Morphology of the spermatheca of Triatoma lecticularia (Hemiptera: Reduviidae) (Stal, 1859) / Morfologia da espermateca de Triatoma lecticularia (Hemiptera: Reduviidae) (Stal, 1859)

Abstract Triatoma lecticularia (Hemiptera: Reduviidae) (Stal, 1859) is a potential vector of Chagas's disease and the comprehension of its reproductive biology is an important tool to control this insect. In the reproductive tract of female insects, the spermatheca plays a crucial role storing male spermatozoa after mating. Whithin insects the spermatheca shows a wide morphological diversity and the analysis of this characteristic can contribute to understand the reproductive biology of the species. This study describes the histology and histochemistry of the spermatheca of T. lecticularia. Females have a pair of elongated spermathecal reservoirs without associated accessory gland. The reservoir opens into the common oviduct via a narrow muscular duct. The reservoir epithelium has single layer of columnar secretory cells. The control of the release of spermatozoa from the spermatheca occurs via the muscular duct. The anatomical features of the spermatheca of T. lecticularia resemble those described of other Reduviidae. However, the histological and histochemical features of spermatheca observed in T. lecticularia were important to explain the maintenance of the viability of the spermatozoa stored.

Resumo Triatoma lecticularia (Hemiptera: Reduviidae) (Stal, 1859) é um potencial vetor da doença de Chagas e a compreensão de sua biologia reprodutiva é um importante fator para seu controle populacional. No aparelho reprodutor feminino dos insetos, a espermateca desempenha a importante funcão de armazenar os espermatozoides após cópula. Nos insetos, a espermateca apresenta uma ampla diversidade morfológica e a análise destas características pode contribuir com o entendimento da biologia reprodutiva das espécies. Este estudo descreve histológica e histoquimicamente a espermateca de T. lecticularia. As fêmeas tem um par de espermatecas alongadas sem glândulas acessórias associadas. O reservatório conecta-se ao oviduto comum através de um ducto muscular curto que controla a liberação dos espermatozoides. O epitélio do reservatório possui uma camada de células secretoras colunares. As características anatômicas da espermateca de T. lecticularia são semelhantes às encontradas em outros Reduviidae. Entretanto, as características histológicas e histoquímicas observadas na espermateca são importantes para explicar a manutenção da viabilidade dos espermatozoides armazenados.

Fragmentación del ADN espermático e infertilidad masculina / Fragmentation of sperm DNA and male infertility

La integridad del ADN de los espermatozoides, es un indicador importante de la fertilidad, se utiliza como una variable adicional para evaluar, junto al espermograma, la calidad de una muestra seminal. En el presente trabajo se describen algunos aspectos de interés relacionados con la fragmentación del ADN espermático, cuya etiología es multifactorial y está relacionada con factores intrínsicos, como el empaquetamiento anormal de la cromatina durante la espermiogénesis, la apoptosis defectuosa antes de la eyaculación y la producción excesiva de especies reactivas del oxígeno; y con factores extrínsecos, como son los cambios en los hábitos de vida, la exposición a agentes tóxicos, así como la edad avanzada. La fragmentación está asociada al deterioro de las variables seminales, además afecta la fertilidad natural y la realizada por tratamientos de reproducción asistida, por lo que la implementación en los laboratorios de Andrología, de la tecnología para detectar si el ADN de los espermatozoides está íntegro o fragmentado, incorpora un nuevo conocimiento en el estudio de los hombres con trastornos de fertilidad, lo que contribuye a mejorar el diagnóstico y pronóstico de la infertilidad masculina. Para realizar este trabajo se revisaron 122 artículos, de los cuales 84 cumplieron con los criterios de calidad esperados. La búsqueda se realizó a través de los buscadores habituales(AU)

Sperm DNA´s integrity is an important indicator of fertility and it is used as an additional variable to evaluate the quality of a seminal sample, together with the spermogram. This paper describes some interesting aspects related to the fragmentation of sperm DNA, whose etiology is multifactorial and is related to intrinsic factors, such as the abnormal packing of chromatin during spermiogenesis, defective apoptosis before ejaculation, and the excessive production of oxygen´s reactive species; and with extrinsic factors, such as changes in life habits, exposure to toxic agents, as well as aging. Fragmentation is associated with the deterioration of seminal variables, it also affects natural fertility and that produced by assisted reproduction treatments, so the implementation in the andrology laboratories of the technology to detect if the DNA of the sperm is intact or fragmented incorporates a new knowledge in the study of men with fertility disorders, which contributes to improve the diagnosis and prognosis of male infertility. To carry out this work, 122 articles were reviewed, of which 84 met the expected quality criteria. The search was made through the usual search engines(AU)

The quantification of testicular cells during the postnatal development in two Caviomorph rodents: the guinea pig (Cavia porcellus) and the cutia (Dasyprocta agouti)

ABSTRACT The germinative, Sertoli and Leydig cells of two caviomorph rodents (Cavia porcellus and Dasyprocta agouti) were counted as well as the estimation of the total volume of the testis and the total volume of seminiferous tubules and interstitium in prepubertal, pubertal and adult animals. The number of spermatogonia, spermatocytes and spermatids cells increased during the pubertal phase in both rodents, notably the spermatid cells. The spermatocyte and spermatid slightly decreased in the adult of both rodents, but the increment in spermatogonia cells number was seen, mainly in cutias. The number of Sertoli cells increased in pubertal rodents, but in the adult the number reduced. Substantial number of Leydig cells was counted in pubertal and adult guinea pigs. In cutias, the number of Leydig cells increased in pubertal phase and decline in adults. The design-based stereological method has proven to be unbiased and reliable to be applied in reproduction studies.

Morfología espermática: comparación de las observaciones realizadas por 10 expertos / Sperm morphology: comparison of observations made by 10 experts

Introducción: La morfología espermática es uno de los parámetros más importantes que se evalúan durante el análisis seminal, aunque es un parámetro que presentan alta variabilidad inter-laboratorios. Por lo tanto el objetivo de este trabajo fue determinar la variación interindividuo al analizar la morfología espermática observada por 10 expertos. Materiales y métodos: En este estudio descriptivo prospectivo se planteo una encuesta de 133 preguntas, cada una presentaba una fotografía de un espermatozoide con el fin de clasificarlo como normal o anormal, y de ser anormal se debía especificar su anormalidad (cabeza, pieza intermedia, cola y/o gota citoplasmática). Esta encuesta se distribuyo a los laboratorios y personal experto en el tema de evaluación seminal, se recolectaron 10 encuestas entre septiembre y agosto de 2016. Resultados: De las 132 fotografías, 23 (17%) representaban células normales, mientras que 109 (83%) representaban espermatozoides con algún tipo de anormalidad. El coeficiente de variación para el numero de aciertos totales fue de 22.4%, para aciertos normales fue de 66% y para los aciertos anormales del 4.5%. La variación para las células normales totales fue de 42.3%, mientras que la variación para los anormales totales fue solo del 6.5%. Conclusión: Aunque la variabilidad entre los observadores es similar a la reportado en otros estudios, es necesario crear redes de cooperación que permitan estandarizar los procedimientos de determinación de la morfología espermática entre los diferentes laboratorios del país.

Introduction: Sperm morphology is one of the most important parameters that are evaluated during the seminal analysis, although it is a parameter that presents high inter-laboratory variability. Therefore the objective of this study was to determine the interindividual variation when analyzing the sperm morphology observed by 10 experts. Material and Methods: In this prospective descriptive study a survey of 133 questions was presented, each photograph show of a spermatozoon in order to classify it as normal or abnormal, and if abnormal, its abnormality (head, intermediate piece, tail and/or cytoplasmic droplet). This survey was distributed to laboratories and experts in the subject of seminal evaluation, 10 surveys were collected between September and August 2016. Results: Of the 132 photographs, 23 (17%) represented normal sperm, while 109 (83%) represented spermatozoa with some type of abnormality. The coefficient of variation for the number of total hits was 22.4%, for normal hits was 66% and for abnormal hits of 4.5%. The variation for the total normal cells was 42.3%, while the variation for the total abnormal cells was only 6.5%. Conclusion: Although the variability among observers is similar to that reported in other studies, it is necessary to create cooperation networks that allow standardization of the procedures for determining the sperm morphology between the different laboratories in the country.

Recovery of sperm after epididymal refrigeration from domestic cats using ACP-117c and Tris extenders / Recuperação de espermatozoides após refrigeração do epidídimo de gatos domésticos utilizando os diluidores ACP-117c e Tris

We aimed to compare fresh sperm and sperm cooled to 4ºC that had been recovered from the epididymides of cats using powdered coconut water (ACP-117c) and Tris extenders. Sixty epididymides were divided into 6 groups: 10 fresh epididymides were recovered using Tris (T0h); 10 were kept at 4°C/2h and recovered using Tris (T2h); 10 were kept at 4°C/4h and recovered using Tris (T4h); 10 fresh were recovered using ACP-117c (A0h); 10 were kept at 4°C/2h and recovered using ACP-117c (A2h), and 10 were kept at 4°C/4h and recovered using ACP-117c (A4h). The testis-epididymis complexes (TEC) control were not cooled. The others were cooled at 4°C for 2 or 4h. The epididymis was separated and the sperm was recovered by the modified flotation method. Sperm kinetic parameters were evaluated by a computer-system analysis, and vigor, viability, concentration, membrane function and morphology of the sperm were assessed under a light microscope. The progressive motility with ACP-117c declined after 2h of cooling, but did not differ between fresh and 4h. The vigor and membrane function were higher in A4h than A0h. The vigor at T2h and T4h were decreased compared to T0h. T0h was higher than A0h for vigor and sperm membrane function. However, after 4h of cooling, ACP-117c maintained a higher percentage of living cells. Feline epididymal sperm quality can be maintained to the degree necessary for artificial breeding programs following cooling and ACP-117c may be successfully used to recover cat sperm that have been cooled for up to 4h.(AU)

Objetivou-se comparar a qualidade de espermatozoides recuperados a fresco e após refrigeração a 4ºC do epidídimo de gatos domésticos utilizando-se os diluidores ACP-117c e Tris. Sessenta epidídimos foram distribuídos em seis grupos: 10 epidídimos a fresco com o Tris (T0h), 10 a 4°C/2h e recuperados com Tris (T2h), 10 a 4°C/4h e recuperados com Tris (T4h), 10 epidídimos a fresco com o ACP-117c (A0h), 10 a 4 °C/2h e recuperados com ACP-117c (A2h), 10 a 4°C/4h e recuperados com ACP-117c (A4h). Os complexos testículo-epidídimo (CTE) do controle não foram refrigerados. Os outros foram refrigerados a 4°C durante duas e quatro horas. Os epidídimos foram separados das demais estruturas, e os espermatozoides recuperados pela técnica de flutuação modificada. Os parâmetros cinéticos foram avaliados em um sistema computadorizado, e o vigor, a viabilidade, a concentração, a funcionalidade de membrana e a morfologia celular foram avaliados em microscopia de luz. A motilidade progressiva com ACP-117c declinou após duas horas de refrigeração, mas não diferiu entre a recuperação a fresco e após refrigeração por quatro horas. Vigor e integridade funcional da membrana celular foram significativamente superiores no grupo A4h em comparação ao A0h. O vigor espermático em T2h e T4h reduziu significativamente em comparação com T0h. T0h foi significativamente superior ao A0h quanto aos parâmetros de vigor e integridade funcional da membrana espermática, entretanto, após quatro horas de refrigeração, o ACP-117c apresentou um maior percentual de células vivas. Os espermatozoides epididimários de felinos domésticos conseguem manter a qualidade necessária para serem utilizados em programas de reprodução artificial após serem refrigerados e recuperados por meio da técnica de flutuação modificada, e o diluidor ACP-117c pode ser utilizado com sucesso para recuperação de células espermáticas refrigeradas de gatos por até quatro horas.(AU)

An easy, reproducible and cost-effective method for andrologists to improve the laboratory diagnosis of non-obstructive azoospermia: a novel microcentrifugation technique

ABSTRACT This study describes a new method of microcentrifugation as an improved, viable, cost-effective option to the classical Cytospin apparatus to confirm azoospermia. Azoospermic semen samples were evaluated for cryptozoospermia by a centrifugation method similar to that of World Health Organization guidelines (2010; entire specimen centrifuged at 3000g for 15 min, and aliquots of the pellet examined). Then, if no sperm were detected, the pellet from that procedure was resuspended in culture medium, centrifuged (2000g for 15 min), and the entire pellet spread on a 4 X 6mm area of a slide and stained using the Christmas tree method (Nuclear-Fast solution and picric acid). The entire stained area was examined for the presence or absence of sperm. A total of 148 azoospermic samples (after standard WHO diagnosis) were included in the study and 21 samples (14.2%) were identified as sperm-positive. In all microcentrifugation slides, intact spermatozoa could be easily visualized against a clear background, with no cellular debris. This novel microcentrifugation technique is clearly a simple and effective method, with lower cost, increasing both sensitivity and specificity in confirming the absence or presence of spermatozoa in the ejaculate. It may represent a step forward of prognostic value to be introduced by andrology laboratories in the routine evaluation of patients with azoospermia in the initial semen analysis.

Estrés oxidativo: ¿un estado celular defectuoso para la función espermática? / Oxidative stress: a defective cell state for sperm function?

En los organismos vivos, las cantidades de radicales libres y especies reactivas del oxígeno (ROS) son controladas por un complejo sistema de homeostasis, capaz de mantener niveles fisiológicos de ROS necesarios para el funcionamiento y regulación de algunas biomoléculas. Paralelamente, los organismos poseen sistemas bioquímicos de protección contra el estrés oxidativo, que consiste en el desbalance entre la producción de especies químicas altamente reactivas y las defensas antioxidantes de la célula. Dicho estrés contribuye de manera importante a la etiología tanto de la senescencia celular como de algunas enfermedades. En el contexto reproductivo, las células espermáticas pasan por una serie de cambios fisiológicos durante los procesos de maduración, capacitación y fecundados, entre los que se incluyen las modificaciones de las proteínas existentes, reguladas por señales procedentes del entorno espermático, donde las ROS modulan importantes vías bioquímicas, involucradas en procesos fundamentales de la función del espermatozoide y que se pueden alterar en estados de estrés oxidativo. El objetivo de esta revisión de literatura es describir algunos de los procesos que contribuyen al estrés oxidativo y sus implicaciones sobre la funcionalidad espermática.

Living organisms regulate the load of free radicals and reactive oxygen species (ROS) by a complex homeostatic system, capable of maintaining physiological levels of ROS, necessary for the action and regulation of some biomolecules. In parallel, organisms harbor biochemical protection systems against oxidative stress, consisting of an unbalance state between oxygen reactive chemical species and antioxidant defense production; this kind of biochemical stress has been shown to contribute to cellular senescence and the development of different diseases. In the reproductive field, the spermatic cells undergo a serial of physiological changes during the maturation, capacitation and fertilization process. Such changes include the modification of proteins regulated by signals from the sperm environment, where ROS modulate important biochemical pathways involved in fundamental processes of sperm function, and that could be altered under oxidative stress conditions. The objective of this review is to describe some of the processes that contribute to oxidative stress and its implications on sperm functionality.

A Correlative Study of Oocytes Morphology with Fertilization, Cleavage, Embryo Quality and Implantation Rates after Intra Cytoplasmic Sperm Injection.

ntroduction: Non-invasive selection of developmentally human oocytes may increase the overall effi ciency of human assisted reproduction. Morphologic abnormalities in the oocyte are relevant for determining its developmental fate. Th e objective is to evaluate the infl uence of MII oocyte morphology on intra cytoplasmic sperm injection (ICSI) outcomes. Material and Methods: 132 patients undergoing ICSI cycles and having female factors of infertility and unexplained infertility. Couples having male factors of infertility were excluded. A total of 1200 oocytes were retrieved from 132 ICSI cycles, of which 1056 MII oocytes were evaluated. Th e criteria for morphological evaluations were: (i) Normal MII oocytes showing clear cytoplasm with uniform texture and homogenous fi ne granularity, a round or ovoid fi rst polar body with a smooth surface, and perivitelline space of normal size. (ii) MII oocytes with extra cytoplasmic abnormalities (fi rst polar body and perivitelline space abnormalities). (iii) MII oocytes with cytoplasmic abnormalities (dark cytoplasm, granular cytoplasm, inclusion body and presents of vacuoles). (iv) MII oocytes with combined abnormalities. Result: From 1056 MII oocytes, 180 (17.04%) had normal morphology while 876 (82.95%) had at least one demonstrable morphological abnormality. Cytoplasmic abnormalities were observed in 516 (58.9%) of the oocytes. Extra cytoplasmic abnormalities were observed in 104 (11.87%) while combined abnormalities were responsible for the remaining 256 (29.22%). Th ere were no signifi cant diff erences in fertilization, cleavage, and embryo quality between the groups but there was a highly signifi cant diff erence in implantation rate which was higher in the group of normal oocytes morphology than abnormal oocytes morphology, oocytes with cytoplasmic, extracytoplasmic and combined abnormality 11.11%, 7.33%, 9.03%, 2.3%, and 4.34% respectively. Conclusion: MII oocyte morphology did not aff ect fertilization, cleavage, and embryo quality, but aff ecting implantation rate.

Semen parameters from 2002 to 2013 in Korea young population: A preliminary report

PURPOSE: To analyze the differences of semen parameters in Korean young population for three periods from 2002 to 2013. MATERIALS AND METHODS: A total of 516 semen samples were collected from Korean men presenting for infertility, varicoceles or other infectious problems for three periods from 2002 to 2012: January 2002-December 2003, January 2007-December 2008, and January 2012-December 2013. A standard World Health Organization procedure for semen analysis was performed for assessment of semen concentration, volume, motility, morphology, and pH. RESULTS: A total of 160, 162, 194 men constituted the study populations in 2002 to 2003, in 2007 to 2008, and in 2012 to 2013, respectively. The overall sperm parameter results suggested a statistically significant difference between 2002 to 2003 and 2012 to 2013 except pH. However, considering the data from 2007 to 2008, there were no trends in changes in overall semen parameters. Negative correlations were observed in all semen parameters with increasing age in all patients, except for pH. In addition, semen volume, motility, and morphology had higher negative correlation coefficients with age, from 2002 to 2013, serially. CONCLUSIONS: There were no significant changes in the semen parameters of Korean men from 2002 to 2013. In addition, semen volume, motility, and morphology showed higher negative correlation coefficients with age from 2002 to 2013, serially.

Validación de sybr-14 y 6-cfda para evaluar la viabilidad e integridad de la membrana plasmática en espermatozoides caninos de raza chihuahua / Validation of sybr-14 and 6-cfda to evaluate the viability and plasma membrane integrity in sperm of chihuahua canine breed

En la aplicación de técnicas reproductivas es importante determinar in vitro la capacidad fecundante de los espermatozoides, para ello se utilizan combinaciones de tinciones para evaluar los diferentes parámetros de función espermática, aumentando así la precisión de la estimación de la muestra. El objetivo del estudio fue comparar la efectividad de la utilización de los fluorocromos 6-CFDA y SYBR-14 combinados con PI para determinar la viabilidad e integridad de la membrana plasmática por citometría de flujo. Se utilizó semen fresco de caninos (n=5) de raza Chihuahua, con una concentración espermática >150x106 esp/ml y motilidad progresiva >80%. Tres protocolos fueron ensayados: grupo 1: SYBR-14/PI, grupo 2: 6-CFDA/PI y grupo 3: PI. La integridad de la membrana plasmática de los espermatozoides fue similar entre grupos 1 y 2, independiente del fluorocromo utilizado (37,26±13,9 y 33,8±14,6, respectivamente; p=0,4601). Asimismo, la viabilidad espermática entre los grupos 1, 2 y 3 (62,7±13,9, 66,1±14,6 y 66,4±13,3, respectivamente; p=0,8987). En conclusión, no se evidenció diferencias en la efectividad para determinar la viabilidad e integridad de la membrana plasmática mediante la utilización de SYBR-14 y 6-CFDA, ambas tinciones pueden ser incorporadas al análisis de rutina de semen canino de raza Chihuahua.

In applying reproductive techniques in vitro it is important to determine the fertilizing capacity of the sperm, for this a combination of dyes were used to assess different parameters of sperm function, thereby increasing the accuracy of the estimation of the sample. In dogs (Canis lupus familiaris) Chihuahua breed there is no precedent for evaluating sperm function parameters. The aim was to assess the viability and plasmatic membrane integrity, basic parameters of sperm function. Propidium iodide (PI) was used, a fluorescent dye-specific DNA, which combined with fluorochromes permeable acts as marker of the sperm membrane integrity. The effectiveness of the use of 6-CFDA and SYBR-14 fluorochromes combined with PI was also compared to determine viability and sperm membrane integrity using flow cytometry. Fresh semen of dogs (n=5) Chihuahua breed was used with a concentration of >200x106 sp/ml and progressive motility >80%. Three protocols were performed: group 1: SYBR-14/PI, group 2: 6-CFDA/PI and group 3: PI. The plasma membrane integrity of sperm was similar, independent of the fluorophore used between groups 1 and 2 (13.9±37.26 and 33.8±14.6, respectively, p=0.4601). This also applied to sperm viability between groups 1, 2 and 3 (62.7±13.9, 66.1±14.6 and 66.4±13.3, respectively, p=0.8987). No difference was demonstrated in effectiveness to determine the viability and integrity of the sperm membrane using SYBR-14 and 6-CFDA, both dyes can be incorporated in to routine analysis of semen in canine Chihuahua breed.

Selection of sperm based on hypo-osmotic swelling may improve ICSI outcome: a preliminary prospective clinical trial

The intra-cytoplasmic sperm injection [ICSI] technique selects sperm according to morphology and motility. However, these parameters cannot predict the chromatin integrity of sperm. Considering the detrimental effects of DNA-damaged sperm on reproductive outcomes, novel sperm selection procedures have been proposed to circumvent the possibility of inseminating DNA-damaged sperm. It has been shown that different potential hypo-osmotic swelling test [HOST] patterns possess the potential to differentiate between sperm that have intact or damaged chromatin. Therefore, for the first time, this preliminary study evaluates the role of HOST as a sperm selection procedure in a clinical setting. In this preliminary prospective clinical trial study, we divided infertile couples diagnosed with male infertility into two groups. In the treatment group [n=39], half of the oocytes were inseminated by sperm selected following density gradient centrifugation [DGC group]. The remaining oocytes from the treatment group were inseminated by sperm chosen according to HOST pattern [c, d or e] following DGC processing [HOST group]. In the control group [n=63], all oocytes were inseminated by sperm chosen after DGC. There was a significantly higher percentage of embryos that had good quality, implantation, and chemical pregnancy rates in the HOST group compared to the DGC group [p

Determinação da dose inseminante e embriogênese na fertilização artificial de tambaqui (Colossoma macropomum) / Determination of insemination dose and embryonic development in the artificial fertilization of tambaqui (Colossoma macropomum)

Determinou-se a dose inseminante para fertilização artificial e descreveu-se o desenvolvimento embrionário de tambaqui (Colossoma macropomum). Os gametas foram coletados de reprodutores induzidos hormonalmente. Foi realizada fertilização artificial nas proporções de espermatozoides/ovócito de D1-50.666; D2-75.999; D3-101.332; D4-126.665; D5-151.998. O desenvolvimento embrionário foi acompanhado por meio de observações periódicas em estereoscópio até a eclosão dos ovos. Na fase de fechamento do blastóporo foi calculada a taxa de fertilização nas diferentes doses inseminantes. A porcentagem de fertilização aumentou de forma linear segundo a equação Ŷ =0,050 + 0,00000773X (R²=97,5), atingindo um platô em 84% na proporção de 102.486 espermatozoides/ovócito. Os embriões apresentaram segmentação meroblástica discoidal, típica de ovos telolécitos, com eclosão ocorrendo aos 357 horas-grau após a fertilização. Conclui-se que o desenvolvimento embrionário de tambaqui obedece ao esperado para peixes com ovos telolécitos e recomenda-se o uso da dose inseminante de aproximadamente 100.000 espermatozoides/ovócito na rotina de fertilização artificial dessa espécie.

The objective of this research was to determine the insemination dose for artificial fertilization and describe the embryonic development of tambaqui (Colossoma macropomun). The gametes were collected from induced breeding hormonally. An artificial fertilization was performed with different sperm/oocyte ratios of D1-50666, D2-75999, D3-101 332, 126 665-D4, D5-151 998 sperm/oocyte. Embryonic development was monitored through periodic stereoscopic observations until hatching. When embryos reached the blastopore closure stage, the rate of fertilization in different insemination doses was calculated. A regression equation was estimated to determine the ideal proportion of the gametes. The fertilization rate increased linearly according to the equation Ŷ = 0.050 + 0.00000773 X (R² = 97.5), up to the proportion of 102.486 spermatozoa/oocyte, and, from this point, the fertilization rate was maintained at 84%. The embryonic development of tambaqui was meroblastic discoidal, as expected from telolecithal eggs and we recommend the use of the insemination dose of approximately 100.000 sperm/oocyte in the artificial fertilization of tambaqui.

Enzymatic digestion improves testicular sperm retrieval in non-obstructive azoospermic patients

In patients with non-obstructive azoospermia [NOA], vital spermatozoa from the tissue is obtained from testes by enzymatic treatment besides the mechanical treatment. To increase the sperm recovery success of testicular sperm extraction [TESE], with enzymatic digestion if no sperm is obtained from testis tissue by mechanical method. Tissue samples were collected from 150 men who presented with clinical and laboratory data indicating NOA by means of TESE and micro dissection TESE methods. Initially, mature spermatozoa were examined for by mechanical extraction technique shredding the biopsy fractions. In cases whom no spermatozoa was observed after maximum 30 min of initial searching under the inverted microscope, the procedure was followed by enzymatic digestion using DNaseI and collagenase type IV. Surgery type, pathology, AZF, karyotype, hormones and testis size were compared in patients. Of 150 cases with NOA, conventional mincing method extended with enzymatic treatment yielded successful sperm recovery in 13 [about 9%] patients. Comparison of parameters revealed that level of FSH and LH were significantly different [p=0.04 and 0.08 respectively] between two groups that response negative and positive to enzymatic digestion. The combination of conventional TESE and enzymatic digestion is an effective method to recover spermatozoa. The benefit of the mincing combined with enzyme to sperm retrieval for NOA firstly shorten the mechanical searching time, leading to minimizing further cellular damage as well as exposure to external conditions, and secondly reduce the number of cases with sperm recovery failures. Also, the serum level of FSH and LH are factors that influence the chance of sperm retrieval

Malathion Affects Spermatogenic Proliferation in Mouse / Malathion Afecta la Proliferación Espermatogénica del Ratón

The restriction of the mechanisms of cell proliferation in murine seminiferous epithelium, in terms of induction of programmed cell death until recently has not been fully analyzed. The aim of this work was to assess the effect of Malathion (MP) on testicular morphology and function in mouse spermatogenesis. For the experiments, male albino mice of strain NMRI-IVIC, weighing between 30-40 g were used, and divided into control and experimental groups of 5 each. The animals of the experimental groups were injected with a single dose of MP: 241mg/kg weight (1/12 LD 50 ) resuspended in 0.9% saline, intraperitoneally. Animals were sacrificed at 8.3, 16.6 and 33.2 days post-injection (first, second and third spermatogenic cycles). Testicular samples were obtained for light microscopy (LM), transmission electron microscopy procedures, and to detect apoptosis and p53 antigen by immunohistochemical methods. Blood was collected to quantify testosterone and plasmatic cholinesterase activity. From 8.3 days, Sertoli cell vacuolization, karyolisis of pachytene spermatocytes and Leydig cells and a decreased in average of the diameter of seminiferous tubules was observed. No damage to inter-Sertoli cells junctions was detected. Percentage of seminiferous tubules showing germ cells apoptosis was increased from 8.3 days, plasmatic acetylcholinesterase activity was reduced in the group treated only 24 hours after administration of MP. Serum testosterone levels were low in treated animals at 16. 6 and 33.2 days. p53 was mostly expressed in pachytene spermatocytes from 8d. The findings of this study indicate that MP alters the testicular function affecting the DNA and interfering with spermatogenesis as well as steroidogenesis.

La restricción de los mecanismos de proliferación celular en epitelio seminífero murino, en términos de inducción de muerte celular programada hasta hace poco no había sido completamente analizada.El objetivo de este trabajo fue evaluar el efecto de malathion (MP) sobre la morfología y la función testicular del ratón.Ratones macho albinos de la cepa NMRI-IVIC, con pesos entre 30-40 g fueron utilizados, se dividieron en grupos control y experimental. Los grupos experimentales fueron inyectados por vía intraperitoneal con una dosis única deMP:241mg/kg de peso (1/12 DL50) resuspendido en 0,9% de solución salina.Los animales fueron sacrificados en el día 8,3, 16,6 y 33,2 después de la inyección (primer, segundo y tercer ciclos de la espermatogénesis).Se obtuvieron muestras de testículo para estudio en microscopía de luz (ML), microscopía electrónica de transmisión, para la detección de apoptosis y el antígeno p53 (proliferación celular), por métodos inmunohistoquímicos.Se recogió sangre para cuantificar la testosterona y la actividad plasmática de colinesterasa.Desde el día 8,3 día se observó vacuolización de células de Sertoli, cariolisis de espermatocitos en paquiteno y células de Leydig, y una disminución en el promedio del diámetro de los túbulos seminíferos. No se detectó daño en las uniones entre células de Sertoli. El porcentaje de túbulos seminíferos que mostraban células germinales en apoptosis se incrementó a los 8,3 días, laactividad de la acetilcolinesterasa plasmática se redujo en el grupo tratado sólo 24 horas después de la administración de MP.Los niveles séricos de testosterona disminuyeron en los animales tratados a los 16,6 y 33,2 días.P53 se expresó sobre todo en los espermatocitos en paquiteno desde los 8,3 días.Los resultados de este estudio indican que MP altera la función testicular, afecta al ADN e interfiere con la espermatogénesis, así como con la esteroidogénesis.

Influence of strict sperm morphology on the results of classic in vitro fertilization

OBJECTIVE: To determine the influence of the concentration of oval spermatozoa according to the strict morphology criterion in men with normal sperm concentration following the World Health Organization criteria on the results of classic IVF. MATERIALS AND METHODS: Based on review of patient charts, this study included infertile couples presenting with female causes for infertility or unexplained infertility, in whom men presented with normal spermogram values for sperm concentration, sperm motility, volume of ejaculate and total sperm count after semen processing greater than 20 million. Based on the value obtained in strict sperm morphology, patients were divided into three groups: in Group A, patients with values between 0% and 4%; in group B, between 5% and 14%, and in group C, patients with sperm morphology greater than 14%. The outcomes analyzed were oocyte fertilization rate, biochemical pregnancy rate, clinical pregnancy rate and rate of liveborns. RESULTS: A total of 244 cases met the inclusion criteria, 27 of them in group A, 165 in group B, and 52 in group C. The mean fertilization rate and the rate of liveborns were, respectively: 71.9% and 33.3% in group A; 80.9% and 24.2% in group B, and 78.8% and 28.8% in group C. There was no statistical difference among the groups in any of the outcomes analyzed. CONCLUSION: The values of strict sperm morphology, as proposed by Kruger and adopted by the World Health Organization, had no influence on the results of classic in vitro fertilization in the studied sample.

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.

Isolating sperm cells by laser capture microdissection technique from mixture sample / 法医学杂志

OBJECTIVE@#To assess the application value of laser capture microdissection (LCM) technique for isolating a small number of sperm cells from mixture sample.@*METHODS@#Mixture samples were prepared with sperm cells and vaginal epithelia at different concentrations. Both LCM technique and the differential lysis method were employed to obtain sperm cells from the mixture samples, and DNA was extracted by magnetic beads method. STR genotyping was determined using Identifiler kit.@*RESULTS@#The successful STR genotype rate of sperm cells isolated from mixture samples with LCM technique was 92.86% (13/14). The rate of differential lysis method was 7.14% (1/14). The successful rates between the two methods were statistically different (P < 0.05).@*CONCLUSION@#LCM technique can effectively exclude the interference of female cell component and isolate a small number of sperm cells to obtain a single male STR genotyping. LCM technique is obviously better than the differential lysis method.

Varicocele time-dependently affects DNA integrity of sperm cells: evidence for lower in vitro fertilization rate in varicocele-positive rats

We designed this study to clarify how varicocele can time-dependently affect sperm morphological parameters and DNA integrity. In this study, we intend to estimate the effect of various periods of varicocele on the in vitro fertilization [IVF] rate in rats. In this experimental study, left varicocele were induced as the test group [n=18] which was further sub-divided into three groups based on the study termination time [4, 6 and 8 months after varicocele induction]. The control-sham group [n=6] consisted of rats who received no treatment. Repopulation index [RI], tubular differentiation index [TDI], sperm viability and motility, morphological maturity, chromatin integrity and ability to undergo IVF were assessed. In addition, the potential impact of varicocele on serum total antioxidant capacity [TAOC] and total thiol molecules [TTM] were examined. Histological results showed that varicocele negatively influenced TDI and RI. All sperm morphological parameters were lower than those in the control-sham group. DNA damage was severely and time-dependently substantiated in all test groups. Varicocele significantly reduced the ability of sperm derived from varicocele rats to undergo IVF. Serum TAOC and TTM levels reduced in a time-dependent manner. Right testes varicocele-induced rats showed remarkably less damaged profile for all investigated parameters compared to the left testes varicocele. Our data suggested that experimentally induced varicocele negatively impacted sperm maturation and chromatin integrity in a time-dependent manner. This consequently caused a remarkable reduction in IVF ability. The detrimental effect of varicocele may be attributed to the significant reduction of antioxidant capacity of the serum

Pattern of semen fluid abnormalities in male partners of infertile couples in Khartoum, Sudan

Normal semen is a mixture of spermatozoa suspended in secretions from the testis and epididymis, which at the time of ejaculation, are combined with secretions from the prostate, seminal vesicles, and pulbourethral glands. Many factors affect the quantity and quality of semen parameters such as cigarette smoking, excessive exercise and alcohol consumption. The objectives of the study were to determine the pattern of semen fluid abnormalities [volume of the ejaculate, sperm concentration, sperm motility, sperm morphology] in male partners of infertile couples in Khartoum, Sudan. This was a descriptive study, 100 records of couples who attended Sudan Assisted Reproduction Centre in Khartoum seeking fertility treatment from July - December 2008 were reviewed for the volume of the ejaculate, the concentration of the sperms, the motility and the morphology. Semen production was obtained in the centre or at home after 3-7 days of abstinence from intercourse. Production of semen was by masturbation in a sterile container. Evaluation of samples was made by a qualified andrology technician. Statistics was done by the computer using SPSS soft ware. Results showed that 41.5%, 53.9%, 3.1%, and 1.5% of men had a volume of < 2, 2-4, 5-7 and >8 ml respectively. 25%, 37% and 38% had normal, oligozoospermia and azoospermia respectively. As regards motility, 27.4% had normal motility 62.9% had sluggish motility and 9.7% their sperms were immotile. There were only 1.8% of men who had normal morphology. The study concluded that, few men had sperm profile that was consistent with reference values with regard to volume, concentration, motility and morphology. Morphology showed the least normality. Results showed that no subject in the cohort fulfilled the full criteria of normozoospermia

Simultaneous assessment of plasmatic, acrosomal, and mitochondrial membranes in ram sperm by fluorescent probes / Avaliação simultânea das membranas plasmática, acrossomal e mitocondrial de espermatozoides de carneiros por sondas fluorescentes

In this experiment, it was defined a protocol of fluorescent probes combination: propidium iodide (PI), fluorescein isothiocyanate-conjugated Pisum sativum agglutinin (FITC-PSA), and JC-1. For this purpose, four ejaculates from three different rams (n=12), all showing motility >80 percent and abnormal morphology <10 percent, were diluted in TALP medium and split into two aliquots. One of the aliquots was flash frozen and thawed in three continuous cycles, to induce damage in cellular membranes and to disturb mitochondrial function. Three treatments were prepared with the following fixed ratios of fresh semen:flash frozen semen: 0:100 (T0), 50:50 (T50), and 100:0 (T100). Samples were stained in the proposal protocol and evaluated by epifluorescence microscopy. For plasmatic membrane integrity, detected by PI probe, it was obtained the equation: v=1.09+0.86X (R²=0.98). The intact acrosome, verified by the FITC-PSA probe, produced the equation: v=2.76+0.92X (R²=0.98). The high mitochondrial membrane potential, marked in red-orange by JC-1, was estimated by the equation: v=1.90+0.90X (R²=0.98). The resulting linear equations demonstrate that this technique is efficient and practical for the simultaneous evaluations of the plasmatic, acrosomal, and mitochondrial membranes in ram spermatozoa.

Neste experimento, foi definida uma combinação de sondas fluorescentes: iodeto de propídio (PI), aglutinina de Pisum sativum conjugada ao isotiocionato de fluoresceína (FITC-PSA) e JC-1. Para esta proposta, quatro ejaculados de três carneiros (n=12), que apresentavam motilidade >80 por cento e alterações morfológicas <10 por cento, foram diluídos em meio TALP e divididos em duas alíquotas. Uma alíquota foi submetida a três ciclos de flash frozen e descongelação, para induzir danos nas membranas celulares e distúrbios na função mitocondrial. Três tratamentos foram preparados com as seguintes proporções preestabelecidas de sêmen fresco: sêmen submetido a flash frozen: 0:100 (T0), 50:50 (T50) e 100:0 (T100). As amostras foram coradas no protocolo proposto e avaliadas por microscopia de epifluorescência. Para integridade de membrana plasmática, detectada pela sonda PI, foi obtida a equação: v=1,09+0,86X (R²=0,98). O acrossomo intacto, verificado pela sonda FITC-PSA, produziu a equação: v=2,76+0,92X (R²=0,98). O alto potencial de membrana mitocondrial, marcada em vermelho-alaranjado pelo JC-1, foi estimado pela equação: v=1,90+0,90X (R²=0,98). As equações lineares resultantes demonstraram que a técnica é eficiente e prática para avaliação simultânea das membranas plasmática, acrossomal e mitocondrial em espermatozoides de carneiros.