, mass and individual motility evaluated in the fresh ejaculate, and individual motility in post-thawed semen straws) were provided by the AI center. Artificial insemination bull fertility was obtained from official field recording as successful or failed insemination. The results show that the average SDF was low (around 3.5%) at 0 and 6 h of incubation. A moderate effect of inbreeding depression was found. Estimated heritability for SDF traits were moderately high (0.41 and 0.29 for SDF_0 and SDF_6, respectively) and estimated repeatability of SDF measures in the same animal were high (0.73 and 0.70 for SDF_0 and SDF_6, respectively). An overall estimated service bull value (ESBV) obtained through statistical modeling that allowed for adjustment of systematic environmental effects not specific to a bull and of the female contribution to fertility, and the estimated genetic values (EGV) were obtained from field-recorded AI information. The ESBV and EGV were also obtained for all semen traits. Moderately large and negative Pearson correlation coefficients were observed between SDF traits and male fertility ranging from (-0.43 to -0.50; P <0.001). Results of stepwise regression analyses showed that SDF_6 had the largest partial r(2) (0.15 to 0.26) among all semen characteristics. Overall, the selected semen traits explained 25% and 31% of the observed variability in bull fertility measured as EGV and ESBV, respectively. When looking at the predictive ability of bull fertility categories, the results of discriminant and logistic regression analyses showed that low-fertility bulls (those in the 10th or lower percentile in the fertility distribution) can be accurately identified by using measures of SDF alone or in combination with sperm motility. Values of SDF around 7% to 10% could be used as indicators of low AI success.