Effects of chelating calcium in cryopreservation extender on frozen-thawed dog semen / [Efeitos da quelação de cálcio no meio de criopreservação no sêmen descongelado de cão]

We evaluated the effect of reducing free calcium in the cryopreservation medium, using the calcium chelator ethylene diamine tetracetic acid (EDTA) at 0.3% and 0.5% concentrations. Three male mixed breed dogs were subjected to semen collection by digital manipulation (n=16). Each ejaculate was divided in three aliquots, and each one was diluted in TRIS-glucose-egg yolk extender with 6% glycerol and 0.5% Equex STM Paste® (TGE, control); and added with 0.3% EDTA (EDTA 0.3) or 0.5% EDTA (EDTA 0.5). Calcium concentration reduced in EDTA 0.3 and all the calcium ions were chelated in EDTA 0.5. The EDTA addition did not affect sperm morphology or plasma membrane integrity; however, by removing all free calcium (EDTA 0.5), the sperm motility reduced (64.7% in TGE and 45% in EDTA 0.5; p<0.05). Acrosome integrity and sperm binding ability were not improved by calcium chelation. The failure to prevent the premature AR may explain why sperm longevity was not affected by calcium removal. Thus, the partial or complete calcium removal, through EDTA addition, is not able to prevent acrosomal damage or premature acrosomal reaction, and therefore does not improve the dog sperm binding ability.(AU)

Avaliou-se o efeito da redução do cálcio livre no meio de congelamento, usando-se o quelante de cálcio etilenodiaminotetracético (EDTA) a 0,3% e 0,5%. Três cães machos sem raça definida foram submetidos à coleta de sêmen por manipulação digital (n=16). Cada ejaculado foi diluído em diluidor controle com TRIS-glicose - gema de ovo (TGE, controle), ou em diluidor TGE enriquecido com 0,3% (EDTA 0,3) ou 0,5% de EDTA (EDTA 0,5). A concentração de cálcio reduziu no meio EDTA 0,3, e todos os íons de cálcio foram quelados no meio EDTA 0,5. A adição do EDTA e a consequente quelação do cálcio não afetaram a morfologia espermática ou a integridade da membrana plasmática, no entanto, ao remover todo o cálcio do meio (EDTA 0,5), a motilidade espermática se reduziu (64,7% no TGE e 45% no EDTA 0,5; P<0,05). A integridade do acrossoma e a capacidade de ligação do espermatozoide não melhoraram com a quelação do cálcio. Apesar da influência da concentração de cálcio sobre a motilidade espermática após o descongelamento, a falha em prever a reação acrossomal prematura pode explicar por que a longevidade espermática não foi afetada pela remoção do cálcio no meio. Dessa forma, a remoção parcial ou total do cálcio, por meio da adição de EDTA, não é capaz de prevenir o dano no acrossoma ou a reação acrossomal prematura e, portanto, não aumenta a capacidade do espermatozoide de se ligar ao oócito.(AU)

Sperm agglutinating activity of Saccharomyces cerevisiae and Candida albicans as a potential causative factor of infertility in mice (Mus musculus)

@#<p style="text-align: justify;"><strong>BACKGROUND AND OBJECTIVE:</strong> Vaginal yeast infections in women are usually caused by Candida albicans and, to a lesser extent, by Saccharomyces cerevisiae. Studies on C. albicans have shown that it can cause sperm agglutination which can lead to lowered fertility. This study was conducted to compare the effect of S. cerevisiae and C. albicans on the fertility of ICR mouse (Mus musculus) through sperm agglutination.</p><p style="text-align: justify;"><strong>METHODOLOGY:</strong> Sperm agglutinating activity was examined by mixing different concentrations of S. cerevisiae (10, 10°, and 10 CFU/mL) and C. albicans (10", 10°, and 10 CFU/mL) separately with semen from male mice of ICR strain. Determination of the effect of S. cerevisiae and C. albicans on the fertility outcome of female mice was done by intravaginal inoculation of 20 uL of 104, 106, and 108 CFU/ml of the two yeast organisms and later allowed to mate.</p><p style="text-align: justify;"><strong>RESULTS AND CONCLUSION:</strong> The study showed a statistically significantly higher percent sperm agglutination by S. cerevisiae than C. albicans at 10* CFU/ml but no difference was observed at 10° and 10 CFU/ml. No significant difference was observed in the number of sperm per agglutinate between the two yeast species at a=0.05. The concentration that exhibited the highest percentage of agglutinated sperm is 10° CFU/mL for both yeast. The most frequent type of agglutination observed in S. cerevisiae is the mixed type, while head-to-head type is most frequent in C. albicans. Both yeasts were able to cause a decline in the number of births in mice starting at 10 CFU/ml. While sperm agglutination could be one of the reasons for the infertility observed in mice, there may be other processes, mechanisms, and/or activities that could contribute to such an outcome.</p>

Insights into the role of cervical mucus and vaginal pH in unexplained infertility / Sobre o papel do muco cervical e do PH vaginal na gênese da infertilidade inexplicada

Unexplained infertility diagnosis is made in the presence of a normal semen analysis when tubal patency and normal ovulatory function are established. Among several potential causes, unexplained infertility could be attributed to vaginal pH and cervical mucus abnormalities. Although the vaginal canal and the cervix generally function as effective barriers to sperm, and although the production of mucus is essential to transport them from the vagina to the uterine cavity, these factors receive little attention in the investigation of couples with unexplained infertility. A substantial reduction in sperm number occurs as they transverse the cervix. From an average of 200 to 300 million sperm deposited in the vagina, only a few hundred achieve proximity to the oocyte. Given this expected high spermatozoa loss, a slight modification in cervical mucus may rapidly transform the cervix into a "hostile" environment, which, together with changes in vaginal environment and cervix structure, may prevent natural conception and be a cause of infertility. In this review, we discuss the physiological role of the vaginal pH and cervical mucus in fertility, and describe several conditions that can render the cervical mucus hostile to sperm and therefore be implicated in the pathophysiology of unexplained infertility.

RESUMO O diagnóstico de infertilidade inexplicada baseia-se na presença de espermograma normal, constatadas também permeabilidade tubária e função ovulatória normais. Entre as várias causas potenciais de infertilidade inexplicada, a presença de muco cervical e pH vaginal anormais devem ser consideradas. Embora a produção adequada de muco cervical seja essencial para o transporte dos espermatozóides da vagina para a cavidade uterina, e tanto o canal vaginal quanto o colo do útero desempenham função importante como barreira à passagem dos espermatozóides, estes fatores recebem pouca atenção na investigação de casais com infertilidade inexplicada. Uma redução substancial do número de espermatozoides ocorre à medida que estes percorrem o trato reprodutivo feminino. Partindo de cerca de 200 a 300 milhões de espermatozoides depositados na vagina, apenas algumas centenas alcançam a proximidade do oócito. Alteracões do muco cervical podem rapidamente transformar o colo do útero num ambiente hostil, que em conjunto com alterações no ambiente vaginal e da estrutura de colo do útero, podem apresentar-se condicões impedientes para a concepção natural; desse modo, convertem-se em causa de infertilidade. Nesta revisão, discutimos o papel fisiológico do pH vaginal e do muco cervical na fertilidade, descrever várias condicões que podem tornar o muco cervical hostil aos espermatozoides e, por fim analisamos como estes fatores interferem na fisiopatologia da infertilidade inexplicada.

Determinación del espermatocrito y efecto del volumen de la dosis seminante sobre la fertilidad en yamú (Brycon amazonicus) / Determination of spermatocrit and effect of volume of insemination dose on fertility in yamú (Brycon amazonicus)

El yamú (Brycon amazonicus . Spix & Agassiz 1829) es un pez omnívoro de rápido crecimiento y carne de alta calidad y aceptación comercial. Para estandarizar el espermatocrito (SPCTO), 23 muestras de semen fueron centrifugadas a 14000 g durante 1, 3, 5, 7 ó 10 min. El tiempo de centrifugación tuvo un efecto significativo sobre el valor del espermatocrito (SPCTO) (p<0.05). Para conocer la concentración espermática (CE) durante la época reproductiva y su relación con el SPCTO, la espermiación de 40 machos fue inducida con extracto de hipófisis de carpa (4.0 mg.kg -1). La CE fue evaluada mediante cámara de Neubauer (dilución 1:1200) y el SPCTO por centrifugación a 14000 g durante 5 min. La relación entre SPCTO y CE fue positiva y altamente significativa (y= 1.69 + 0.59x; r2= 0.74; p<0.0001), pero no se observaron diferencias estadísticas (p>0.05) entre los meses evaluados (marzo, abril y mayo). Finalmente, para determinar el efecto del volumen de la dosis seminante sobre la fertilidad, 2 g de oocitos fueron seminados con 3, 15, 45, 90 o 180 mL de semen (n= 12), cuya CE fue previamente ajustada para contener 50000 espermatozoides (sptz)/oocito, utilizando como diluyente plasma seminal homólogo. Los porcentajes de fertilidad fueron 83, 86, 86, 85 y 87 por ciento, respectivamente (p>0.05).

Evaluation of fish sperm concentration using two counting chambers

A concentraçäo espermática do sêmen do peixe anostomídeo Leporinus elongatus (Valenciennes, 1849) foi estimada nas câmaras de Makler e de Neubauer, tendo-se obtido, respectivamente, 10,9 ± 2,8 ´ 10(9) e 11,9 ± 3,7 ´ 10(9) espermatozóides/ml de sêmen sem que ocorresse diferença significativa entre essas médias (P > 0,05). A estimativa da concentraçäo espermática do sêmen na câmara de Makler apresentou vantagens em relaçäo à da câmara de Neubauer. A contagem na câmara de Makler pôde ser feita sem a necessidade de se esperar pela deposiçäo dos espermatozóides no seu fundo. A baixa diluiçäo do sêmen (1:1.000) necessária para se estimar a concentraçäo espermática pode ser considerada também como vantagem pois altas diluiçöes (como na câmara de Neubauer) säo fontes consideráveis de erro. Dessa forma, a câmara de Makler pode ser utilizada na determinaçäo da concentraçäo espermática do sêmen de peixes e, possivelmente, de outros vertebrados, incluindo a de animais domésticos

Efeito da concentraçäo espermática e do número de inseminaçöes artificiais sobre a fertilidade de éguas inseminadas com sêmen fresco diluído / Effects of sperm concentration and number of artificial inseminations on fertility of inseminated mares with diluted fresh semen

Sessenta e duas fêmeas eqüinas (tipo militar) foram distribuídas, ao acaso, em dois grupos experimentais para estudar o efeito da concentraçäo espermática (200I 10(6) e 400I 10(6) de espermatozóides progressivamente móveis/dose inseminante) e do número de inseminaçöes/ciclo (duas, três e quatro ou mais inseminaçöes) sobre a fertilidade. As éguas foram rufiadas e inseminadas às segundas, quartas e sextas-feiras, a partir de um folículo de 3,0 a 3,5cm de diâmetro, com sêmen de apenas um garanhäo com fertilidade comprovada, diluído para um volume inseminante de 10ml com diluidor de mínima contaminaçäo. As taxas de concepçäo ao primeiro ciclo para as concentraçöes de 200 e 400 milhöes foram de 66,7 por cento (20/30) e 65,5 por cento (19/29), e as taxas de concepçäo/ciclo, após quatro ciclos, de 52,0 por cento (26/50) e 57,8 por cento (26/45), respectivamente (P>0,05). As taxas de concepçäo ao primeiro ciclo para os grupos com duas, três e quatro ou mais inseminaçöes/ciclo foram, respectivamente, 72,0 por cento (18/25), 65,2 por cento (15/23) e 54,6 por cento (6/11), sem que se observassem diferenças entre elas (P>0,05). Após quatro ciclos, as taxas de concepçäo foram de 59,0 por cento (23/39), 52,5 por cento (21/40) e 50,0 por cento (8/16), respectivamente, na mesma ordem de citaçäo (P>0,05). Com base nos resultados, recomendam-se inseminaçöes às segundas, quartas e sextas-feiras, utilizando-se a concentraçäo de 200I10(6) SPTZ/dose inseminante, sem que haja perda da fertilidade, independente do número de inseminaçöes/ciclo

Relationship between bicarbonate and cyclic nucleotide in the promoting effects on head-to-head agglutination in boar spermatozoa / 亚洲男科学杂志(英文版)

<p><b>AIM</b>To clarify the relationship between bicarbonate and cAMP in the promoting effects on the sperm agglutination.</p><p><b>METHODS</b>Spermatozoa were collected from mature boars, washed and resuspended in a modified Krebs-Ringer HEPES lacking calcium chloride (mKRH). The sperm suspensions were incubated in a water bath (38.5 degrees C) for 60 min and then the percentage of head-to-head agglutinated spermatozoa was determined.</p><p><b>RESULTS</b>Supplementation of the mKRH with sodium bicarbonate (5-10 mM) significantly raised the percentage of head-to-head agglutinated spermatozoa in the samples. The addition of selective inhibitors for calcium/calmodulin-dependent phosphodiesterases (type 1: 8-methoxymethyl-IBMX and vinpocetine, 25-50 micro M) or for cAMP-specific phosphodiesterases (type 4: Ro20-1724 and rolipram, 25-50 microM) enhanced the effect of bicarbonate on sperm agglutination as highly as did the addition of non-selective inhibitors for phosphodiesterases (IBMX and papaverine, 25-50 microM). A calmodulin antagonist (W-7, 2 microM), that potentially blocks the stimulator of the calcium/calmodulin-dependent phosphodiesterases, significantly enhanced the effect of bicarbonate on sperm agglutination. Moreover, a phosphodiesterase-resistant cAMP analogue (cBiMPS, 0.1 mM) markedly induced agglutination in more spermatozoa (76%) after the incubation without bicarbonate and phosphodiesterase inhibitors than did a less potent cAMP analogue (dibutyryl cAMP, 1 mM) (21%), while three kinds of cGMP analogues (0.1-1 mM) had no effect on sperm agglutination. In addition, a cAMP antagonist (Rp-cAMPS, 1 mM) significantly reduced the sperm agglutination resulting from the actions of bicarbonate and IBMX. On the other hand, the effect of bicarbonate was abolished by a change of incubation temperature from 38.5 degrees C to 25 degrees C.</p><p><b>CONCLUSION</b>These findings demonstrate that the bicarbonate-induced agglutination of boar spermatozoa is controlled via the cAMP-mediated, temperature-dependent signaling cascade. This cascade is suppressed by the action of the phosphodiesterase (at least types 1 and 4).</p>

Varicocele en adolecentes y jóvenes, su correlación clínica y su incidencia en los parámetros seminales / Varicocele in adolescent and young, clinical correlation and its incidence in alteration of seminal parameters

Se estudian 294 pacientes entre 18 y 24 años con varicocele clínico de los cuales 164 tienen espermograma físico y químico en un período de 5 años previo a cualquier tratamiento. Se obtuvo una correlación altamente significativa entre los parámetros clínicos de los diferentes grados de varicocele. Comparando los parámetros clínicos con los del espermograma, concentración, motilidad y morfología, se obtienen correlaciones estadísticamente significativas en los varicoceles grado 3 izquierdos, lo que confirma que en éstos hay una mayor incidencia de alteración de los parámetros seminales. Esto justificaría tratar al grado 3 sólo con los parámetrso clínicos: grado, doppler y orquidometría, sin la estricta necesidad de un espermograma previo. Encontramos en estos jóvenes con varicocele, parámetros seminales alterados estadísticamente respecto de los valores de la O.M.S.

Análise da associaçäo entre os resultados do teste hiposmótico e da morfologia seminal humana / Analysis of the association between the results of the hypoosmotic swelling test and the morphology of human spermatozoa

O estudo realizado teve como objetivo avaliar a associaçäo entre os resultados do teste da hiposmolaridade e as características da morfologia espermática em dois grupos de pacientes. Foram incluídas neste estudo 91 amostras de sêmen. Destas amostras, 50 pertenciam ao grupo infértil, e 41 pertenciam a doadores férteis. As amostras foram analisadas pelo espermograma convencional, pelo teste da hiposmolaridade e pela morfologia. Quanto à avaliaçäo dos testes de interesse, ambos se mostraram associados ao diagnóstico de infertilidade, com medidas de acuidade razoáveis. No entanto, uma melhor acuidade foi observada para a combinaçäo destes testes no diagnóstico. O ponto de corte obtido pelo estudo desta amostra, para a hiposmolaridade (55 por cento de formas reativas), näo alterou em nada os resultados já conhecidos, com a utilizaçäo do ponto de corte usual (60 por cento). Porém, para a morfologia, a classificaçäo dos pacientes, mediante o ponto de corte encontrado (48 por cento), leva a resultados mais satisfatórios, conferindo um aumento significativo da sensibilidade do teste, com valores de falso-positivo e falso-negativo, inteiramente aceitáveis. Além disso, a associaçäo da morfologia com a hiposmolaridade passa a ser significativa. Acredita-se que este novo ponto de corte para a morfologia tenha sido o principal achado deste estudo.

Relationship between the direct mixed antiglobulin reaction (MAR) test and spontaneous sperm agglutination in men from infertile couples.

To determine the relationship between the direct MAR test and spontaneous sperm agglutination in men from infertile couples, semen samples from 160 men were studied. Spontaneous sperm agglutination was present in 80 samples, and absent in the others. Routine semen analysis and the direct MAR test were performed on all samples. The incidence of positive MAR tests in samples with and without spontaneous sperm agglutination were 15 and 0 per cent respectively (p < 0.05). There were no significant differences in mean sperm concentration, motility, and morphology between the samples with and without spontaneous sperm agglutination, and between the positive and negative MAR test groups. It can be concluded that spontaneous sperm agglutination relates significantly to antisperm antibodies detected by the direct MAR test, and this factor could be used as indication for antisperm antibody testing of infertile men.

Unexplained or idiopathic infertility: a possible role of some immune mechanisms

Fourty one patients with unexplained or idiopathic infertility were chosen from a total of 294 women with primary infertility. A complete medical history, a thorough physical examination and a full investigation of reproductive function were done. A control group consisting of 40 women of proven fertility were included in the study. Determination of circulating immune complexes [CICs] and sperm-agglutinating antibodies [SAA] in the sera of patients and controls were performed. The results of the study showed that the CICs were positive in 16 [39%] of 41 infertile women while none of the fertile women had positive test result, furthermore the duration of infertility was positively correlated with the CICs level in the sera of patients with unexplained or idiopathic infertility. Of the 41 infertile women 18 [43.9%] were positive for SAA, whereas only 6 [15%] of the 40 fertile women were positive [a statistically significant difference], also presence of SAA was positively correlated with the duration of infertility. There was a positive correlation between the presence of CICs and SAA in the sera of women with unexplained or idiopathic infertility of 3 years duration or more, of the 18 infertile women with positive SAA, 10 women had positive CICs

Eficacia terapeutica en pacientes ambulatorios con infertilidad masculina / Therapeutic efectiveness in ambulatory pacients with male infertility

Fueron examinados los resultados de los seminogramas de 97 pacientes (ambulatorios, externos al Instituto de Genetica) que fueron sometidos a diferentes esquemas de tratamiento para infertilidad msculina. Ochenta y dos de los pacientes (84.54 por ciento ) continuaron presentando anormalidades en los indicadores seminologicos soleccionados, luego de un periodo variable de tratamiento. La motilidad y la aglutinacion espermatica parecen constituir las alteraciones mas frecuentes en los seminogramas anormales, en este grupo de pacientes

Sperm antibodies and unexplained infertility in females

Immunological factors as a possible cause for unexplained infertility was investigated in 50 infertile patients and a Similar number of women taken as a control The investigated [positive sperm immobilizing antibody test [SIT] was Significantly higher among the infertile group than the control. This significant Association was not found with the sperm agglutinating antibody test [SAT]

Resultado del tratamiento de la infertilidad masculina en pacientes ambulatorios / Results of treatment of male infertility in ambulatory patients

Fueron examinados los resultados de los semiogramas de 97 pacientes (ambulatorios/externos al Instituto de Genetica) que fueron sometidos a diferentes esquemas de tratamiento para la infertilidad masculina. 82 de los pacientes (84,54 //) continuaron presentando anormalidades en los indicadores seminologicos seleccionados, luego de un periodo variable de tratamiento. La motilidad y la aglutinacion espermatica parecen ser las alteraciones mas frecuentes en los seminogramas anormales en este grupo de pacientes

Anticorpos antiespermatozóides: Métodos para identificaçåo / Antiesperm antibodies: Methods for identification

Este trabalho apresenta uma rvisåo sobre métodos laboratoriais para detecçåo de anticorpos antiespermatozóides. Såo descritos os métodos de microaglutinaçåo e imobilizaçåo espermática e as técnicas que såo usadas para identificar anticorpos fixados na superfície do espermatozóide. Såo também feitas algumas consideraçöes sobre estes métodos

Sperm autoimmunity and abnormal semen parameters in insulin-dependent diabetic men

To evaluate sperm autoimmunity in IDDM, 20 men with IDDM [mean age 30 +/- 7.1 years] and ten healthy fertile men of comparable age, were studied. Fasting blood glucose, serum fructosamine, LH, FSH, testosterone and insulin were measured. Complete semen analysis was performed. Insulin was also measured in seminal plasma. The presence of antisperm antibodies was detected in both blood serum and seminal plasma using the slide agglutination method. Antisperm antibodies were detected in serum and seminal plasma in 60% and 50% of men with IDDM, respectively, and in none of healthy men. The presence of these antibodies did not correlate with the degree of metabolic control. In diabetic men, the volume of the ejaculate and the sperm motility were reduced, while the abnormal forms of sperms were more common in comparison to healthy subjects. None of these abnormalities correlated with the presence of sperm antibodies, with the degree of metabolic control, or with the level of seminal plasma insulin which was lower than normal in diabetics. Serum LH, FSH and testosterone were normal in diabetic men, thus sperm autoimmunity is in association with IDDM

Immune response to syngeneic spermatozoa & its effect on target organs in mice.

Two groups of adult Swiss mice were immunised with washed syngeneic spermatozoa without any adjuvant for a period of two months or four months respectively. The presence of antibodies to spermatozoa was measured by micro sperm-agglutination and micro sperm-immobilization tests. The development of cell mediated immune response (CMIR) was measured by leucocyte migration inhibition test (LMIT) using spermatozoal antigens solubilized by 3M KCl, Nonidet P-40 or by subjecting the cells to ultrasonication. SDS-PAGE analysis of these proteins indicated that extraction of spermatozoa with 3 M KCl was a better method for solubilization of antigens present on sperm membrane. Almost all immunized mice had varying titers of sperm agglutinating antibodies. Nearly 40-50 per cent of the mice had a titre of 1:128 in both groups whereas only 33 per cent had sperm immobilizing antibodies. CMIR, as assessed by LMIT, was detected in immunized mice. However, this had not resulted in the infiltration of immune cells into the target organs perhaps due to the lower magnitude of immune response.