Light Microscopic, Ultrastructure Analysis and Functional Morphology of Cornu aspersum Spermatozoa Contained in the Frozen Hermaphroditic Duct / Análisis de Microscopía de Luz, Ultraestructura y Morfología Funcional de Espermatozoides de Cornu aspersum Contenidos en el Conducto Hermafrodita Congelado

SUMMARY: In this study we describe the functional morphology of Cornu aspersum (Helix aspersa), spermatozoa using light, scanning (SEM) and transmission electron (TEM) microscopies. The studies were performed with sperm located in the frozen hermaphroditic duct. Our results showed that the head presents an elongated conical shape slightly coiled in a corkscrew, with the nucleus partially covered by an acrosome, where an apical vesicle is located at the lateralized apex. This peculiar shape suggests the helical displacement movement of the spermatozoa. The head and the nucleus are slightly larger size compared to those of other gastropod species. The intermediate tract is surrounded by a mitochondrial complex and a glycogen helix. The glycogen helix is coiled helically along the intermediate tract, presenting at least five twists of glycogen helices. The complexity of both the mitochondrial complex and the glycogen helix suggests a high metabolic consumption considering the long period of time until fertilization occurs. Our findings on the detailed characterization of Cornu aspersum spermatozoa, obtained from a frozen hermaphroditic duct can contribute to a better understanding of the functional morphology of sperm and serve as a reference for future studies.

En este estudio describimos la morfología funcional de Cornu aspersum (Helix aspersa), espermatozoides utilizando microscopías de luz, barrido (SEM) y electrónica de transmisión (TEM). Los estudios se realizaron con espermatozoides localizados en el conducto hermafrodita congelado. Nuestros resultados mostraron que la cabeza presenta una forma cónica alargada ligeramente enrollada en un tirabuzón, con el núcleo parcialmente cubierto por un acrosoma, donde se ubica una vesícula apical en el ápice lateralizado. Esta peculiar forma sugiere el movimiento de desplazamiento helicoidal de los espermatozoides. La cabeza y el núcleo son de un tamaño ligeramente mayor en comparación con los de otras especies de gasterópodos. El tracto intermedio está rodeado por un complejo mitocondrial y una hélice de glucógeno. La hélice de glucógeno se enrolla helicoidalmente a lo largo del tracto intermedio, presentando al menos cinco giros de hélices de glucógeno. La complejidad tanto del complejo mitocondrial como de la hélice de glucógeno sugiere un alto consumo metabólico considerando el largo período de tiempo hasta que ocurre la fecundación. Nuestros hallazgos sobre la caracterización detallada de los espermatozoides de Cornu aspersum, obtenidos de un conducto hermafrodita congelado, pueden contribuir a una mejor comprensión de la morfología funcional de los espermatozoides y servir como referencia para futuros estudios.

Ultraestrutura e criopreservação de sêmen de jundiá amazônico (Leiarius marmoratus) em cativeiro / Ultrastructure and sperm cryopreservation of the amazon catfish (Leiarius marmoratus) in captivity

Os objetivos do presente estudo foram analisar a ultraestrutura do espermatozoide do jundiá amazônico e avaliar a sua criopreservação com três agentes crioprotetores (metanol 10%, DMSO 10% e etilenoglicol 10%) e duas soluções ativadoras (NaCl 0,29% e NaHCO3 1%). Como diluente, foi utilizada uma solução de glicose a 5%, sendo o sêmen envasado em palhetas de 0,25mL e congelado em vapor de nitrogênio (botijão dry shipper). No sêmen fresco, o espermatozoide apresentou comprimento de 25,46±2,54µm, cabeça esférica (1,51±0,18µm), ausência de acrossoma, peça intermediária com formato cônico (0,93±0,17µm), ligeiramente assimétrica, com presença de vesículas, e flagelo único (21,48±2,45µm). O sêmen descongelado apresentou valores mais altos (P<0,05) para duração, vigor e taxa de motilidade espermática com os crioprotetores metanol 10% e DMSO 10%. A duração da motilidade espermática foi maior (P<0,05) com o ativador NaHCO3 1% (21-96 s). O sêmen de Leiarius marmoratus criopreservado com DMSO e metanol apresentou, respectivamente, 7,32±4,21% e 8,94±6,69% de taxa de motilidade. No entanto, os resultados não foram satisfatórios para estabelecer um protocolo para a espécie.(AU)

The aims of this study were to describe the spermatozoon ultrastructure and to evaluate the sperm cryopreservation of the amazon catfish with three cryoprotectant agents (10% methanol, 10% DMSO, and 10% ethylene glycol) and two activator agents (0.29% NaCl and 1% NaHCO3). Glucose 5% extender was used as a diluent solution and sperm loaded in 0.25 straws was frozen in nitrogen vapor (dry shipper). Fresh spermatozoon was 25.46±2.54µm long, the head was spherical (1.51±0.18µm) with no acrosome, the midpiece was cone shaped (0.93±0.17µm) with presence of vesicles, slightly asymmetric, and the flagellum was single (21.48±2.45µm). Post-thawed semen presented higher values (P< 0.05) for duration, vigor and sperm motility rate with cryoprotectants 10% methanol and 10% DMSO. The duration of sperm motility was longer (P< 0,05) when triggered in 1% NaHCO3 (96-21 s). Leiarius marmoratus semen cryopreserved with DMSO and methanol, presented respectively 7.32±4.21% and 8.94±6.69% of motility. However, the results were not satisfactory to establish a protocol for the specie.(AU)

Characterization of the Campylobacter fetus subsp. venerealis adhesion to bovine sperm cells / Caracterización de la adhesión de Campylobacter fetus subsp. venerealis a espermatozoides bovinos

Campylobacter fetus is extracellular bacteria of the genital tract of cattle. They cause infertility and abortion, but there is no documented information on the susceptibility of bovine sperm cells to this bacteria. The aim of this present work was to study the effects provoked by Campylobacter fetus subsp. venerealis when in interaction with bovine sperm cells. The bovine spermatozoa were obtained frozen bovine semen pooled from uninfected bulls, and were exposed to living campylobacter over different periods of time. Light microscopy and scanning electron microscopy first revealed a tropism, then a close proximity followed by tight adhesion between these two different cells. A decrease in the spermatozoa motility was observed. Motile bacteria were observed during the next 3 h, this process began with a tight membrane­membrane adhesion. The adhesion between Campylobacter fetus to the sperm cell occurred either by the flagella or by sperm head. Results from this study demonstrated with light microscopy scanning electron microscopy allowed us to characterize some aspects of the interaction of Campylobacter fetus subsp. venerealis and bovine sperm while preserving the cellular and bacterial structure. This ex vivo model might be useful for studies on adhesion and cytopathogenicity of different field strains of Campylobacter fetus.

Campylobacter fetus subsp. venerealis es un patógeno extracelular del tracto genital de bovinos. En las hembras causa subfertilidad y aborto, mientras que los toros son portadores en el esmegma prepucial y se desconoce si provoca daño en los espermatozoides. El objetivo del presente trabajo fue estudiar los efectos de Campylobacter fetus subsp. venerealis sobre espermatozoides bovinos. Los espermatozoides obtenidos a partir de pajuelas de semen pertenecientes a toros no infectados, se coincubaron con una cepa de Campylobacter fetus subsp. venerealis por diferentes períodos de tiempo. Por microscopía óptica y electrónica de barrido se observó el tropismo inicial de la bacteria hacia los espermatozoides y la adhesión bacteriana, de forma colateral se observó su efecto en el espermograma. Post incubación los espermatozoides presentaron menor motilidad progresiva y mayor porcentaje de muertos con respecto al control. Se comprobó la viabilidad de la bacteria a las 3 h. Se registró la adhesión de Campylobacter fetus subsp. venerealis a la membrana celular de distintas porciones del espermatozoide: cabeza, pieza media, cuello y cola. Los resultados de este estudio permitieron caracterizar la interacción entre Campylobacter fetus subsp. venerealis y espermatozoides bovinos por microscopía óptica y electrónica de barrido. La aplicación de este modelo ex vivo permitirá profundizar los conocimientos referentes a los procesos de adhesión y citopatogenicidad de Campylobacter fetus.

Identification of ultrastructural and functional damages in sperm from six-banded armadillos (Euphractus sexcinctus) due to cryopreservation / Identificação de danos ultraestruturais e funcionais em espermatozoides de tatus-peba (Euphractus sexcinctus) oriundos da criopreservação

The aim of the study was to cryopreserve the semen of six-banded armadillos (Euphractus sexcinctus) in Tris-yolk and glycerol diluent, and to determine the damage caused by the freezing-thawing process, using fluorescent markers and ultrastructural analysis. Semen samples (n=11) collected from 4 adult six-banded armadillos by electroejaculation were cryopreserved in Tris diluent plus 20% egg yolk and 3% glycerol, in a fast freezing curve. Classical analysis of samples was performed after dilution, refrigeration and thawing, followed by fluorescence analysis, using a combination of fluorescent probes to assess membrane integrity (propidium iodide - PI and Hoechst - H342), and mitochondrial activity (CMXRos - Mito Tracker Red®). We also used the ultrastructural analysis to verify possible morphological alterations caused by cryoinjuries. When compared with fresh samples, we verified a significant decline in all the armadillos' semen parameters after thawing, in which only 6.1% motile sperm were found. However, the percentage of sperm which remained with viable (13%) and functional (24.7%) membranes after thawing suggests that some cells could be live but immotile. Analysis using fluorescent markers revealed that the mitochondria of armadillos' sperm is highly sensible to the freezing protocol and the findings through ultrastructure analysis proved this statement. Additionally, the images obtained by transmission electron microscopy revealed that frozen-thawed sperm presented damaged plasma membrane, nuclear modifications as changes in chromatin and acrossomal changes relative to sperm capacitation. In conclusion, this study is the first attempt to cryopreserve the semen of an armadillo species, and to help us to identify critical points on the freezing-thawing procedure in order to improve the protocol.(AU)

O objetivo deste estudo foi criopreservar o sêmen de tatus-peba (Euphractus sexcinctus) em diluente Tris-gema e glicerol, e determinar os danos causados pelo processo de congelação-descongelação, utilizando marcadores fluorescentes e análise ultraestrutural. As amostras de sêmen (n=11) coletadas de 4 tatus-peba adultos por eletroejaculação foram criopreservadas em diluente Tris acrescido de 20% de gema de ovo e 3% de glicerol, em curva rápida de congelação. A análise clássica das amostras foi realizada após a diluição, refrigeração e descongelação, seguida por análise de fluorescência, utilizando uma combinação de sondas fluorescentes para avaliar a integridade da membrana (Iodeto de Propídio - PI e Hoechst - H342), e a atividade mitocondrial (CMXRos - Mito Tracker RED®). Foi também utilizada a análise ultraestrutural para verificar possíveis alterações morfológicas causadas pela crioinjúria. Quando comparadas com as amostras a fresco, verificou-se uma queda significativa em todos os parâmetros seminais dos tatus após a descongelação, em que apenas 6,1% de espermatozoides móveis foram encontrados. No entanto, o percentual de espermatozoides que permaneceu com membrana viável (13%) e funcional (24,7%) após a descongelação sugere que algumas células podem estar vivas, mas imóveis. Análises utilizando marcadores fluorescentes revelaram que as mitocôndrias dos espermatozoides de tatus são altamente sensíveis ao protocolo de congelação e os achados através da análise ultraestrutural comprovaram esta afirmação. Além disso, as imagens obtidas por microscopia eletrônica de transmissão revelaram que espermatozoides congelados-descongelados apresentaram membranas plasmáticas danificadas, modificações nucleares como alterações na cromatina, e alterações acrossomais relativas à capacitação espermática. Em conclusão, este estudo é a primeira tentativa de criopreservação de sêmen em uma espécie de tatu, e nos auxiliou a identificar pontos críticos no processo de congelação-descongelação, a fim de melhorar o protocolo.(AU)

Efecto de la vitrificación de ovocitos humanos sobre la capacidad de unión y el estado acrosomal de espermatozoides humanos / Effect of the vitrification of human oocytes on the binding capacity and acrosome status of human sperm

Conocer los aspectos moleculares que acontecen en el proceso de unión de los espermatozoides humanos a la zona pelúcida (ZP) humana es uno de los grandes retos de la biología de la Reproducción. Por otra parte conocer si el proceso de fecundación puede verse afectado por la criopreservación de los gametos femeninos sigue siendo otra cuestión debatida en la literatura. En base a esto, el objetivo principal de este trabajo fue conocer si la vitrificación ovocitaria puede alterar la interacción de los espermatozoides con el glicocáliz de la ZP y demostrar si la ZP de estos ovocitos pierde la capacidad de inducir la reacción acrosómica en los espermatozoides. Según nuestros resultados el método de vitrificación ovocitaria cerrado (S3) no altera la capacidad de unión de los espermatozoides a la zona pelúcida, ni la capacidad de ésta para inducir la reacción acrosómica.

To know the molecular aspects that occur in the process of human sperm binding to the human zona pellucida (ZP) is one of the great challenges of reproduction biology. Moreover knowing if the fertilization process may be affected by cryopreservation of female gametes is still another issue discussed in the literature. Based on this, the main objective of this study was to determine whether the oocyte vitrification may alter the interaction of sperm with the glycocalyx of ZP and show whether these oocytes lost the ability to induce the acrosome reaction in sperm. According to our results the oocyte closed vitrification method (S3) does not alter the ability of the sperm binding to the zona pellucida, and their ability to induce the acrosome reaction.

Impaired protamination and sperm DNA damage in a Nellore bull with high percentages of morphological sperm defects in comparison to normospermic bulls / Deficiências de protaminação e danos de DNA no sêmen de um touro Nelore com elevada porcentagem de patologias espermáticas em comparação a touros normospérmicos

The routine semen evaluation assessing sperm concentration, motility and morphology, does not identify subtle defects in sperm chromatin architecture. Bulls appear to have stable chromatin, with low levels of DNA fragmentation. However, the nature of fragmentation and its impact on fertility remain unclear and there are no detailed reports characterizing the DNA organization and damage in this species. The intensive genetic selection, the use of artificial insemination and in vitro embryo production associated to the cryopreservation process can contribute to the chromatin damage and highlights the importance of sperm DNA integrity for the success of these technologies. Frozen-thawed semen samples from three ejaculates from a Nellore bull showed high levels of morphological sperm abnormalities (55.8±5.1%), and were selected for complementary tests. Damage of acrosomal (76.9±8.9%) and plasma membranes (75.7±9.3%) as well as sperm DNA strand breaks (13.8±9.5%) and protamination deficiency (3.7±0.6%) were significantly higher compared to the values measured in the semen of five Nellore bulls with normospermia (24.3±3.3%; 24.5±6.1%; 0.6±0.5%; 0.4±0.6% for acrosome, plasma membrane, DNA breaks and protamine deficiency, respectively) (P<0.05). Motility and percentage of spermatozoa with low mitochondrial potential showed no differences between groups. This study shows how routine semen analyses (in this case morphology) may point to the length and complexity of sperm cell damage emphasizing the importance of sperm function testing.(AU)

O exame de rotina de sêmen, o qual avalia a concentração de espermatozoides, a motilidade e a morfologia, pode não identificar defeitos sutis na arquitetura da cromatina de espermatozoides. Os touros parecem ter cromatina estável com baixos níveis de fragmentação do DNA. No entanto, a natureza da fragmentação e o seu impacto sobre a fertilidade ainda não estão claros e não há relatos que caracterizam a organização do DNA e os danos nessa espécie com mais detalhes. A seleção genética intensiva e o uso da inseminação artificial e da produção in vitro de embriões, além do processo de criopreservação, podem contribuir para o dano da cromatina, e sabe-se a importância da integridade do DNA espermático para o sucesso dessas tecnologias. Amostras de sêmen de três ejaculados de um touro Nelore com altos níveis de alterações morfológicas (55,8±5,1%) foram selecionadas para realização de exames complementares. Os danos de acrossoma (76,9±8,9%) e das membranas plasmáticas (75,7±9,3%), bem como quebras de fita de DNA de espermatozoides (13,8±9,5) e deficiência de protamina (3,7±0,6) foram significativamente maiores em comparação aos valores avaliados no sêmen de cinco touros Nelore com normospermia (24,3±3,3%; 24,5±6,1%; 0,6±0,5%; 0,4±0,6% para acrossoma, membrana plasmática, quebras de DNA e deficiência de protamina, respectivamente) (p<0,05). Motilidade e porcentagem de espermatozoides com baixo potencial mitocondrial não diferiram estatisticamente. Essas avaliações mostram que análises de sêmen de rotina (neste caso, morfologia) podem apontar para a extensão e a complexidade dos danos na célula espermática, o que indica que a deficiência de protamina e os danos no DNA podem ocorrer simultaneamente a defeitos morfológicos. Tal ocorrência enfatiza a importância das análises de sêmen clássicas e dos testes complementares.(AU)

Revisión sistemática de los factores asociados a la presentación de gotas citoplásmicas en porcinos / Systematic Review of Factors Associated with the Presence of Cytoplasmic Droplets in Boars

Estimar la percepción de los pacientes sobre la atención médica en el primer nivel de atención. Metodología Se aplicó una encuesta telefónica a pacientes atendidos en dos meses diferentes del 2012, indagando por variables sociodemográficas, relaciones médico-paciente y acerca del proceso de atención médica. Resultados Se encuestaron 804 pacientes. El tiempo promedio de acceso a la atención fue de 9,6 días. El 78% refiere haber podido contar todo lo que sentía al médico, el 60% que el médico le explicó lo que tenía y, uno de cuatro,que indagó por su familia. El 30% sintió alivio completo luego de la atención médica. La calificación promedio de la atención médica fue de 7,9 (DE ±1,7). Las variables relacionadas con las calificaciones más altas fueron: Poder contarle todo al médico (OR 7,5IC 95% 1,8-31), ser examinado (OR7,5 IC 95% 1,5-38,5, explicarle quétiene (OR 5,2IC 95% 1,8-15), preguntar por la familia (OR 5,8 IC 95% 2,1-16,1)y haberlo atendido antes (OR 3,5 IC 95% 1,4-8,6).Conclusiones La comunicación extensa con el paciente es tan importante como el enfrentar la enfermedad en el acto médico...

To assess outpatients' perceptions of medical care. Methodology A telephone survey was administered to patients treated in two different months in 2012 with a focus on socio-demographic variables, access to care, physician-patient relationships, and on the process of medical care. Results 804 patients were surveyed. The average time of access to care was 9.6 days. 78 % reported having been able to tell the doctor everything that they felt, 60 % reported that the doctor explained what they had, and one in four patients said that the doctor asked about their families. 30 % felt complete relief after medical care. The average rating of care was 7.9 (SD ±1.7). Variables related to the highest ratings were: having been able to tell that doctor everything that they felt (OR 7.5 CI 95 % 1.8-31), having been examined (OR 7.5 CI95 % 1.5-38.5), that the doctor explained what they had (OR 5.2 CI 95 % 1.8-15), that the doctor asked about the family (OR 5.8 CI95 % 2.1-16.1), and if doctor had treated them formerly (OR 3.5 CI95 % 1.4-8.6). Conclusions Extensive communication with the patient is as important as dealing with the disease in the medical act...

Histology and ultrastructure of male reproductive system of the Indian Spiny lobster Panulirus homarus (Decapoda: Palinuridae) / Histología y ultraestructura del sistema reproductor masculino de la langosta espinosa de la India Panulirus homarus (Decapoda: Palinuridae)

The spiny lobster Panulirus homarus, distributed along the Southeast and Southwest coasts of India, is an important commercial species having mariculture potential. Despite its importance, the structural and ultrastructure features of male gonads from this species have received scarce attention. Hence this study was aimed to describe the male reproductive tract of the species, using standard histological and electron microscopy techniques. Gonads from 94 specimens of P. homarus ranging in carapace length 37mm-92mm from vizhinjam (Southwest coast of India.) were obtained and processed for the study (Histology-70 numbers & ultrastructure-24 numbers). The male reproductive system consists of paired testis and vas deferens located in the cephalothoracic region. Macroscopically, the reproductive tract was observed in lobsters >35mm carapace length. In immature testis, spermatogonia were seen which measured 6.9-13.8µm in diameter and in the mature testis primary (5.4-5.9µm) and secondary spermatocytes (2.8-3µm) and spermatids (2.2-2.4µm) were present. Each vas deferens consists of proximal and distal portions. The spermatophoric mass begins formation in the proximal vas deferens. In the distal vas deferens the spermatophoric mass containing the spermatozoa are arranged in packets towards the periphery by the gelatinous matrix produced by the typhlosole. Ultrastructurally, the spermatogonia have lamina, nucleus and mitochondria like bodies, the primary spermatocytes have nucleus, dense chromatin and vacuolated cytoplasm and the spermatids have mitochondria, endoplasmic reticulum and centrioles. The endoplasmic reticulum and the nuclear envelope in the spermatids form the acrosome. The radial arms with microtubules are formed in association with the dense endoplasmic reticulum, near the nucleus. The sperm has a spherical structure with the nucleus, lamellar region, spikes and acrosome. This is the first comprehensive report of the structure of the male gametes and spermatogenesis in P. homarus from Indian waters.Rev. Biol. Trop. 62 (2): 533-541. Epub 2014 June 01.

La langosta espinosa Panulirus homarus, distribuida a lo largo de las costas sudeste y sudoeste de la India, es una especie de importancia comercial con gran potencial para la mari-cultura. A pesar de su importancia, las características estructurales y ultraestructurales de las gónadas masculinas de esta especie han sido poco estudiadas. Debido a esto, el objetivo de este estudio fue describir el aparato reproductor masculino de dicha especie, utilizando técnicas convencionales de microscopía histológica y electrónica. Se procesaron 94 ejemplares de P. homarus de vizhinjam (costa suroeste de la India) (70 individuos para histología y 24 para ultraestructura), cuyos caparazones variaron de 37 mm a 92 mm de longitud. El sistema reproductor masculino de esta especie consistió en un par de testículos y un conducto deferente situados en la región céfalo-torácica. Macroscópicamente, el aparato reproductor se observó en langostas con una longitud de caparazón >35mm. En testículos inmaduros, la espermatogonia midió 6.9-13.8μm de diámetro y se encontró presente en los testículos maduros primarios (5.4-5.9μm), espermatocitos secundarios (2.8 a 3 μm) y espermátidas (2.2-2.4μm). Cada conducto deferente consistió de porciones proximales y distales. La formación de la masa espermatofórica comienza en los conductos deferentes proximales. En el conducto deferente distal espermatofórico, la masa que contiene los espermatozoides está dispuesta en paquetes hacia la periferia, en una matriz gelatinosa producida por el tiflosol. Ultraestructuralmente, las espermatogonias presentan una lámina, núcleo y mitocondrias, los espermatocitos primarios tienen núcleo, cromatina densa y citoplasma vacuolado, mientras que las espermátidas tienen mitocondrias, retículo endoplasmático y centríolos. En las espermátidas, el retículo endoplásmico y la envoltura nuclear forman el acrosoma. Los brazos radiales con microtúbulos se forman en asociación con el retículo endoplásmico denso, cerca del núcleo. El esperma presenta una estructura esférica con el núcleo, la región laminar, las espinas y el acrosoma. Este documento constituye el primer informe exhaustivo de la estructura de los gametos masculinos y espermatogénesis en P. homarus de la India.

Ultrastructure of Spermatogenesis in the Testis of Paragonimus heterotremus

Lung fluke, Paragonimus heterotremus, is a flatworm causing pulmonary paragonimiasis in cats, dogs, and humans in Southeast Asia. We examined the ultrastructure of the testis of adult P. heterotremus with special attention to spermatogenesis and spermiogenesis using scanning and transmission electron microscopy. The full sequence of spermatogenesis and spermiogenesis, from the capsular basal lamina to the luminal surface, was demonstrated. The sequence comprises spermatogonia, spermatocytes with obvious nuclear synaptonemal complexes, spermatids, and eventual spermatozoa. Moreover, full steps of spermatid differentiation were shown which consisted of 1) early stage, 2) differentiation stage representing the flagella, intercentriolar body, basal body, striated rootlets, and electron dense nucleus of thread-like lamellar configuration, and 3) growing spermatid flagella. Detailed ultrastructure of 2 different types of spermatozoa was also shown in this study.

Assessment of Spermatozoa Morphology under Light Microscopy with Different Histologic Stains and Comparison of Morphometric Measurements / Evaluación de la Morfología de los Espermatozoides bajo Microscopía Óptica con Diferentes Tinciones Histológicas y Comparación de las Mediciones Morfométricas

The aim was to examine the morphology of spermatozoa with different staining methods and aimed to find the better staining methods for morphology of spermatozoa in our study. Randomized 67 patients taken for the study who were admitted to Assisted Reproductive Techniques Unit. In the first part of the study, smears were stained with Hematoxylin Eosin (HE), Toluidin Blue (TB), Giemsa, Wright, ferrous Weigert haematoxylin stain, Orange G, eosin-aniline blue dye, Shorr Method, Papanicolau, Berg Method, Light Green stain, Acridine Orange (AO) and Janus Green dyes. In the second part of the study, smear preparations of 10 patients with normozoospermic were stained with HE, Toluidin Blue (TB), Shorr Method and Papanicolau. Four measurements were made including the middle piece, head length- head width and tail length for 200 spermatozoa with normal morphology. Comparisons were made between the stains that which showed a better morphology. Condensation assessment was not possible in smears stained with Shorr, Berg, Method, AO. Better assessment of condensation could be made in other stains. In the second part the smallestvalues belonged to of TB stain according to measurements of head of the spermatozoa. There was a significant difference at the head length with TB stain. Although measurements of Shorr and Papanicolau areclose to each otherand the largestvalues belonged to Papanicolau dye. It was concluded that measurement values in human sperm morphology could alter with the used staining method.

El objetivo fue examinar la morfología de los espermatozoides con diferentes métodos de tinción y encontrar los mejores métodos para su estudio. Fueron seleccionados para el estudio, de manera aleatoria 67 pacientes, quienes ingresaron a la Unidad de técnicas de reproducción asistida. En la primera parte del estudio, se realizaron y tiñeron frotis con hematoxilina eosina (HE), Azul de Toluidina (AT), Giemsa, tinción de Wright, Hematoxilina Férrica de Weigert, Anaranjado G, tinción eosina-anilina, método de Shorr, Papanicolau, método Berg, tinción verde brillante, anaranjado de acridina (AO) y tinción verde Janus. En la segunda parte del estudio, se realizaron frotis de 10 pacientes con normozoospérmicos y se tiñeron con HE, AT, Método Shorr y Papanicolau. Se realizaron cuatro mediciones: ancho de la cabeza, longitud de la cabeza, parte media y cola, sobre 200 espermatozoides con morfología normal. Se compararon las tinciones que mostraban mejor la morfología. La evaluación de la compactación no fue posible en los frotis teñidos con los métodos de Shorr, Berg y AO. Una mejor evaluación de la copactación podría hacerse en otras tinciones. En la segunda parte los valores menores correspondieron a la tinción de AT en relación a la medición de la cabeza de los espermatozoides. Hubo una diferencia significativa en la longitud de la cabeza con tinción de AT. Las mediciones en los frotis con técnicas de Shorr y Papanicolau fueron similares, con valores más altos bajo tinción de Papanicolau. Se concluyó que los valores de la medición morfológica en espermatozoides humanos podrían ser alterados según el método de tinción utilizado.

DAZL 260A>G and MTHFR 677C>T variants in sperm DNA of infertile Indian men.

DAZL (deleted in azoospermia-like) 260A>G and MTHFR (methylene tetrahydrofolate reductase) 677C>T are two important autosomal variants associated with impaired spermatogenesis. In this study, we investigated DAZL 260A>G and MTHFR 677C>T variants in sperm DNA and their frequency in oligozoospermic infertile men of Indian origin. The study on sperm DNA was performed, since it is more prone to oxidative stress-induced damage and mutation. One hundred oligozoopsermic infertile men having normal chromosomal complement with intact Y chromosome and 100 age- and ethnically-matched fertile controls were investigated for these variants in their sperm genome. Spermatozoa were separated by gradient centrifugation and DNA was isolated and analyzed for the single nucleotide polymorphisms (SNPs) by polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP). The results showed no significant differences in the frequency of DAZL AG (P = 0.58) and MTHFR CT (P = 0.44) between oligozoospermic infertile men and controls. However, 8% (8/100) oligozoospermic infertile men harbored both the variants and showed significantly (P<0.0001) lower sperm count (3.28 ± 1.1 vs 12.50 ± 4.09) compared to infertile men with either of the single variant. None of the fertile controls showed the presence of the both variants. In conclusion, the combined effect of both DAZL 260A>G and MTHFR 677C>T variants may have role in compromised sperm count. However, further studies are required to find the pathological role of these combined variants in male infertility.

Congelación ultra rápida de espermatozoides humanos: efecto sobre la función espermática y producción de especies reactiva de oxígeno / Ultra rapid freezing in human spermatozoa: effect on sperm function and reactive oxygen species production

El incremento del número de pacientes que desean mantener su fertilidad, ya sea por motivos oncológicos o de fertilidad, como son los pacientes con enfermedades infecciosas virales trasmitidas por vía sexual, o que se someten en forma voluntaria a la esterilización quirúrgica, requieren de métodos de congelación que preserven en forma adecuada la función de los espermatozoides. En el área de la criobiología, la utilización de técnicas de congelación ultrarrápida ha permitido preservar en forma exitosa ovocitos, embriones y tejido ovárico. Este método se ha incorporado recientemente para preservar el gameto masculino. El presente estudio evalúa el efecto de la congelación ultrarrápida (vitrificación) sobre la función espermática de 10 donantes normozoospérmicos. Los espermatozoides se seleccionaron por Swim-up y la solución espermática se dividió en dos subfracciones. Una fracción se vitrificó sumergiéndola directamente en nitrógeno líquido mientras que la segunda se utilizó como control. En ambas fracciones se determinaron viabilidad, movilidad, potencial de membrana mitocondrial (YMMit), integridad del ADN, reacción de acrosoma espontánea e inducida, y superóxido intracelular (O2.-). Se observó que la vitrificación preserva una adecuada función celular en un alto número de espermatozoides, siendo además un método simple, rápido y de menor costo, ya que no necesita equipo de congelación. No obstante, existe una significativa activación de la producción de especies reactivas de oxígeno, que conlleva a una prematura capacitación espermática, evento que es necesario de modular, especialmente si se utilizan estas células en técnicas de inseminación intrauterina. Futuros estudios con adición de antioxidantes a los medios de congelación parecen necesarios para optimizar esta técnica.

The number of patients who wish to maintain their fertility is ever increasing. This group of patients includes cancer patients, those with fertility problems or viral infectious diseases acquired through sexual contact and others submitting to voluntary surgical sterilization; all of the above requiring freezing methods to adequately preserve sperm function. In the field of cryobiology the use of ultra-rapid freezing techniques has successfully preserved oocytes, embryos and ovarian tissue. This method has recently been incorporated in preserving male gametes. This study evaluates the effect of ultra-rapid freezing (vitrification) on sperm function of 10 normozoospermic donors. The sperm were selected by swim-up technique and the solution divided into two fractions. One fraction is vitrified by dipping directly into liquid nitrogen and the second fraction is used as control. In both fractions, viability, motility, mitochondrial membrane potential (YMMit) DNA integrity, spontaneous and induced acrosome reaction and intracellular superoxide (O2.-) were determined. It was noted that vitrification preserves cell function in a great number of spermatozoon, and is also simple, rapid and cost effective as this method does not require freezing equipment. There is however, significant activation of the production of reactive oxygen species, which leads to premature sperm capacitation, an event necessary to modulate particularly when using these cells in intrauterine insemination techniques. Future studies with addition of antioxidants to freezing media are necessary to further improve this technique.

Aumento del daño en el ADN espermático en varones mayores de 40 años / Association of age with sperm DNA fragmentation

Background: There is an association between aging ana an increased number ofsperms with alterations in nuclear DNA. Aim: To study the association between age and fragmentation of sperm DNA. Material andMethods: Sixty two volunteers provided semen for analysis. These were separated in a group aged less than forty years and a second group aged more than forty years. Sperm DNA fragmentation was studied by TÚNEL (terminal deoxynucleotidyl transferase-mediated2'-deoxyuridine 5'-triphosphate nick end-labeling) and SCD (sperm chromatin dispersión test) assays. Results: Compared with theiryounger counterparts, patients aged more than 40years had a higher proportion ofsperms with DNA fragmentation by TÚNEL (20 ±1.3 and24 ± 1.9 percent respectively, p < 0.05) and SCD (22 ± 1.4 and26 ± 1.6 percent respectively, p < 0.05). The results ofboth assays had a correlation coefficientofO.8. No differences between groups were observed for other seminal parameters. Conclusions: Sperm DNA fragmentation increases with age in males.

Characteristics of the Male Reproductive System and Spermatozoa of Leptophlebiidae (Ephemeroptera)

This study describes morphological changes in the male reproductive system of Miroculis amazonicus (Savage & Peters) from mature nymphs to subimago stages. The sperm ultrastructure of Massartela brieni (Lestage), Farrodes carioca (Domínguez et al) and Miroculis mourei (Savage & Peters), as well as aspects of cell fragments observed in these species' subimagos deferent ducts were described. Sperm from the three species studied are aflagellated and immotile, while those from F. carioca and Ma. brieni are approximately spherical with a homogenous nucleus and acrosome. Sperm of F. carioca present two or three mitochondria located between the nucleus and the acrosome. In Ma. brieni, only one lateral mitochondria was found. Sperm from Mi. mourei are shaped as a number 'eight', with electron lucent spots inside the nucleus and two mitochondria above the acrosome. Large cell fragments containing degenerative vesicles and some sperm were observed in the deferent duct lumen of the three species. Testes of Mi. amazonicus are extremely reduced in the subimago stage, which suggests that these cell fragments originated from testes degeneration.

Comparison of spermatozoa quality in male smokers and nonsmokers of Iranian infertile couples

Smoking has a negative effect on fertility and sperm quality. This study was designed to evaluate the effects of smoking on sperm quality and the related parameters such as sperm concentration, morphology and motility. This cross-sectional study was conducted on 180 infertile men with at least one year history of idiopathic infertility, who admitted to the Avicenna Infertility Center, Tehran, Iran. A complete history including smoking habits and other diseases was obtained and semen analysis was performed for all participants. Statistical analysis was performed using SPSS software version 16 and t test and Mann-whitney tests with a significance level of alpha = 0.05. Comparison of sperm parameters in the two groups of smoker and nonsmoker subjects showed that active smoking [p=0.04] and cigarette consumption even in small amounts [p=0.03] decreased sperm concentration, However, no significant correlation was detected between smoking status and morphology or motility of sperms. This study failed to find a significant correlation between sperm analysis and smoking status except for sperm concentration, which was significantly decreased in the active smokers ,even in those consuming small amounts of tobacco. This finding propounds that tobacco consumption may negatively affect fertility

influence of sperm morphology, total motile sperm count of semen and the number of motile sperm inseminated in sperm samples on the success of intrauterine insemination

The present study aimed to analyze the prognostic value of sperm morphology, total motile sperm count [TMSC] and the number of motile sperm inseminated [NMSI] on the outcome of intrauterine insemination [IUI]. This cross sectional study was carried out 445 women undergoing 820 IUI cycles. All of the patients underwent controlled ovarian hyper stimulation with clomiphen citrate and human menopausal gonadotropin [HMG] followed by intrauterine insemination with the husband's sperm. Pregnancy rate [PR] per cycle in correlation to sperm morphology, TMSC and NMSI was obtained. Statistical analysis of the data was done by the SPSS version 13 [Chicago, USA]. A total of 81 clinical pregnancies were obtained for a pregnancy rate per cycle of 9.9%. When the TMSC was 5x10[6] to <10x10[6], the PR per cycle was significantly higher than the subgroups <1x10[6], 1x10[6] to <5x10[6] and >/= 10x10[6] [15%, 5.6%, 5.1%, 10.8%, respectively]. Sperm morphology was in itself a significant factor that affected the likelihood of IUI success. Nonetheless, the most significant difference of the PR per cycle with sperm morphology was in the subgroup <5% [2.1% vs. 97.9%].When the NMSI was >/= 10x10[6], the PR per cycle was significantly higher than the subgroups<5x10[6] and 5x10[6] to< 10x10[6] [11.2%, 4.1%, 5.2%, respectively]. The study showed that TMSC 5x10[6] to < 10x10[6] and normal sperm morphology >/= 5% and NMSI >/= 10x10[6] are useful prognostic factors of IUI cycles

Morfología testicular del yaque Leiarius marmoratus (Pisces: Siluridae) en estadio de madurez reproductiva / Testicular morphology of yaque Leiarius marmoratus(Pisces: Siluridae) in maturity reproductive stage

El conocimiento de la morfología y fisiología del testículo, permite generar bases para el entendimiento del comportamiento reproductivo de las especies, ya que diferencias anatómicas están relacionadas con el desempeño reproductivo. Con el fin de contribuir al conocimiento biológico y reproductivo del yaque Leiarius marmoratus, fueron analizadas las características anatómicas, morfológicas y funcionales del testículo en animales durante el estado de madurez reproductiva. En L. marmoratus los testículos están ubicados ventralmente a la vejiga gaseosa y presentan numerosas y largas digitaciones que terminan en un conducto espermático. Microscópicamente se encontró que el testículo es espermatogonial irrestricto de tipo tubular, donde la porción anterior presenta actividad espermatogénica, que disminuye en la porción media y desaparece en la región distal, dando paso a tejido glandular con actividad secretora, que actúa como vesícula seminal. Al interior de cada túbulo testicular, se observan cistos que contienen células espermáticas en el mismo estadio de desarrollo. Basados en las características microscópicas, fueron identificados espermatocitos, espermátides y gran cantidad de espermatozoides libres en el lumen. También fueron identificadas células glandulares, tejido muscular liso y tejido epitelial. En el ducto espermático (región media y distal), se encontró abundante secreción acidofílica que en algunas regiones estaba acompañada de espermatozoides libres.

Knowledge of the morphology and physiology of the testis, leads to generate bases for understanding the reproductive behavior of species, because the anatomical differences are related to reproductive performance. To contribute to biologic and reproductive knowledge of Yaque Leiarius marmoratus, anatomic, morphologic and functional characteristics of the testis in animals in a state of reproductive maturity were analyzed. In L. marmoratus, the testicles are located ventrally to the gas bladder and have many long finger-like structures that finish in spermatic duct. Microscopically it was found that the testis is espermatogonial unrestricted and tubular type, where the anterior portion presented spermatogenic activity, which diminishes in the medium portion and disappears in the caudal portion, leading to tissue with glandular secretory activity serving as a seminal vesicle. Inside each tubule of the testis, there are cysts containing sperm cells within the same stage of development. Based on microscopic features, were identified spermatocytes, spermatids and a large amount of free sperm into the lumen. Glandular cells were also identified, as well as smooth muscle tissue and epithelial tissue. In the spermatic duct (medium and distal portion), acidophilous secretion was abundant and in some regions were accompanied by free spermatozoa.

Sperm morphology and natural biomolecules from marine snail Telescopium telescopium: a phylogenetic perspective / Morfología de los espematozoides y biomoléculas naturales del caracol marino Telescopium telescopium: una perspectiva filogenética

Biochemical analysis of the cytosol fraction isolated from the ovotestis/spermatheca glands of marine mollusc Telescopium telescopium and it's sperm microtubular structure revealed that relatively similar biomolecules like different enzymes, hormones, minerals and structures of the sperm are also exist in humans. Moreover, antiserum of the cytosol fraction was found to cross-react with the human sperm antigen indicated presence of a common sperm surface antigenicity between these two diversified species. These findings might support and / or hypothesize about the origin and diversification of the vertebrate molecules from its ancestral form (s) from the invertebrates, and basic physiological functions of these ancestral biomolecules including some of the cellular structures plausibly remain the same regardless their structural changes even after evolution.

El análisis bioquímico de la fracción aislada del citosol desde las glándulas ovotestes/espermateca del molusco marino Telescopium telescopium y su estructura tubular espermática revelaron biomoléculas relativamente similares como tales como diferentes enzimas, hormonas, minerales y estructuras de los espermatozoides que también existen en los seres humanos. Por otra parte, en el antisuero de la fracción citosólica se encontró una reacción cruzada con los antígenos del esperma humano indicando la presencia de una superficie espermática de antigenicidad común entre estas dos diversificadas especies. Estos hallazgos pueden apoyar y/o hipotetizar sobre el origen y la diversificación de las moléculas de los vertebrados desde su forma (s) ancestral desde los invertebrados, y funciones básicas fisiológicas de estas biomoléculas ancestrales incluyendo algunas de las estructuras celulares siendo plausiblemente las mismas, independientemente de sus cambios estructurales incluso después de la evolución.

Morphological study of the cytoplasmic droplets as an index of sperm maturation in Black Bengal buck (Capra hircus) / Estudio morfológico del droplet citoplasmático como un índice de maduración de espermatozoides en cabras Black Bengal (Capra hircus)

Location of the cytoplasmic droplets (CD) and their dimensions varied significantly (p<0.01) when sperm cells traverse through the regions of caput, corpus and cauda epididymis and vasdeferens respectively. The gradual diminution in the morphology of CD between the epididymal regions were related significantly (p<0.01, p<0.05). Caudal shift of the CD, along with regression in size and finally their exclusion from the sperm cells reflected one of the most important events in the maturation process of male gametes in Black Bengal buck.

La ubicación de los droplets citoplásmicos (CD) y sus dimensiones variaron significativamente (p <0,01) cuando las células espermáticas atraviesan a través de las regiones de cabeza, cuerpo y cola de epidídimo y vas deferens respectivamente. La disminución gradual en la morfología de los CD entre las regiones del epidídimo se relacionaron de forma significativa (p <0,01, p <0,05). El desplazamiento caudal de las CD, junto con la regresión en el tamaño y, finalmente, su exclusión desde los espermatozoides refleja uno de los eventos más importantes en el proceso de maduración de los gametos masculinos en la cabra Black Bengal.