Evaluation of stallion sperm motility with ImageJ using a cell phone camera and a light microscope / [Avaliação da motilidade espermática de garanhões com ImageJ usando-se câmera de celular e microscópio óptico]

This study aimed to determine the accuracy of assessing stallion sperm motility using a light microscope, a cell phone camera, and a free computer-assisted semen analysis (FCASA) package for ImageJ. The total motility of frozen (n=22) and cooled (n=48) equine semen was determined by FCASA and compared to the results of subjective visual analysis (SVA) by two technicians. Frozen samples were also evaluated by a commercial computer-assisted semen analysis (CCASA) system. The Friedman test revealed no significant differences (P>0.05) between cooled samples analyzed by FCASA (38.0) and SVA (technician 1, 40.0; technician 2, 40.0), nor between frozen samples analyzed by FCASA (23.36 ± 15.9), SVA (25.5 ± 18.8 and 25.8 ± 18.5), and CCASA (25.2 ± 18.3). However, mean FCASA results were underestimated by 7.2% compared with CCASA. The correlation between FCASA and CCASA was significant and strong (P<0.0001, r=0.95). Chi-squared tests indicated that FCASA provided similar results (P=0.14) to the reference method (CCASA), but SVA had lower accuracy (P=0.04). ImageJ analysis of cell phone videos captured under a light microscope can be used for estimation of stallion sperm motility with comparable accuracy to commercial systems.(AU)

O objetivo deste estudo foi testar as configurações necessárias para avaliar a motilidade espermática total de garanhões, mediante o uso de ImageJ, microscópio óptico e câmera de celular. Os valores de motilidade total das amostras de sêmen equino congeladas (22) e refrigeradas (48) foram comparados por análise visual (SVA) e pelo plugin do ImageJ (CASAF). Amostras congeladas também foram comparadas por um CASA comercial (CCASA). O teste de Friedman não resultou em diferença estatística (P>0,05) entre as 48 amostras analisadas com CASAF (38,0) e SVA de dois avaliadores (40,0 e 40,0). A comparação das 22 amostras congeladas entre CASAF (23,36±15,9), SVA (25,5±18,8 e 25,8±18,5) e CCASA (25,2±18,3) também não resultou em diferença estatística, sendo que a média dos resultados obtidos com CASAF subestimou a obtida com o CCASA em 7,2%. A correlação entre CASAF e CCASA foi significativamente elevada (r=0,95, P<0,0001). O teste de qui-quadrado resultou em proporção de acertos semelhantes entre o CASAF e o CCASA (P=0,14), enquanto SVA resultou em proporção diferente (P=0,04), indicando menor acurácia. O uso de microscópio óptico e câmera de celular foi útil para obter vídeos de sêmen de garanhões a serem analisados com ImageJ, proporcionando resultados de motilidade total equiparáveis a sistemas comerciais.(AU)

Are specialized sperm function tests clinically useful in planning assisted reproductive technology?

CASE STUDY 40-year-old male patient and 32-year-old female partner, with a history of primary infertility of two years duration. The workup revealed idiopathic mild oligoasthenotheratozoospermia, and no apparent female infertility factors. The couple has failed three intrauterine insemination (IUI) cycles, planning more IUI cycles but also considering in vitro fertilization/intracytoplasmic sperm injection (IVF/ICSI).

Diluente Tris suplementado com óleo de Mauritia flexuoxa sobre a qualidade do sêmen caprino após a criopreservação / Tris diluent supplemented with Mauritia flexuoxa oil on the quality of goat semen after cryopreservation

Objetivou-se verificar os efeitos, nos parâmetros espermáticos, na integridade mitocondrial, acrossomal e de membrana em células espermáticas, desencadeados pelo uso do Tris (Tris hidroximetil aminometano) suplementado com óleo de Mauritia flexuoxa como diluente para criopreservação de sêmen caprino. Quatro caprinos clinicamente saudáveis foram utilizados. Os animais eram alimentados diariamente com volumoso (Pennisetum purpureum, Schum.), concentrado (ração peletizada com teor de 20% proteína, 300 g/animal/dia) e sal mineral específico para Caprinos (Caprinofós®), à vontade. Dois ensaios foram realizados: I – Teste de toxicidade; II – Criopreservação do sêmen com concentrações ideais. No teste de toxicidade as concentrações avaliadas foram: 5%, 10%, 15% e 20% de diluente a base de óleo de Mauritia flexuoxa. Após o teste de toxicidade, foi escolhido a concentração que apresentou o melhor resultado (5%). Logo após, foram realizadas mais 32 coletas, que foram diluídas em Tris-gema-glicerol (grupo controle) ou diluente contendo óleo vegetal (Mauritia flexuoxa). As amostras foram criopreservadas com auxílio do aparelho Tk3000®. Após o período mínimo de uma semana as palhetas foram descongeladas em banho-maria a 37 °C por 30 segundos, acondicionadas em microtubos de centrifugação e homogeneizadas para a análise imediata de motilidade, vigor espermático e morfologia. Em seguida, por meio de sondas fluorescentes foram avaliadas a integridade de acrossomo, membrana plasmática (Diacetato de Carboxifluresceína e Iodeto de Propídeo) e função mitocondrial sob microscopia de epifluorescência. Quanto a motilidade e vigor, integridade mitocondrial e acrossomal, o grupo buriti foi inferior ao grupo controle. O Tris suplementado com óleo de Mauritia flexuoxa na concentração de 5% não influenciou significativamente a qualidade espermática, porém, observouse morfologia e integridade de membrana favoráveis. Dessa forma, sendo uma alternativa para substituição de diluentes a base de produtos de origem animal.

The objective was to verify the effects, sperm parameters, mitochondrial, acrosomal and membrane integrity in sperm cells, triggered by the use of Tris (Tris hydroxymethyl aminomethane) supplemented with Mauritia flexuoxa oil as a diluent for cryopreservation of goat semen. Four goats clinically healthy were used. The animals were fed daily with bulky (Pennisetum purpureum, Schum.), concentrate (pelleted feed with 20% protein content, 300 g / animal / day) and mineral salt Specific for Goats (Caprinofós®), ad libitum. Two tests were carried out: I - Toxicity test; II - Semen cryopreservation with ideal concentrations. In the toxicity test as selected were: 5%, 10%, 15% and 20% of Mauritia flexuoxa oil-based diluent. After the toxicity test, the concentration that showed the best result (5%) was chosen. Soon after, a further 32 samples were obtained, which were diluted in Tris-glycerol (control group) or diluent containing vegetable oil (Mauritia flexuoxa). The samples were cryopreserved using the Tk3000® machine. After a minimum of one week, the samples were thawed in a 37 ° C water bath for 30 seconds, packed in centrifugation microtubes and homogenized for immediate analysis of motility, sperm vigor and morphology. Then, by means of fluorescent probes, the integrity of the acrosome, plasma membrane (Carboxyflurescein diacetate and Propidium Iodide) and mitochondrial function under epifluorescence microscopy were evaluated. As for motility and vigor, mitochondrial and acrosomal integrity, the buriti group was inferior to the control group. Tris supplemented with Mauritia flexuoxa oil at a concentration of 5% did not significantly influence sperm quality, however, favorable motility, morphology and membrane integrity were observed. Thus, being an alternative to replace diluents based on products of animal origin.

Acute exposure to hyperosmotic conditions reduces sperm activation by urine in the yellowtail tetra Astyanax altiparanae, a freshwater teleost fish / Exposição aguda a condições hipereosmoticas reduz ativação do sêmen por urina em lambari do-rabo-amarelo (Astyanax altiparanae), um teleósteo de água doce

In freshwater fish with external fertilization, sperm sampling can be contaminated with urine, which triggers motility and gives rise to decreased fertilization success. The maintenance of freshwater fish in hyperosmotic conditions may reduce urine production and improve sperm quality. Thus, the aim of this work was to verify if acute exposure to various NaCl concentrations improves sperm quality in the yellowtail tetra Astyanax altiparanae. Spermiation was induced using a single dose of carp pituitary gland (5 mg kg-1) and the males were maintained at various NaCl concentrations: NaCl 0.00% (control), NaCl 0.45% (hypoosmotic), NaCl 0.9% (isosmotic) and NaCl 1.0% (hyperosmotic) for 6 h at 26 °C. Sperm was collected and verified for activation by urine and motility traits. At 0.00%, 0.45%, and 0.90%, the sperm was motile just after sampling, indicating activation by urine. Surprisingly, at hyperosmotic conditions, no activation was observed. Other sperm and motility parameters did not show any statistical differences, including sperm viability (P = 0.7083), concentration (P = 0.9030), total motility (P = 0.6149), VCL (curvilinear velocity; P = 0.1216), VAP (average path velocity; P = 0.1231) and VSL (straight-line velocity; P = 0.1340). Our results indicate that acute maintenance at hyperosmotic conditions eliminates sperm activation by urine and maintains sperm quality. Such a new procedure is interesting for both basic and applied sciences, including reproductive practice in fish.(AU)

Em peixes de água doce com fertilização externa, a amostragem de espermatozoides pode ser contaminada pela urina, o que desencadeia motilidade e gera menor sucesso na fertilização. A manutenção de peixes de água doce em condições hiperosmóticas pode reduzir a produção de urina e melhorar a qualidade do esperma. Assim, o presente trabalho foi delineado para verificar se a exposição aguda a várias concentrações de NaCl melhora a qualidade do esperma no tetra-amarelo Astyanax altiparanae. A espermiação foi induzida usando uma dose única de hipófise da carpa (5 mg kg-1) e os machos foram mantidos em várias concentrações de NaCl: NaCl 0,00% (controle), NaCl 0,45% (hipoosmótico), NaCl 0,9% (isosmótico) e NaCl 1,0% (hiperosmótico) por seis horas a 26 °C. O esperma foi colhido e verificado quanto à ativação por urina e traços de motilidade. Em 0,00%, 0,45%, 0,90% os espermatozóides eram móveis logo após a amostragem, indicando ativação pela urina. Surpreendentemente, em condições hiperosmóticas, nenhuma ativação foi observada. Outros parâmetros espermáticos e de motilidade não mostraram diferenças estatísticas, incluindo viabilidade espermática (P = 0,7083), concentração (P = 0,9030), motilidade total (P = 0,6149), VCL (Velocidade Curvilinear; P = 0,1216), VMD (Velocidade Média de Deslocamento; P = 0,1230) e VLR (Velocidade em linha Reta; P = 0,1340). Nossos resultados indicam que a manutenção aguda em condições hiperosmóticas elimina a ativação do esperma pela urina e mantém a qualidade do esperma. Esse novo procedimento é interessante para as ciências básicas e aplicadas, incluindo a prática reprodutiva em peixes.(AU)

Bovine semen thermoresistance tests and their correlation with pregnancy rates after fixed-time artificial insemination / Testes de termorresistência do sêmen bovino e sua correlação com taxas de prenhez após inseminação artificial em tempo fixo

The objective of the study was to determine whether or not there is a correlation between thermoresistance tests (TT) after semen thawing and pregnancy rate (PR) after fixed-time artificial insemination (FTAI). Four different TT were performed on ten samples used for AI; a rapid test (RTT) (30min / 46°C) and three slow tests (STT): STT1 (60min/38°C), STT2 (180min/38°C), and STT3 (300min/38°C). Two hundred and fifteen multiparous crossbred cows were submitted to FTAI under the following protocol: on day zero (d0) the animals received a P4 device +EB; on d7 PGF2α; on d8 P4 was removed and eCG+EC were administered; IATF was performed on d10. Three gestational diagnoses (G D) were performed on d40, d70 and d120. The mean sperm motility (%) in RTT and STTs were 19.84±6.13, 28.55±10.48, 17.62±5.87 and 8.63±3.46, respectively, and TP in the three DG 61.86%, 57.67%, and 55.81%, respectively. Through Person test a significant negative correlation (P< 0.05) was found between STT2 and PR at 60 days (r= -0.644) and between STT3 and all TPs (r= -0.774, -0.752, 0.748). It was concluded that TT parameters are not able to determine correlation between semen quality and TP.(AU)

O objetivo do presente estudo foi determinar se há ou não correlação entre testes de termorresistência (TT) após descongelamento do sêmen e taxa de prenhez (TP) após inseminação artificial em tempo fixo (IATF). Quatro diferentes TT foram realizados nas 10 amostras utilizadas para a IA; um teste rápido (RTT) (30min/46°C) e três testes lentos (STT): STT1 (60min/38°C), STT2 (180min/38°C) e STT3 (300min/38°C). Duzentas e quinze vacas cruzadas multíparas foram submetidas à IATF sob o seguinte protocolo: no dia zero (d0), os animais receberam um dispositivo de P4+EB; em d7, PGF2α; em d8, retirou-se P4 e eCG+EC administrados; no d10, foi realizada IATF. Três diagnósticos gestacionais (DG) foram feitos, em d40, d70 e d120. As médias de motilidade espermática (%) em RTT e STTs foram 19,84±6,13, 28,55±10,48, 17,62±5,87 e 8,63±3,46, respectivamente, e TP nos três DG 61,86%, 57,67% e 55,81%, respectivamente. Por meio do teste de Person, uma correlação negativa significativa (P<0,05) foi encontrada entre os resultados de STT2 e PR aos 60 dias (r=-0,644) e entre STT3 e todas TPs (r=-0,774, -0,752 e -0,748). Concluiu-se que parâmetros de TT não são capazes de determinar correlação entre qualidade do sêmen e TP.(AU)

Reference values (RV), MNS and MTS for morphological characteristics of normal and abnormal human spermatozoon describing Teratozoospermic Index (TZI) & Sperm Deformity Index (SDI) in Sudanese infertile couples using CASA System / Valores de referencia (RV), MNS y MTS para las características morfológicas de espermatozoides humanos normales y anormales que describen el Índice Teratozoospérmico (TZI) y el Índice de Deformidad del Esperma (SDI) en parejas infértiles de Sudán que utilizan el sistema CASA

A scientific determination of fertilization ability of the sperm can be made by motility, viability and morphological examinations that are called as potential fertility criteria. Some previous studies documented only the total morphological defect rates without including the morphologic subjects. As it is recorded in many studies, the success of IUI, ICSI and IVF depends mainly on the quality of the collected semen. 1) Determine and classify the common morphological defects and rates of sperms. 2) Analyze the effect of age on the sperm abnormalities and to assess the impact of environment on changing normal sperm characteristics. 3) Determine the common types of semen abnormalities (Asthenoterato-zoospermia, Asthenozoospermia, Azoopyospermia, Azoospermia, Hypospermia, Oligoasthenoteratozoospermia, Oligoasthenozoopyospermia, Oligoasthenozoospermia and Teratozoospermia). 4) Determine the range of teratozoospermic index (TZI) and sperm deformity index (SDI). A prospective case series study for 566 men of infertile couples who were requesting semen analysis was applied to those came to the ULTRALAB laboratory center main campus and ULTRALAB branch of the Sudan Assisted Reproduction Center (SARC), Khartoum, Sudan. Data collection was done for 20 months from April 1st, 2010 to December 31st, 2011. analysis of semen samples was done using Computer Assisted Semen Analysis (CASA). 1) The study demonstrates that there are differences in normality and abnormality of sperm morphology parameters of sperm between Sudanese population and other studies including WHO standards. 2) SDI and TZI were higher in overall semen as well as in abnormal semen samples. 3) Common semen abnormalities were oligoasthenoteratozoospermia, asthenoteratozoospermia and asthenozoospermia. There are differences in sperm morphology of both normal and abnormal parameters between Sudanese population and other studies including WHO standards.

Se realizó una determinación científica de la capacidad de fertilización de los espermatozoides mediante motilidad, viabilidad y exámenes morfológicos denominados criterios de fertilidad potencial. Algunos estudios previos documentan solamente el total de las tasas de defectos morfológicos sin incluir a los sujetos morfológicos. Como se registra en muchos estudios, el éxito de IUI, ICSI y FIV depende principalmente de la calidad del semen recolectado. Los objetivos de este trabajo fueron: 1) Determinar y clasificar los defectos morfológicos comunes y las tasas de espermatozoides; 2) Analizar el efecto de la edad en las anomalías de los espermatozoides y evaluar el impacto del medio ambiente en el cambio de las características normales de los espermatozoides; 3) Determinar los tipos comunes de anomalías en el semen (astenoterato-zoospermia, astenozoospermia, azoopiepermia, azoospermia, hipospermia, oligoastenoteratozoospermia, oligoastenozoopermia, oligoastenozoospermia y teratozoospermia); 4) Determinar el rango del índice teratozoospérmico (TZI) y el índice de deformidad del esperma (IDE). Se realizó un estudio prospectivo de series de casos en 566 hombres, de parejas infértiles, que solicitaban análisis de semen en laboratorios ULTRALAB y la sucursal de ULTRALAB del Centro de Reproducción Asistida de Sudán (SARC), Jartum, Sudán. La recolección de datos se realizó durante 20 meses, desde el 1 de abril de 2010 hasta el 31 de diciembre de 2011. El análisis de las muestras de semen se realizó mediante el análisis de semen asistido por computadora (CASA). Existen diferencias en la normalidad y anormalidad de los parámetros de la morfología de los espermatozoides entre la población sudanesa y otros estudios, incluidas las normas de la Organización Mundial de la Salud. Los parámetros SDI y TZI fueron más altos en el semen general, así como en muestras de semen anormales. Las anomalías comunes del semen fueron oligoastenatoatoospermia, astenoteratozoospermia y astenozoospermia. Existen diferencias en la morfología de los espermatozoides de los parámetros normales y anormales entre la población sudanesa y otros estudios que incluyen las normas de la Organización Mundial de la Salud.

Análise da integridade funcional dos espermatozoides de Tayassu pecari por diferentes soluções hiposmóticas e osmolaridades / Analysis of the functional integrity of Tayassu pecari sperm by different hyposmotic solutions and osmolarities

Queixada (Tayassu pecari) é um mamífero neotropical, classificado como vulnerável devido à caça e à destruição de seu habitat. Em razão das dificuldades na reprodução em cativeiro, técnicas de reprodução assistida podem ser aplicadas em programas de repovoamento da espécie. Dessa forma, objetivou-se identificar o melhor protocolo de teste hiposmótico (HOST) para avaliar a integridade funcional dos espermatozoides de queixada. O sêmen de quatro machos adultos foi coletado com auxílio de eletroejaculador após contenção física e protocolo de sedação e anestesia. O sêmen foi avaliado quanto às características macro e microscópicas e diluído nas seguintes soluções hiposmóticas: água destilada (0mOsmol/L), sacarose (50, 100, 150mOsm/L) e frutose (50, 100, 150mOsm/L). Cada amostra foi incubada em duplicata, e uma sofreu fixação em solução de citrato de sódio formolizado a 4%. Duzentos espermatozoides foram avaliados por amostra e classificados em reativos ou não ao HOST. Todas as soluções testadas foram semelhantes em identificar o percentual de espermatozoides reativos, independentemente de a amostra ser ou não fixada (P>0,05). Dessa forma, pode-se usar água destilada como HOST por este apresentar resultados similares e por ser um teste mais barato.(AU)

White-lipped peccary (Tayassu pecari) is a neotropical mammal classified as vulnerable due to overhunting and habitat destruction. Due to the low reproduction success in captivity, assisted reproduction techniques can be used in re-stocking programs. Therefore, we aimed to identify the best hyposmotic swelling test (HOST) to evaluate the functional integrity of peccary sperm. Semen samples of four adult males were collected with the aid of an electroejaculator after physical contention, sedation and anesthesia protocols. The semen was evaluated for macro and microscopic characteristics and diluted in the following hyposmotic solutions: distilled water (0mOsmol/L), sucrose (50, 100, 150mOsm/L) and fructose (50, 100, 150mOsm/L). Each sample was incubated in duplicate and one was fixed in 4% formalized sodium citrate solution. Two hundred spermatozoa were evaluated from each sample and classified as reactive or not- reactive to HOST. There were similar proportions of reactive and functional sperm regardless of whether the sample was fixed or not fixed (P> 0.05). Therefore, distilled water can be used as HOST to evaluate the functional integrity of white-lipped peccary sperm because it shows similar results and it is a cheaper test.(AU)

Use of infrared thermography to evaluate the inflammatory reaction in cat testis after intratesticular injection of 0. 9% NaCl or 20% CaCl2 with 1% lidocaine / O uso da termografia infravermelha para avaliar a reação inflamatória após a injeção intratesticular de NaCL 0, 9 % e cloreto de cálcio 20% com lidocaína 1%

Due to the doubts and questions about the inflammatory reaction caused by chemical castration, this study aimed to use infrared thermography to detect, evaluate and monitor the inflammatory reaction caused by the intratesticular injection of calcium chloride (CaCl2) 20% with lidocaine 1%. For this, thermographic measurements were taken before (M0), 10 minutes (M1), 1 and 6 hours (M2 and M3), for 7 consecutive days (M4 to M10), at 15 (M11), 30 (M12) and 60 (M13) days after intratesticular injection. Additionally, changes to testicular tissue and effects over spermatogenesis were evaluated by andrological exam before (M0) and 60 days (M13) after intratesticular injection. All cats were orchiectomized at M13, and testicles were submitted to histological analysis. CaCl2 (20%) with lidocaine (1%) administration produced testicular tissue damage and interfered with the spermatogenesis in 70% of treated cats without exacerbating the inflammatory reaction or impairing the cat's welfare. It was concluded that thermographic evaluation is a useful, efficient, easy and quick method to diagnose and monitor cat testicular inflammatory reactions.(AU)

Devido a constantes dúvidas e questionamentos sobre a reação inflamatória ocasionada pela castração química, este estudo objetivou o uso da termografia infravermelha para detectar, avaliar e monitorar a reação inflamatória causada pela injeção intratesticular de cloreto de cálcio (CaCl 2 ) 20% associada à lidocaína 1%. Para isso, medidas termográficas foram aferidas antes (M0), 10 minutos (M1), uma e seis horas (M2 e M3), por sete dias consecutivos (M4 a M10), aos 15 (M11), 30 (M12), e 60 (M13) dias após injeção intratesticular, nos grupos tratado e controle. Todos os gatos foram orquiectomizados no M13, e os testículos submetidos à análise histológica. A injeção CaCl 2 a 20% associada com lidocaína a 1% produziu lesão testicular e interferiu na espermatogênese de 70% dos gatos tratados, sem exacerbar a reação inflamatória ou prejudicar o bem-estar do animal. Foi concluído que a avaliação termográfica é uma ferramenta útil, eficiente, rápida e fácil para o diagnóstico e o monitoramento da reação inflamatória em gatos.(AU)

Relación entre los antioxidantes y la calidad seminal / Relationship between antioxidants and semen quality

Los suplementos dietarios tales como vitaminas, minerales y antioxidantes mejoran la ingesta de nutrientes. Recientemente se ha descrito que, especialmente aquellos que contienen altas propiedades antioxidantes también mejoran la capacidad fértil. Se presenta el caso de un voluntario de 37 años con posible infertilidad masculina y se desea determinar el efecto del consumo de antioxidantes sobre la calidad seminal. Se realizó evaluación de los parámetros seminales convencionales y funcionales antes y después del uso del suplemento dietario Male Fertility. Se observó que el uso del suplemento dietario incrementó la concentración espermática, el potencial de membrana mitocondrial alto y la capacidad antioxidante del semen; además disminuyó la producción de 1as reactivas de oxígeno, la lipoperoxidación y la fragmentación de la cromática espermática. El suplemento dietario Male Fertility contiene altas concentraciones de vitamina A, C, E, B2, B3, B12, folato, zinc, selenio, acetil L-carnitina, coenzima Q10, L-metionina y licopeno. Se ha descrito que la ingesta de cada uno de estos compuestos tiene efectos positivos sobre la calidad seminal. El reporte de este caso permitió observar que el uso de suplementos dietarios ricos en vitaminas y antioxidantes puede mejorar la calidad seminal a través de la disminución del efecto adverso de las especies reactivas del oxígeno y por el incremento de las moléculas antioxidantes en el plasma seminal(AU)

Dietary supplements such as vitamins, minerals and antioxidants improve nutrient intake. Recently it has been described that, especially those containing high antioxidant properties also improve fertility. We report here the case of a 37-year-old volunteer with possible male infertility and we want to determine the effect of antioxidant consumption on semen quality. Evaluation of the conventional and functional seminal parameters was performed before and after the use of the Male Fertility dietary supplement. The use of this supplement was observed to increased the sperm concentration, the mitochondrial membrane potential and the antioxidant capacity of the semen. In addition, the production of oxygen reactants, lipoperoxidation and fragmentation of the spermatic chromatin decreased. The dietary supplement Male Fertility contains high concentrations of vitamin A, C, E, B2, B3, B12, folate, zinc, selenium, acetyl L-carnitine, coenzyme Q10, L-methionine and lycopene. The ingestion of each of these compounds has been described to have positive effects on seminal quality. The report of this case allowed to observe that the use of dietary supplements rich in vitamins and antioxidants can improve the seminal quality through the decrease of the adverse effect of the reactive oxygen species and by the increase of the antioxidant molecules in the seminal plasma(AU)

Efecto de la disminución del índice de masa corporal sobre la calidad seminal / Effect of Decreasing Body Mass Index Seminal Quality

El estilo de vida saludable incluye una alimentación saludable, un peso adecuado y en casos de exceso de peso, su pérdida. Estos son nuevos factores asociados a la fertilidad. Se presenta el caso de un voluntario fértil aparentemente sano que se sometió a un régimen de pérdida de peso. Con el propósito de evaluar el efecto de la pérdida de peso sobre la calidad seminal se realizó un análisis retrospectivo de los parámetros seminales convencionales y funcionales según los lineamentos establecidos en el Manual de análisis seminal de la Organización Mundial de la Salud. Los parámetros seminales evaluados fueron: volumen seminal, concentración espermática, concentración espermática total, movilidad, viabilidad, potencial de membrana mitocondrial e índice de fragmentación del ADN. La reducción del índice de masa corporal (19 por ciento) incrementó los parámetros espermáticos: volumen seminal (400 por ciento), concentración espermática (96 por ciento), concentración espermática total (220 por ciento), viabilidad (38 por ciento), movilidad (122 por ciento), potencial de membrana mitocondrial (40 por ciento) y además disminuyó el índice de fragmentación del ADN en un 71 por ciento. A partir de estas observaciones se infiere que la obesidad está inversamente relacionada con la calidad seminal. Los resultados obtenidos en este caso nos inducen a recomendar que los hombres con sobrepeso y obesidad deben perder peso con el fin de mejorar su calidad seminal(AU)

Healthy lifestyle includes healthy diet, adequate weight and in cases of excess weight, its loss. These are new factors associated with fertility. We present the case of an apparently healthy fertile volunteer who underwent a weight loss regimen. In order to evaluate the effect of weight loss on seminal quality, a retrospective analysis of the conventional and functional seminal parameters was carried out according to the guidelines established in the Manual of Seminal Analysis of the World Health Organization. Semen parameters evaluated were seminal volume, sperm concentration, total sperm concentration, mobility, viability, mitochondrial membrane potential, and DNA fragmentation index. The reduction of the body mass index (19 percent) increased the sperm parameters: seminal volume (400 percent), sperm concentration (96 percent), total sperm concentration (220 percent), viability (38 %), mobility (122 percent), mitochondrial membrane potential (40 percent) and decreased the DNA fragmentation rate by 71 percent. From these observations, it is inferred that obesity is inversely related to seminal quality. The results obtained in this case lead us to recommend overweight and obese men should lose weight in order to improve their seminal quality(AU)

Effect of sperm selection techniques in frozen/thawed cat spermatozoa on sperm motility analyzed by CASA system / Efecto de técnicas de selección espermática en espermatozoides congelados/descongelados de gato sobre la motilidad espermática analizada mediante sistema CASA

SUMMARY: Freeze/thawing process reduces sperm survival and fertilizing ability of cat spermatozoa, with sperm motility being the most sensitive sperm parameter altered, due to cryo-damage. In this context, swim-up and density gradient processing methods can help to recover high motile and normal spermatozoa. Maximizing the use of frozen semen sample is essential, especially in endangered felids or high value cats in which sample size, number of samples or access to semen collection is reduced. To our knowledge, there is no previous report describing an in depth analysis of sperm motility improvement, after sperm selection techniques in frozen cat semen. Accordingly, we evaluated the effect of percoll gradient (PG) and swim up (SU) sperm selection techniques on sperm motility parameters and sperm recovery rate in frozen/thawed spermatozoa of domestic cat. Next, we evaluated the individual effect of the cat over sperm motility after PG sperm selection of frozen/thawed spermatozoa. SU and PG improved significantly all sperm motility parameters of frozen/thawed cat spermatozoa compared to simple washing. However, PG allows better sperm recovery from the original frozen sample and works mostly homogeneously among individual cats. This new information could help to maximize the use of frozen semen in endangered felids or high value domestic cats for its subsequent application on in vitro fertilization and artificial insemination.

RESUMEN: El proceso de congelación/descongelación reduce la sobrevivencia espermática y la habilidad para fertilizar en los espermatozoides de gato, siendo la motilidad espermática el parámetro más sensiblemente alterado debido al daño por frío. En este contexto, los métodos de procesamiento de swim-up y gradiente de densidad pueden ayudar a recuperar los espermatozoides normales y de alta motilidad. Maximizar el uso de una muestra de semen congelado es esencial, especialmente en felinos amenazados o en gatos de alto valor en los cuales el tamaño de muestra, número de muestras o el acceso a la colecta de semen son reducidos. Para nuestro conocimiento, no hay reportes previos que describan un análisis profundo del mejoramiento de la motilidad luego de técnicas de selección espermática en semen congelado de gato. De acuerdo a esto, evaluamos el efecto de las técnicas de selección espermática gradiente de percoll (PG) y swim up (SU) sobre los parámetros de motilidad y porcentaje de recuperación de espermatozoides congelados/descongelados de gato doméstico. Luego, evaluamos el efecto individual del gato sobre la motilidad espermática luego de la selección espermática con PG en espermatozoides congelados/descongelados. SU y PG mejoraron significativamente todos los parámetros de motilidad espermática de los espermatozoides congelados/descongelados comparado con el lavado simple. Sin embargo, PG permitió una mejor recuperación de espermatozoides desde la muestra congelada original y funcionó en su mayoría de manera homogénea entre los gatos individualmente. Esta nueva información puede ayudar a maximizar el uso del semen congelado en felinos amenazados o en gatos de alto valor para su posterior aplicación en fecundación in vitro e inseminación artificial.

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.

La importancia clínica del ADN espermático en el análisis seminal cotidiano / The clinical importance of spermatic DNA in seminal analysis

El objetivo de este reporte es resaltar la importancia de las pruebas funcionales para evaluar la calidad seminal durante el análisis seminal de rutina. Se analizó mediante citometría de flujo el potencial mitocondrial, la integridad de la membrana espermática, la producción de especies reactivas del oxígeno y la suceptibilidad de fragmentación del ADN espermatico, en un paciente que consultó por infertilidad al Grupo Reproducción de la Universidad de Antioquia. Se encontró una producción basal de especies reactivas del oxígeno junto con una actividad mitocondrial alto y un porcentaje elevado de espermatozoides con su membrana plasmatica integra, siendo estos indicadores de una buena calidad espermática; no obstante, se observó una elevada suceptibilidad de fragmentación del material genético (DFI=42%). A pesar que un hombre aparentemente manifiesta características espermáticas de un individuo sano puede que la calidad del material genético de sus espermatozoides esté incidiendo sobre su éxito reproductivo.

The aim of this report is highlighting the importance of functional tests to evaluate semen quality during routine semen analysis. Mitochondrial function, membrane integrity, intracellular reactive oxygen species production and sperm DNA fragmentation were evaluated by flow cytometry in a infertil patient from Reproduction Group at the University of Antioquia.A basal production of reactive oxygen species and mitochondrial activity, and a high percentage of sperm with integrated plasma membrane was found, all indicators of good sperm quality; however, a high susceptibility of genetic material fragmentation (DFI=42%) was observed. Although a men apparently manifested sperm characteristics of a healthy individual may that the quality of genetic material in the spermcells is impacting on their reproductive success.

Efeito da radiação vermelha de baixa intensidade sobre o sêmen canino criopreservado / Effect of low intensity red light on the canine semen cryopreservation

O objetivo geral deste trabalho foi analisar o efeito da radiação vermelha de baixa intensidade sobre alguns parâmetros cinéticos do espermatozoide canino criopreservado. Ejaculados de oito cães adultos foram centrifugados, rediluídos em meio tris-gema de ovo com 6% de glicerol, e, posteriormente, fracionados em: T1: incidência de radiação vermelha (660 NM) (Fisioled - Mmoptics - 100mW) por 60 segundos, antes do resfriamento e após a descongelação; T2: incidência somente antes do resfriamento; T3: incidência somente após a descongelação; e T4: sem incidência. Após a descongelação, as amostras foram submetidas ao TTR utilizando-se Sperm Class Analyzer(r). No TTR0, TTR60 e TTR90, não houve diferença entre as variáveis analisadas pelo CASA. Somente no TTR30 os efeitos da incidência da radiação vermelha foram evidentes e significativos em T1 e T2; T1 resultou em baixa MT (12,5 + 10,6%) e T2 determinou o melhor resultado de MT 40,3 + 26,1%. De forma similar T1 apresentou maior número de espermatozoides estáticos (77,5±28,9%) em relação ao T2 (50,6±28%). Concluiu-se que a dupla incidência de radiação vermelha de baixa intensidade antes do resfriamento e após a descongelação teve efeito deletério sobre a motilidade do espermatozoide canino, expressa principalmente aos 30 minutos após descongelação.

The aim of this study was to analyze the effect of low intensity red light on some kinetic parameters of cryopreserved canine sperm. Ejaculates from 08 adult dogs were centrifuged, diluted in Tris-egg yolk with 6% glycerol, and subsequently separated into: T1: incidence of red light (660 nm) (Fisioled -MMOptics - 100mW) for 60 seconds before the cooling and after thawing, T2: just before cooling, T3: only after thawing, and T4: no incidence. After thawing the samples were subjected to TTR using Sperm analyzer(r). In TTR0, TTR60 and TTR90 there were no differences between the variables analyzed by CASA. Only in TTR30 the effects of the incidence of red light were visible and significant in T1 and T2. T1 resulted in low MT (12.5 ±10.6%) and T2 determined the best result of MT 26.1%±40.3. Similarly T1 showed a higher number of static spermatozoa (77.5±28.9%) compared to T2 (50.6±28%). We concluded that the double incidence of low intensity red light, before cooling and after thawing, had a deleterious effect on canine sperm motility expressed at 30 minutes after thawing.

Metodologia da análise seminal para pacientes azoospérmicos no laboratório fleury / Methodology of seminal analysis for azoospermicos patients in the laboratório fleury

A azoospermia é definida como a ausência de espermatozoide no líquido seminal ejaculado pelo homem depois de aplicada a técnica de centrifugação em pelo menos duas amostras. Dada a importância de um diagnóstico correto da análise seminal para os casais, toda amostra que não apresentar espermatozoides no exame a fresco deve seguir em avaliação laboratorial. Com isso, o presente estudo tem como objetivo analisar os resultados de centrifugação de uma alíquota do sêmen ejaculado ou de todo o volume ejaculado de pacientes com diagnóstico de azoospermia para determinar qual o melhor método a ser empregado na análise seminal para esse grupo de pacientes.

The azoospermia is defined as the absence of sperm in the ejaculate by the seminal fluid man after centrifugation technique conducted in at least two samples. Given the importance of a correct diagnosis of the seminal analysis for couples, all sample no sperm present in fresh examination should follow in laboratory tests. Thus the present study aims to analyze the results of a spin rate of ejaculate or all of the ejaculate volume of patients with azoospermia to determine the best method to be used in semen analysis for this group of patients.

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.

Clinical relevance of routine semen analysis and controversies surrounding the 2010 World Health Organization criteria for semen examination

Semen analysis is the corner stone of infertility evaluation as it provides information on the functional status of the seminiferous tubules, epididymis and accessory sex glands. The methods on how the human semen should be evaluated are provided by the World Health Organization, which periodically releases manuals that include specific protocols and reference standards. In 2010, the WHO published new criteria for human semen characteristics that were markedly lower than those previously reported. In this review initially it is discussed the limitations of semen analysis as a surrogate measure of a man’s ability to father a pregnancy. Secondly, it is analyzed methodology issues that could explain why the newly released reference values were different from those earlier reported. Thirdly, it is speculated on the likely effects of the 2010 WHO criteria in the management of male infertility. Due to the several inherent limitations of semen analysis as a surrogate marker of male infertility, physicians should exercise caution when interpreting results. A template for semen analysis reports that incorporates the distribution of the semen characteristics of recent fathers in centiles rather than solely the minimum thresholds could aid clinicians to better understand how a given patient results compare with the reference population. Importantly, a male infertility evaluation must go far beyond a simple semen analysis, as it has to be complemented with a proper physical examination, a comprehensive history taking, and relevant endocrine, genetic, and other investigations.

Establishing the hypoosmotic swelling test for sperm analysis in collared peccaries (Pecari tajacu) / Estabelecimento do teste hiposmótico para a análise de sêmen de catetos (Pecari tajacu)

Soluções hiposmóticas com diferentes concentrações (0, 50, 100, 150, 200mOsm/L) foram testadas para a avaliação funcional da membrana espermática de catetos (n=13). Foi verificado que o número de espermatozoides reagidos diminuía (P<0,05) de acordo com o aumento da osmolaridade do meio. A maior porcentagem (71,8%) de espermatozoides reagidos, bem como a menor variação nas respostas osmóticas, foi detectada com o uso de água destilada (0mOsm/L) (P<0,05), a qual também apresentou a menor variação nos resultados, de acordo com os erros padrão verificados. Em conclusão, a água destilada aparenta ser uma solução adequada para o uso no teste hiposmótico para sêmen de catetos.

Diferencias del estado redox entre espermatozoides y plasma seminal en adultos jóvenes / Difference of redux status between spermatozoa and seminal plasma in young adults

Introducción: Las especies reactivas del oxígeno y el estrés oxidativo desempeñan un papel fundamental en la infertilidad masculina. Sin embargo, estas especies oxidantes también han sido asociadas con los procesos de capacitación de los gametos masculinos cuando son generadas a bajos niveles y de manera controlada. Actualmente los biomarcadores redox son introducidos en el diagnóstico clínico de la infertilidad masculina en el mundo, como una herramienta complementaria a los parámetros del espermiograma. Sin embargo, en Cuba, esta metodología aún no se encuentra extendida a los servicios de salud. Objetivo: El objetivo del presente trabajo fue caracterizar el estado redox de espermatozoides y el líquido seminal de sujetos aparentemente sanos, a través de la determinación de una serie de biomarcadores de estrés oxidativo. Los niveles de malonildialdehído, la actividad de las enzimas antioxidantes superóxido dismutasa y catalasa, así como los niveles de glutatión reducido, el potencial de peroxidación y la capacidad reductora fueron determinados mediante técnicas espectrofotométricas. Métodos: Se analizaron 40 muestras de semen de sujetos aparentemente sanos, las cuales fueron obtenidas mediante masturbación sin el empleo de lubricantes y con al menos tres días de abstinencia eyaculatoria. En el estudio se incluyeron sujetos de 20 a 35 años, aparentemente sanos según exámenes de laboratorio clínico y con paternidad probada. Resultados: Los resultados demostraron que existen diferencias significativas (p< 0,05) entre los marcadores evaluados en el líquido seminal con respecto al espermatozoide, lo cual sugiere la existencia de un estado redox diferenciado entre ambos compartimentos. Conclusiones: Estos hallazgos demuestran la necesidad de evaluar el nivel de estrés oxidativo tanto en las células sexuales como en el fluido que las contiene. Ello contribuirá, sin lugar a dudas, a un diagnóstico más eficaz e integral de la capacidad fértil del hombre

Background: Reactive oxygen species and oxidative stress play a fundamental role in male infertility. However, these oxidizing species have also been associated with the process of capability of male gametes when they are generated at low levels and in a controlled manner. Currently, redox biomarkers are introduced in the clinical diagnosis of male infertility in the world as a complementary tool to the spermiogram parameters. However, in Cuba, this methodology is not yet extended to health services. Objective: The objective of this study was to characterize the redox status of spermatozoa and seminal fluid of apparently healthy subjects through the identification of a number of biomarkers of oxidative stress. Methods: 40 samples of semen of apparently healthy subjects were analyzed, which were obtained by masturbation without the use of lubricants and with at least 3 days of ejaculatory abstinence. The study included subjects from 20 to 35 years of age, who were apparently healthy according to both laboratory tests and paternity test results. Results: The results showed that there are significant differences (p<0, 05) between the markers evaluated in the seminal fluid and the spermatozoon which suggests the existence of a differentiated redux status between the two compartments. Conclusions: These findings demonstrate the need to evaluate the level of oxidative stress in both sexual cells and the fluid that contains them. It will contribute, with no doubt, to a more effective and comprehensive diagnosis of man's fertility