Effect of plasma separation techniques and sperm selection on sperm recovery and viability of cooled pony stallion semen for 48h at 5°C / [Efeito das técnicas de separação de plasma e de seleção de esperma na recuperação e viabilidade do sêmen de garanhão pônei resfriado por 48h a 5°C]

Separation techniques of seminal plasma [centrifugation (SC) and Sperm Filter® (SF)] and sperm selection [Androcoll-E (SCA) and filtration glass wool (GW)] were used in 24 ejaculates from 6 stallions. In experiment 1, the ejaculates were allocated into control (no spin), centrifugation at 600 g x 10min, SF and GW. In experiment 2, semen was submitted to SC, SGA and filtered through GW. Following the treatments in both experiments, samples were kept chilled at 5°C to 50 x 106 sperm/ml for 48h. The variables measured on fresh and cooling semen were pH, motility, membrane viability function by 6-carboxyfluorescein diacetate and propidium iodide (CFDA / PI), viability or vitality (eosin / nigrosine) and mitochondrial activity. In experiment 1, centrifugation to remove seminal plasma resulted in greater damage to sperm than separation by sperm filter, and selection by glass wool was more efficient in separating viable cells and maintaining viability during cooling. In experiment 2 Androcoll-E and glass wool treatments resulted in higher (P <0.0001) motility, membrane function, mitochondrial activity, and viability than centrifuged semen. Both selection by Androcoll- E and glass wool improved the quality of semen pony stallions for preservation for up to 48h to 5ºC.(AU)

As técnicas de separação do plasma seminal (centrifugação, SpermFilter) e de seleção espermática (Androcoll-E e filtração por lã de vidro) foram aplicadas em 24 ejaculados de seis garanhões da raça Pônei Brasileiro. Após coleta e separação da fração gel, os ejaculados foram diluídos 1:1 com diluente à base de leite em pó. No experimento 1, os ejaculados foram distribuídos em controle (sem centrifugação), centrifugação a 600g x 10min, SpermFilter e filtração por lã de vidro. No experimento 2, o sêmen foi submetido aos procedimentos: centrifugado (SC), centrifugado com Androcoll-E e filtrado por lã de vidro. Após os procedimentos de ambos os experimentos, as amostras foram mantidas refrigeradas a 5ºC, com 50 x 106 espermatozoides/mL, por 48h. As variáveis mensuradas a fresco, 24h e 48h foram: pH, motilidade, funcionalidade de membrana, viabilidade por diacetato de carboxifluoresceína e iodeto de propídio (CFDA/PI, vitalidade (eosina/nigrosina) e atividade mitocondrial. Já osmolaridade e morfologia espermática foram avaliadas somente imediatamente após a coleta. No experimento 1, a centrifugação para retirada do plasma seminal resultou em maiores danos aos espermatozoides do que a separação por SpermFilter. A filtração por lã de vidro mostrou-se mais eficiente em separar células viáveis e manter a viabilidade durante o resfriamento. No experimento 2, os tratamentos com Androcoll-E e filtrado por lã de vidro foram superiores (P<0,0001) ao sêmen centrifugado quanto à motilidade, à funcionalidade de membrana, à atividade mitocondrial e à viabilidade, tanto nas amostras de sêmen fresco como de sêmen refrigerado. O Androcoll-E e a lã de vidro permitiram manter por 48h, a 5ºC, o sêmen de garanhões pôneis utilizando-se diluente à base de leite.(AU)

Diferentes protocolos de congelação de sêmen canino para melhoria de sua cinética e viabilidade / Different canine semen freesing protocols to improve kinetics and viability / Diferentes protocolos de congelación de semen canino para mejoría de su cinética y viabilidad

A maioria dos protocolos utilizados para a criopreservação de sêmen canino, se baseiam em metodologias descritas para outras espécies. Assim, este estudo tem como objetivo avaliar diferentes protocolos de congelação para sêmen desta espécie doméstica. Para tanto, foram utilizados 3 machos, adultos, da raça Buldogue Campeiro, com idades entre 2 a 5 anos e fertilidade comprovada. Foram realizadas 5 colheitas de sêmen de cada animal, pelo método de manipulação digital do bulbo peniano, priorizando a segunda fração do ejaculado. As amostras colhidas foram divididas em 2 grupos, com concentração de 100 x 106 espermatozoides por mL. No grupo 1, as amostras foram diluídas diretamente em meio de congelação comercial Botudog® (Botupharma Biotecnologia Animal). No grupo 2, as amostras foram centrifugadas a 600 g por 10 minutos e em seguida, o pellet foi ressuspendido em meio de congelação comercial Botudog®. As amostras foram envasadas em palhetas de 0,5 mL com concentração de 50 x 106 espermatozoides viáveis. Em seguida, as amostras permaneceram por 1 hora em estabilização a 5ºC. Logo após, transferidas para o vapor de nitrogênio durante 10 minutos, e por fim, mergulhadas em nitrogênio e armazenadas em botijão criogênico. As palhetas foram descongeladas a 46ºC por 15 segundos. Foram avaliados os parâmetros de cinética espermática e integridade de membrana plasmática e acrossomal (IMPA, %). Verificou-se que os parâmetros de motilidade total (%), velocidade linear progressiva (VSL; µm/s), velocidade curvilínea (VCL; µm/s), linearidade (%), percentagem de espermatozoides rápidos (%) e integridade de membrana plasmática e acrossomal avaliados por citometria de fluxo foram superiores no grupo 1, em que as amostras não foram centrifugadas. Estes dados demonstram que, o protocolo para congelação de sêmen canino, utilizando o diluente Botudog®, não preconiza a centrifugação do ejaculado, previamente a congelação.(AU)

Most protocols used for canine semen cryopreservation are based on methodologies described for other species. Thus, this study aims at evaluating different freezing protocols for semen of this domestic species. To this end, 3 adult Bulldog Campeiro males aged 2 to 5 years and proven fertility were used. Five semen samples were collected from each animal using the digital manipulation of the penile bulb method, prioritizing the second fraction of the ejaculate. The collected samples were divided into 2 groups, with a concentration of 100 x 106 sperm per mL. In group 1, the samples were diluted directly into Botudog® commercial freezing medium (Botupharma Animal Biotechnology). In group 2, the samples were centrifuged at 600 g for 10 minutes and then the pellet was resuspended in commercial Botudog® freezing medium. The samples were packed in 0.5 mL straws with a concentration of 50 x 106 viable sperm. Then, the samples remained for 1 hour in stabilization at 5ºC. Afterwards, they were transferred to nitrogen vapor for 10 minutes, and finally, dipped in nitrogen and stored in cryogenic cylinder. The straws were thawed at 46ºC for 15 seconds. Parameters for spermatic kinetics, and plasma and acrosomal membrane integrity (IMPA, %) were evaluated. Total motility (%), progressive linear velocity (VSL; µm/s), curvilinear velocity (VCL; µm/s), linearity (%), percentage of rapid sperm (%) and membrane integrity were found. Plasma and acrosomal samples evaluated by flow cytometry were higher in group 1, where samples were not centrifuged. These data demonstrate that the protocol for canine semen freezing using Botudog® diluent does not recommend centrifugation of the ejaculate prior to freezing.(AU)

La mayoría de los protocolos utilizados para la criopreservación de semen canino se basan en metodologías descritas para otras especies. Por lo tanto, esta investigación tiene como objetivo evaluar diferentes protocolos de congelación para el semen de esta especie doméstica. Con este fin, se utilizaron 3 machos, adultos, de la raza Bulldog Campero, con edades entre 2 a 5 años y fertilidad comprobada. Se realizaron cinco recolecciones de semen de cada animal mediante el método de manipulación digital del bulbo del pene, priorizando la segunda fracción de la eyaculación. Las muestras recolectadas se dividieron en 2 grupos, con una concentración de 100 x 106 espermatozoides por mL. En el grupo 1, las muestras se diluyeron directamente en medio de congelación comercial Botudog® (Botupharma Animal Biotechnology). En el grupo 2, las muestras fueron centrifugadas a 600 g durante 10 minutos y luego el pellet fue resuspendido en medio de congelación comercial Botudog®. Las muestras se envasaron en paletas de 0,5 mL con una concentración de 50 x 106 espermatozoides viables. Luego, las muestras permanecieron durante 1 hora en estabilización a 5ºC. Posteriormente, se transfirieron al vapor de nitrógeno durante 10 minutos y, finalmente, se sumergieron en nitrógeno y se almacenaron en un cilindro criogénico. Las paletas se descongelaron a 46ºC durante 15 segundos. Se han evaluado los parámetros de la cinética espermática y la integridad de la membrana plasmática acrosomal (IMPA, %). Se verificó que los parámetros de motilidad total (%), velocidad lineal progresiva (VSL; µm/s), velocidad curvilínea (VCL; µm/s), linealidad (%), porcentaje de espermatozoides rápidos (%) e integridad de la membrana plasmática y acrosomal evaluadas por citometría de flujo, fueron mayores en el grupo 1, donde las muestras no fueron centrifugadas. Estos datos demuestran que, el protocolo para la congelación de semen canino, usando el diluyente Botudog®, no preconiza la centrifugación del eyaculado, previamente a la congelación.(AU)

Osmolality and composition of the extender during the cold storage of Prochilodus lineatus (Characiformes: Prochilodontidae) sperm

This study evaluates the effects of extender osmolality and composition on the cooling of Prochilodus lineatus sperm. Sperm was diluted in six extenders: two compositions (powdered coconut water(tm) = ACP; Beltsville Thawing Solution(tm) = BTS) x three osmolalities (285, 325, and 365 mOsm/kg) plus an undiluted control, and stored at 6-8°C. Motility rate and velocities (curvilinear, straight line, and average path) were determined every other day. Osmolality did not affect the quality of cooled sperm, thus data were pooled. Motility was higher on d 0 compared to the other days and diluted samples (85-90%) yielded higher motility than control (75%). On d 2, motility was higher in BTS-diluted samples and control, but on d 4 and 6, control yielded the highest motility. Velocities decreased from d 0 to 6 in diluted samples, but not in control. On d 0, velocities were higher in BTS-diluted sperm, but, on d 2, 4, and 6, control yielded higher velocities despite of the large variation among males. Thus P. lineatus sperm should be stored in BTS or without dilution, for a maximum of two days at 6-8°C. Extender osmolality between 285 and 365 mOsm/kg does not affect sperm quality during cold storage...

Neste trabalho avaliou-se os efeitos da osmolalidade e da composição do diluidor no sêmen de Prochilodus lineatus, após o resfriamento. O sêmen foi diluído em seis diluidores: duas composições (água de coco em pó(r) = ACP; Beltsville Thawing Solution(r) = BTS) x três osmolalidades (285, 325 e 365 mOsm/kg) mais uma alíquota sem diluição como controle e armazenadas a 6-8°C. A taxa de motilidade e velocidades (curvilinear, retilinear e média de percurso) foram determinadas a cada dois dias. A osmolalidade não afetou a qualidade do sêmen resfriado, dessa forma foi feito um 'pool' desses dados. A motilidade foi maior no d 0 comparado aos outros dias e as amostras diluídas (85-90%) apresentaram as maiores motilidades do que o controle (75%). No d 2, a motilidade foi maior nas amostras diluídas em BTS e controle, mas nos d 4 e 6, o sêmen controle apresentou as maiores motilidades. As velocidades diminuíram do d 0 para o d 6 nas amostras diluídas, mas não no controle. No d 0, as velocidades foram maiores nas amostras diluídas em BTS, mas, nos d 2, 4 e 6, o controle apresentou as maiores velocidades apesar da grande variação entre os machos. Assim, o sêmen de P. lineatus deve ser resfriado em BTS ou sem diluição (controle), por no máximo dois dias a 6-8°C. A osmolalidade do diluidor entre 285 e 365 mOsm/kg não afeta a qualidade do sêmen durante o resfriamento...

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.

Ejaculados individuais e pools de sêmen: diferenças em condições experimentais / Individual ejaculates and pooled semen: differences under experimental conditions

Avaliaram-se ejaculados caninos individuais e pools de sêmen submetidos a dois tratamentos de renovação do meio diluidor. Sêmen de seis cães foi coletado, na forma de ejaculados individuais e pools de sêmen, diluído na proporção de 1:1 em meio Tris-Gema, centrifugado a 500g/10min, e o pellet ressuspendido até concentração final de 50x10(6) espermatozoides/mL. O sêmen foi resfriado a 0,26ºC/min, entre 37 e 16ºC, e 0,08ºC/min, entre 16 a 8ºC, e mantido em geladeira a 5ºC por 14 dias. No Tratamento 1, o meio diluidor foi renovado a cada seis dias, e no Tratamento 2 aos 12 dias. O sêmen foi avaliado, a cada 48 horas, quanto à motilidade espermática, utilizando-se o Sperm Class Analyser® (SCA), e quanto à integridade de membranas pelo teste hiposmótico e coloração com PI/CFDA. A formação de pools de sêmen simplificou sua manipulação, principalmente com relação ao aumento do volume da amostra disponível; no entanto, resultados obtidos a partir de ejaculados individuais mostraram diferenças entre tratamentos, não identificadas nos pools de sêmen.

Individual ejaculates and pooled dog semen submitted to two treatments of medium exchange were evaluated. Semen was collected from six dogs, as individual ejaculates and pooled semen, diluted in a 1:1 ratio in Tris-Yolk medium, centrifuged at 500g/10min and ressuspended to the final concentration of 50x10(6) sptz/mL. The samples were cooled at rates of 0.26 o C/min between 37 and 16 o C, and 0.08ºC/min from 16 to 8ºC, and then kept in a refrigerator for 14 days. In Treatement 1 medium was exchanged every six days and in Treatment 2 after twelve days. The cooled samples were evaluated every 48 hours for sperm motility using the Sperm Calss Analyser® (SCA), and membrane integrity with hiposmotic swelling test and PI/CFDA stain. Pooled semen was easier to handle, mainly considering the decreased work due to volume. When submitted to medium exchange, pooled semen behaved similarly to individual ejaculates; however, results obtained from individual ejaculates showed differences between treatments, which were not apparent in pooled semen results.

Sperm characteristics as additional evidence of close relationship between lebiasina and piabucina (characiformes: lebiasinidae: lebiasininae)

Spermiogenesis and spermatozoa of representatives of the genera Lebiasina and Piabucina are described. Spermiogenesis is quite similar among all analyzed species of these genera and is identified as Type II. In this type of spermiogenesis, the flagellum of earliest spermatids lies lateral to the nucleus. A slight movement of the nucleus towards the centriolar complex is observed. In late spermatids, the nucleus slightly elongates towards the flagellum. The formation of two striated rootlets apparently occurs in early/intermediate spermatids. The spermatozoa of species of Lebiasina and Piabucina share (1) a drop-shaped slightly elongated nucleus that is laterally positioned relative to the flagellum; (2) a superolateral centriolar complex; (3) two striated rootlets; (4) a basolateral midpiece; (5) oblong mitochondria and (6) a few spherical vesicles. The spermatic characteristics of Lebiasina and Piabucina indicate that spermiogenesis is a homologous process among all species of both genera examined to date and can be considered, along with the spermatozoa morphology, additional evidence indicating a close relationship between Lebiasina and Piabucina.

A espermiogênese e os espermatozoides de representantes dos gêneros Lebiasina e Piabucina são descritos. O processo de espermiogênese é muito semelhante entre todas as espécies analisadas, sendo descrita como do Tipo II. Nesta variação de espermiogênese o flagelo das espermátides iniciais localiza-se lateralmente ao núcleo. O núcleo movimenta-se ligeiramente sobre o complexo centriolar. Nas espermátides finais, o núcleo alonga-se ligeiramente em direção ao eixo flagelar. A formação de duas raízes estriadas ocorre nas espermátides iniciais/intermediárias. Os espermatozoides das espécies de Lebiasiana e Piabucina compartilham principalmente (1) o núcleo em forma de gota, lateralmente posicionado em relação ao eixo flagelar, e ligeiramente alongado em direção ao flagelo, (2) o complexo centriolar superolateral, (3) duas raízes estriadas surgindo a partir do centríolo distal, (4) peça intermediária basolateral, (5) mitocôndrias oblongas e (6) algumas vesículas esféricas. As características espermáticas de Lebiasina e Piabucina foram observadas comparativamente e permitem concluir que o processo de espermiogênese é homólogo em todas as espécies destes dois gêneros examinadas até o momento. Os espermatozoides e principalmente os dados de espermiogênese são apresentados aqui como evidências adicionais que indicam uma relação estreita entre os gêneros Lebiasina e Piabucina.

Avaliação de diferentes crioprotetores intra e extracelulares na criopreservação de sêmen de touros / Evaluation of different intra and extracellular cryoprotectants on bull semen cryopreservation

Em um delineamento experimental usando o fatorial 3x2, três crioprotetores internos, glicerol (GLI), etilenoglicol (EG) e dimetilformamida (DMF), e dois externos, gema de ovo (GEMA) e lipoproteína de baixa densidade (LDL), avaliaram-se a motilidade ao descongelamento de GLI-GEMA 53,9±1,96, sendo superior aos demais tratamentos (P<0,05). Na avaliação de morfologia ao descongelamento, não houve diferença (P>0,05) entre os tratamentos EG-GEMA 68,3±1,58, EG-LDL 72,2±2,39 e DMF-GEMA 68,7±1,67 que foram mais altos que os demais (P<0,05). A avaliação de integridade de membrana por fluorescência ao descongelamento GLI-GEMA 34,2±2,28 e EG-GEMA 30,9±1,32 não diferiram entre si (P>0,05), mas foram mais elevados que os demais (P<0,05), enquanto que a HOST dos tratamentos DMF-GEMA 13,6±1,30 e DMF-LDL 9,8±0,78 diferirem entre si (P<0,05) e foram mais baixas que as demais (P<0,05). O uso de etilenoglicol associado à gema de ovo pode ser uma alternativa ao uso de glicerol nos protocolos de congelamento de sêmen de touros.

The experiment was designed as 3 x 2 factorial design, with three internal cryoprotectants, glycerol (GLY), etileneglycol (EG) and dymethilformamide (DMF) and two external, egg yolk (YOLK) and density low lipoproteina (LDL). The motility at thawing for GLY-YOLK (53.9±1.96) was higher than other treatments (P<0.05). The percentage of cells with normal morphology at thawing was not different between EG-YOLK (68.3±1.58), EG-LDL (72.2±2.39) and DMF-YOLK (68.7±1.67), but they were higher than the others (P<0.05). The evaluation of membrane integrity through fluorescent probes at thawing indicate that GLY-YOLK (34.2±2.28) and EG-YOLK (30.9±1.32) were not different (P>0.05), but were higher than the others (P<0.05). The evaluation of membrane integrity through hypoosmotic swelling test (HOST) indicate that DMF-YOLK (13.6±1.30) and DMF-LDL (9.8±0.78) were different (P<0.05) and lower than the others (P<0.05). The use of ethylene glycol associated to egg yolk can be a viable alternative to the use of glycerol in bull semen freezing protocols.

Initiation and s uppression of sperm motility is osmolality dependent in two South American fish species: streaked prochilod (Prochilodus lineatus) and piracanjuba(Brycon orbignyanus)

Solutions to induce or suppress the initiation of sperm motility in fish ha ve been used to improve reproductive success during artificial fertilization and preservation techniques . The aim of the present study was to evaluate the effects of three solutions (NaCl, glucose , and BTS™) - each prepared with 10 different osmolalities - on the initiation and suppression of fresh sperm motility in Prochilodus lineat us and Brycon orbignyanus . Sperm was diluted in each of the 30 solution s and immediately observed under a light microscope to determine which solution s trigger ed or suppress ed the initiation of sperm motility. When present, motility rate ( % motile sperm ) w as determined at 0, 30 , and 120 s post - activation and the motility quality score ranging from 0 ( no movement ) to 5 ( rapidly swimming sperm) was determined at 0 and 30 s post - activation . Osmolality , but not solution composition , significantly affected both motility rate and quality score . Solutions at osmolali ties up to 270 mOsm/kg in P. lineatus and up to 180 mOsm/kg in B. orbignyanus induced motility in at least 60% of sperm , with a minimum quality score of 3.0 , and were therefore classified as activating agents. The greatest motility at 0 , 30 , and 120 s post - activation was observed with solutions ranging from 135 to 225 mOsm/kg for P. lineatus and at 135 mOsm/kg for B. orbignyanus . On the other hand, solutions ranging from 360 to 450 mOsm/kg in P. lineatus and 270 to 450 mOsm/kg in B. orbignyanus suppressed motility in at least 95% of sperm and were classified as immobilizing media . The osmolality of the surrounding medium is the key factor in the initiation or suppression of sperm motility in P. lineatus a nd B. orbignyanu.

Deterioration of testicular functions in adult male rabbit after exposure to mobile phone radiation with impending recovery

The accumulate effects of electromagnetic field [EMF] release from mobile phones have many effects on multiple organs. Nevertheless, its effect on testicular function is still debated. The objective of the study is to clarify the alterations in testicular functions after exposure to electromagnetic radiation of mobile phone and to investigate the possibility of recovery. Eighteen adult male rabbits are enrolled into 3 groups: control, exposed and recovery group. Tine exposed and recovery groups are exposed to mobile phones in standby position for 18 hours /day and six day/week for 14 weeks. After that, the recovery group was monitored for another 14 weeks. exposure to EMR induced a significant drop in sperm count, sperm motility and sperm fast forward motility at the 6[th], 12[th] and 9[th] week respectively and get maximum inhibition at the 14[th] week. These finding were concomitant with degenerative changes in seminiferous tubules and interstitial cells of Leydig. These negative effects may be attributed to the detectable decrease in the serum level of testosterone, gonadotophic hormones, increase the level of oxidative stress and direct deterioration of testicular tissue. The other study points [body and testicular weight, body temperature and percentage of sperm morphology and live sperm] did not show any alteration. Recovery period significantly ameliorated the suppressed testicular functions and also, restored the hormonal and oxidative biomarkers within the 14 weeks. the longitudinal exposure to EMR causes testicular dysfunction that may be mediated by hormonal disturbances, oxidative stress or direct damage on testicular tissue that could reverse and improve within the recovery period

Qualidade espermática e perfil plasmático de lipídeos em garanhões suplementados com óleo de arroz semirefinado com alto teor de gama-orizanol / Spermatic quality and plasmatic lipids in stallions supplemented with rice bran oil semirefined containing high level of gamma-oryzanol

O gama-orizanol é uma substância natural contida no óleo de arroz, que por suas características hipocolesterolêmicas e antioxidantes, têm sido objeto de estudo em humanos e, mais recentemente, em equinos. Foram utilizados seis garanhões, de várias raças, com idade de 10±5,4 anos e peso inicial de 472,67±90,48 kg. Objetivou-se verificar os efeitos da suplementação da dieta com óleo de arroz semi-refinado, contendo 1,1% de gama-orizanol, sobre os níveis de lipídeos plasmáticos (colesterol total, HDL-C, LDL-C, VLDL-C e triglicérides), de testosterona e da qualidade espermática. Os garanhões foram divididos em dois grupos, recebendo em cada refeição 150 mL de óleo de soja ou de arroz durante 60 dias. Realizaram-se colheitas amostrais de sangue e sêmen a cada 15 dias do período experimental. O delineamento experimental foi inteiramente casualizado, com medidas repetidas no tempo, e as médias comparadas pelo teste F, considerando-se o nível de 5% de significância. Os valores médios de testosterona, colesterol total, HDL-C, LDL-C, VLDL-C e triglicérides foram respectivamente 75,93 ng/dL; 92,73; 61,47; 26,99 e 4,28 mg/dL para o tratamento com óleo de soja; e de 62,13 ng/dL; 110,20; 66,73; 38,44 e 5,02 mg/dL para o tratamento com óleo de arroz. Em relação à qualidade espermática, não foi observada diferença (p<0,05) entre os tratamentos nas variáveis: volume, motilidade, concentração e defeitos. Podemos concluir que a suplementação supracitada não influencia a qualidade espermática nem as concentrações plasmáticas de testosterona, VLDL-C, HDL-C e triglicérides, porém, pode elevar as concentrações plasmáticas de colesterol total e de LDL-C.

Gamma-oryzanol is a natural substance contained in rice bran oil that has been studied for years in humans due to its hypocholeterolemic and antioxidant properties, and in recent years in horses. Using six stallions from different breeds in a randomized experimental design, weighing 472.67±90.48 kg and aging 10±5.4 years, this study was aimed to evaluate the effects of supplementation with rice bran oil containing 1.1% of gamma-oryzanol in plasmatic lipids levels (total cholesterol, HDL-C, LDL-C, VLDL-C and triglycerides), testosterone and sperm quality. Stallions were divided in two groups, one receiving 150 mL of soybean oil, and the other 150 mL of rice bran oil twice a day, during 60 days. Blood and sperm samples were collected every 15 days of the trial period. Data obtained was processed, and means compared using F test, at 5% of confidence level. Mean values of total cholesterol, HDL-C, LDL-C, VLDL-C, triglycerides and testosterone were respectively 92.73; 61.47; 26.99; 4.28 mg/dL and 75.93 ng/dL in the soybean oil supplemented group, and 110.20; 66.73; 38.44; 5.02 mg/dL and 62.13 ng/dL in the rice bran oil one. No difference among treatments (p<0,05) were observed in sperm quality parameters volume, motility, concentration and defects. Supplementation of stallions with rice bran oil containing 1.1% of gamma-oryzanol do not promote any change in sperm quality, testosterone, HDL-C, VLDL-C or triglycerides plasmatic levels, but an increase in total cholesterol and LDL-C.

Evaluación y estandarización de la calidad del espermiograma: nuevos límites inferiores de referencia / Evaluation and standardization of the quality of semen: new lower reference limit

El análisis seminal o espermiograma es uno de los parámetros más usados en la evaluación de la fertilidad masculina. La OMS (WHO, 2010), presentó el 5 Manual para el examen y procesamiento del semen humano, documento que fue analizado durante el primer taller de estandarización del análisis seminal (PLEAS), realizado en Santiago de Chile, mayo del 2010. Posteriormente se aplicaron los nuevos valores indicados como "límite de referencia inferior" (LIR), en el estudio del análisis seminal realizados por varios autores (2003 al 2010). Los resultados obtenidos indican que el 81 por ciento de los investigadores latino americanos creen que el nuevo manual estandariza mejor la concentración espermática, un 96 por ciento está de acuerdo con la nueva subclasificación en la motilidad espermática en progresiva (A), no progresiva (B) e inmóviles (C). El 68 por ciento estima que el mejor instrumental de recuento es la cámara de Neubauer. Respecto a los controles de calidad solo el 18 por ciento realiza controles de calidad externa. El 100 por ciento de los investigadores estima conveniente realizar continuos talleres de estandarización. Respecto a la aplicación de los LIR en las poblaciones en estudio, todos ellos cumplirían con los estándares actuales para ser considerada una población con capacidad de fertilidad. Sin embargo varios autores opinan que una nueva versión del manual OMS, debe realizarse urgentemente para estandarizar mejor la concentración espermática (15 millones por mL) y la morfología según criterios estrictos (4 por ciento), valores de referencia que consideran muy bajos.

Spermogram or semen analysis is one of the most used parameters in the evaluation of male fertility. WHO (2010) presented the 5th Manual for review and processing of human semen, a document that was discussed during the first workshop of standardization of semen analysis (PLEAS), held in Santiago de Chile, May 2010. Subsequently applied the new values expressed in "lower referencelimit" (LRL) for semen in several analysis studies conducted by various authors (2003 to 2010). The results indicate that 81 percent of Latin American researchers believe the new manual standardizes best sperm concentration, 96 percent agree with the new subclassification in progressive sperm motility (A), non-progressive (B) and immobile (C). 68 percent determined that the best instruments for the sperm countis the Neubauer haemocytometerchamber. Regarding quality control only 18 percent performed external quality control. 100 percent of researchers believe it is appropriate to conducton going standardization workshops. Regarding the application of LRL in the study populations (2003-2010), 100 percent comply with the standards to be considered a population with fertility capacity. However, several authors argue that a new version of the WHO manual, must be re-done urgently to better standardize sperm concentration (15 million/mL) and morphology according to strict criteria (4 percent), reference values considered very low.

Variação estacional na qualidade do sêmen de carneiros Lacaune no Brasil / Seasonal variation in semen quality of lacaune rams in Brazil

Little is known regarding the seasonal semen production of Lacaune rams in southern Brazil. Thus, the aim of this study was to evaluate semen quality and production of Lacaune rams maintained in Rio Grande do Sul (RS). Semen was collected using an artificial vagina, from 12 rams (1 to 4 years of age), kept under intensive conditions. The quantitative and qualitative characteristics of the semen and scrotal circumference of each ram were recorded, from the winter of 2002 to the spring of 2003. Seasonal variations were recorded in almost all the monitored semen characteristics and in scrotal circumference. Sperm concentration was the only semen characteristic evaluated that did not show seasonal variation. Sperm volume varied between 1.1 ± 0.4 mL (winter of 2002) and 1.5 ± 0.4 mL (autumn of 2003). Total number of sperm per ejaculate varied between 4.0 ± 3.3x109 (winter of 2002) and 5.7 ± 3.3x109 (summer of 2003). For scrotal circumference records, in 2002 significant differences between seasons were observed. Significant differences were also found in accordance with the age of rams (P < 0.05) in the winter of 2002. Results showed improvement in motility, concentration and volume of the sperm during summer and autumn when compared to spring and winter. So, in this region of Brazil summer and autumn are the more indicated seasons to maximize the utilization of Lacaune rams for reproductive practices. Further studies must be done to evaluate Lacaune semen freezability in these seasons of the year.

Existem poucos dados sobre a produção de sêmen da raça Lacaune, no sul do país. Assim, o objetivo deste estudo foi o de avaliar a produção e a qualidade do sêmen de carneiros desta raça, criados no estado do Rio Grande do Sul (RS). Foram utilizados 12 carneiros, de idades entre 1-4 anos, criados em condições intensivas. O sêmen foi colhido por vagina artificial e foram avaliadas as características quantitativas e qualitativas e a circunferência escrotal, do inverno de 2002 até a primavera de 2003. Com exceção da concentração espermática, foram observadas variações estacionais em todas as características estudadas. O volume variou de 1.1 ± 0.4 mL, no inverno de 2002, a 1.5 ± 0.4 mL no outono de 2003. O número total de espermatozóides por ejaculado variou de 4.0 ± 3.3x109 (inverno de 2002) a 5.7 ± 3.3x109 (verão de 2003). Observou-se variação estacional significativa (P < 0,05) na circunferência escrotal em 2002, bem como variação significativa entre as idades dos animais, no inverno do mesmo ano. Os valores médios da produção espermática observados no verão e o outono foram superiores aos obtidos no inverno e primavera. O verão e outono foram considerados as estações mais indicadas para a utilização de machos da raça Lacaune em programas reprodutivos, no RS. Mais estudos devem ser conduzidos, para a verificação de efeitos estacionais sobre o congelamento do sêmen.

Efeitos do diluidor na viabilidade espermática pós-criopreservação em tourinhos da raça Gir Leiteiro aos 25 meses de idade, pré-selecionados pela classificação andrológica por pontos / Effects of the extender in the semen post - thaw viability in young dairy Gyr bulls (Bos taurus indicus), pre - selected by breeding soundness evaluation

Procedeu-se à criopreservação do sêmen de oito tourinhos Gir Leiteiro, com idade média de 25 meses, pré-selecionados para elevada pontuação (média 84,4±5,6) na classificação andrológica por pontos (CAP), em dois diferentes diluidores: um à base de lactose-gema-glicerol e outro à base de lecitina de soja. As curvas de resfriamento e congelação foram padronizadas com o auxílio da máquina CRYOGEN®. Os parâmetros pós-congelação avaliados no sêmen submetido aos dois diluidores - motilidade, vigor, defeitos maiores, menores e totais, defeitos de acrossoma, cauda dobrada, reação ao teste hiposmótico (Thos) e células normais - foram comparados aos do sêmen fresco, exceto para Thos e entre eles. O sêmen de todos os animais foi congelado com êxito no diluidor lactose-gema-glicerol. Houve diferença (P<0,05) em todas as variáveis analisadas no sêmen fresco e pós-congelado, exceto para defeitos maiores. Entre diluidores, houve diferença (P<0,05) para motilidade, vigor, cauda dobrada e Thos. Estes resultados indicam que a seleção pelo CAP médio >80 é um bom índice para selecionar touros com maior taxa de espermatozoides viáveis pós-congelação.

Semen cryopreservation from eight young dairy Gyr bulls was performed using two different semen extenders. Bulls aging 25 months old and pre-selected for a high average score (84.4±5.6 in a 0-100 scale) in Zebu breeding soundness evaluation (BSE) composed the experimental group. Extenders were based on lactosis-egg yolk-glycerol and soya lecithin. Chilling and freezing curves were standardized by CRYOGEN® machine. Post-thaw features evaluated in semen frost in both extenders - motility, vigor, major, minor and total deffects, morphological alteration in acrossome, bent tail, reative cells to hyposmotic sweeling test (Thos), and normal cells - were compared to the ones in the fresh ejaculate (except Thos) and among them. It was possible to freeze semen from all animals in the lactosis-egg yolk-glycerol extender. There were difference (P<0.05) in all analyzed features between fresh and cryopreserved semen, except for major deffects. Between extenders, there were differences (P<0.05) in motility, vigor, bent tail, and Thos. All bulls had successfull semen freezability. These results sustain that pre-selection for high BSE values (average >80 points) is a good index to identify bulls with good post semen - thaw features. However, the choice of the extender is critical for obtaining acceptable results.

Avaliação computacional da compactação da cromatina e de características morfométricas da cabeça de espermatozoides de coelho (Oryctolagus cuniculus) / Computational evaluation of chromatin condensation and morphometric characteristics of rabbit (Oryctolagus cuniculus) sperm head

Investigou-se a correlação entre a morfometria da cabeça e a intensidade da condensação e heterogeneidade da cromatina em espermatozoides de coelho (Oryctolagus cuniculus). Para tal, utilizaram-se 35 esfregaços de sêmen de coelhos da raça Nova Zelândia, corados com azul de toluidina e avaliados por análise de imagem computacional. As imagens foram obtidas digitalmente em tons de cinza e avaliadas por algoritmos desenvolvidos em ambiente de programação Scilab. As mensurações obtidas da cabeça dos espermatozoides foram área, perímetro, comprimento, largura, relação comprimento largura, elipsidade, fator de forma, descritores Fourier e simetria lateral e anteroposterior. Também foram avaliadas a intensidade da compactação e a heterogeneidade da cromatina espermática. Os espermatozoides de coelho apresentaram compactação e heterogeneidade cromatínica mais intensas do que os de touro e observou-se correlação significativa entre características morfométricas da cabeça e compactação e heterogeneidade cromatínica. Conclui-se que a cromatina é importante para a constituição morfológica da cabeça de espermatozoides de coelho e que a cromatina espermática de coelho é naturalmente mais heterogênea e menos compactada que a de touro.

The correlation between the head morphometry and the intensity of condensation and heterogeneity of sperm chromatin were investigated in rabbit (Oryctolagus cuniculus). To this, 35 semen smears from New Zealand rabbits were stained with toluidine blue and evaluated by computer image analysis. The images were obtained in digital grayscale and analyzed by algorithms developed in the Scilab programming environment. The measurements obtained from the sperm heads were area, perimeter, length, width, length:width ratio, ellipticity, shape factor, lateral and anterior-posterior symmetries, and Fourier descriptors. The intensity and heterogeneity of the compaction of sperm chromatin was also evaluated. The rabbit spermatozoa showed chromatin heterogeneity and condensation more intense than the bull spermatozoa, and it was observed correlation between morphometric characteristics of the head and chromatin compaction and heterogeneity. The results suggest that chromatin is important for the morphological constitution of the head morphology spermatozoa head, as well as, rabbit sperm chromatin is inherently more heterogeneous and less condensed than the bull sperm chromatin.

Validation of Differential Extraction Kit in forensic sexual assault cases / 法医学杂志

OBJECTIVE@#To evaluate the validity of Differential Extraction Kit in isolating spermatozoa and epithelial cell DNA from mixture samples.@*METHODS@#Selective lysis of spermatid and epithelial cells combined with paramagnetic particle method were applied to extract the DNA from the mock samples under controlled conditions and forensic case samples, and template DNA were analyzed by STR genotype method.@*RESULTS@#This Differential Extraction Kit is efficient to obtain high quality spermatid and epithelial cell DNA from the mixture samples with different proportion of sperm to epithelial cell.@*CONCLUSION@#The Differential Extraction Kit can be applied in DNA extraction for mixed stain from forensic sexual assault samples.

best stain for morphological study of human seminal fluid's smears

There is a high need for proper evaluation of the morphological features of human sperms. The importance of this lies in the field of andrology, male fertility and in vitro fertilization. The wet smears can give rough clue about the shape of the sperms, but it is neither accurate nor reproducible. This study aimed to determine the best stain which can be used for seminal fluid cytology. This study was conducted in Port Sudan, Red Sea State, Sudan in the period from October 2006 to September 2007. The total number of patients was 50. Samples which were collected from normospermic patients [NSP] were prepared by direct smear technique. Samples which were collected from oligospermic patients [OSP] and azoospermic patients [ASP] were prepared by direct smear technique and also by indirect smear techniques [concentration method]. Smear samples were stained by freshly prepared Harris's Haematoxylin, Papanicolaou stain, May-Grunwald Giemsa stains [MGG], supra vital stain, Giemsa stain and leishman's stain. In this study, the best stain was Harris's Haematoxylin [80% excellent for the head of sperm, 70% good for the neck, 59% excellent for the tail, 42% very good for cells in background]. Harris's stain was followed by papanicolaou stain and the third best stain was supra vital stain. MGG was better than Giemsa in staining of semen smears [75% good versus 25% good] in overall performance. The worst stain was Leishman's stain. Stained smears must be used for the morphological study of semen samples. Harris's Haematoxylin is the best stain for semen cytological features. Stains which used for the semen samples should be freshly prepared

Sperm chromatin integrity: etiologies and mechanisms of abnormality, assays, clinical importance, preventing and repairing damage

The standard semen analysis is the first line and the most popular laboratory test in the diagnosis of male fertility. It evaluates sperm concentration, motility, morphology and their vitality. However, it is well-known that normal results of semen analysis can not exclude men from the causes of couples' infertility. One of the most important parameters of sperm in its fertilizing potential is [Sperm chromatin integrity] that has direct positive correlation with Assisted Reproductive Techniques [ART] outcomes including; fertilization rate, embryo quality, pregnancy and successful delivery rate. It seems that sperm DMA chromatin integrity provides better diagnostic and prognostic approaches than standard semen parameters. For these reasons understanding the sperm chromatin structure, etiology of sperm chromatin abnormality, identification factors that disturbs sperm chromatin integrity and the mechanism of their action can help in recognizing the causes of couples' infertility. Various methods of its evaluation, its importance in male fertility, clinical relevance in the outcomes of ART and application of laboratory and medical protocols to improve this integrity have valuable position in diagnosis and treatment of male infertility. There has recently been interest in the subject and its application in the field of andrology. Therefore, with regard to the above mentioned importance of sperm chromatin integrity, this review article describes details of the useful information pertaining to sperm DMA damage including the origins, assessments, etiologies, clinical aspects, and prevention of it

Factors influencing quality of semen: a two year prospective study.

In the recent years, male infertility and subfertility has increased, which is attributed to many factors. So our study focuses the effects of age, occupation, smoking, alcohol and varicocele on the semen quality. Detailed history and examination of 93 cases (fulfilled inclusion criterion) was done. Semen analysis of these cases were compared with above parameters using statistical tools like mean, standard deviation, standard error of mean and significance was tested by student's 't' test. The mean sperm density, total motility and rapid progressive motility in control group were 68.95 x 10(6)/ml, 59.9% and 30.5% respectively. Reduction of sperm density was statistically significant (p value < 0.05) in both tobacco users + alcoholics and in varicocele patients in comparison with controls. Age and occupation did not alter semen quality significantly. Our study concluded that semen quality is decreasing in the past few decades and combined tobacco + alcohol use, and varicocele have more detrimental effect on semen quality.

Usefulness of testicular fine needle aspiration cytology in cases of infertility.

Testicular fine needle aspiration cytology (FNAC) is an important investigation in management of male infertility, especially to differentiate between obstructive and non obstructive causes of azoospermia. It is less invasive and associated with no or minimal complications. Nowadays when assisted fertilization techniques are being practiced, fibrosis after biopsy may further hamper in sperm extraction for intra cytoplasmic sperm injection (ICSI). Present study describes a detailed analysis of aspiration cytology in 546 cases and also compared 48 cases of testicular biopsies with cytology. The cytological diagnoses correlated well with histological diagnoses and helped in management of infertility. FNAC can help in management of surgical and medical causes of infertility and can save unnecessary expensive investigations in cases of sertoli cell only syndrome and atrophic patterns. FNAC in combination with semen analysis and serum follicle stimulating hormone levels are of great help in management of male infertility.

Comparative analysis of spermiogenesis and sperm ultrastructure in Callichthyidae (Teleostei: Ostariophysi: Siluriformes)

In Corydoradinae, the presence of spermatids in the lumen of the testicular tubules together with spermatozoa suggests that spermatogenesis is of the semicystic type, whereas in Callichthyinae, sperm production occurs entirely within spermatocysts in the germinal epithelium, characterizing cystic spermatogenesis. Spermiogenesis in Callichthyinae is characterized by an initial lateral development of the flagellum, the presence of nuclear rotation to different degrees, an eccentric or medial formation of a nuclear fossa, formation of a cytoplasmic channel, and presence of centriolar migration, being more similar to type I spermiogenesis. In Corydoradinae, spermiogenesis is characterized by eccentric development of the flagellum, the absence of nuclear rotation, an eccentric nuclear fossa formation, formation of a cytoplasmic channel, and absence of centriolar migration, differing from the types previously described. The process of spermatogenesis and spermiogenesis in Corydoradinae and Callichthyinae revealed unique characters for each of these subfamilies, corroborating the hypotheses that they constitute monophyletic groups. In relation to sperm ultrastructure, the comparative analysis of the callichthyid species shows that the general characteristics found in the spermatozoa were similar, thus, reinforcing the hypothesis that the family is monophyletic

Em Corydoradinae, a presença de espermátides junto com espermatozóides no lúmen dos túbulos testiculares sugere uma espermatogênese do tipo semicística, enquanto que em Callichthyinae a produção do esperma ocorre inteiramente dentro dos espermatocistos no epitélio germinativo, caracterizando a espermatogênese cística. A espermiogênese em Callichthyinae é caracterizada por um desenvolvimento inicial lateral do flagelo, pela presença de rotação nuclear em diferentes graus, formação de uma fossa nuclear excêntrica ou medial, formação de um canal citoplasmático, e presença de migração centriolar, sendo mais similar à espermiogênese do tipo I. Em Corydoradinae, a espermiogênese é caracterizada pelo desenvolvimento excêntrico do flagelo, ausência de rotação nuclear, fossa nuclear excêntrica, formação de um canal citoplasmático, e ausência de migração centriolar, diferindo dos tipos descritos previamente. O processo de espermatogênese e espermiogênese em Corydoradinae e Callichthyinae revelaram caracteres únicos para cada subfamília, corroborando a hipótese de que as mesmas constituem grupos monofiléticos. Em relação à ultraestrutura do esperma, a análise comparativa das espécies de Callichthyidae mostra que as características gerais encontradas nos espermatozóides foram similares, reforçando a hipótese de monofilia da família