Mini-percoll gradient may be used for frozen-thawed sperm selection in sheep / Gradiente de mini-Percoll pode ser utilizado na seleção espermática de sêmen congelado de ovinos

This study evaluated the effect of increasing centrifugal force and reducing centrifugation time and volume in Percoll protocols on ram sperm parameters. Commercial semen of Santa Inês rams were used and five treatments were performed: traditional Percoll and mini-Percoll (MP) techniques (I- 5000 x g, 5min; II- 2500 x g, 5min; III- 1250 x g, 5min; IV- 700 x g, 10min). At post-thawing (PT) and post-selection protocols (0h), samples were assessed for spermatozoa recovery rate, motility, plasma membrane (PM) integrity, sperm capacitation and morphology and incubated at 37 C for 1, 2 and 3h. The sperm recovery rate averaged 9.1±1.4%, and most motility parameters were similar (P> 0.05) among protocols. VCL (µm/s) was higher (P< 0.05) after MP-II, III and IV (66.1±4.5) than traditional Percoll (46.3±4.9). Capacitation status and PM integrity were similar (P> 0.05) among treatments. For the first time, we have demonstrated the reduction of the gradient volume and centrifugation time associated with an increase on centrifugation force at Percoll can be successfully used for frozen-thawed ram sperm selection. MP may be used instead of traditional Percoll, decreasing costs and semen handling time.(AU)

O presente estudo avaliou o efeito do aumento da força de centrifugação, bem como da redução do tempo de centrifugação e do volume do gradiente de Percoll em diferentes protocolos nos parâmetros espermáticos de ovinos. Foi utilizado sêmen comercial de carneiros da raça Santa Inês, e cinco tratamentos foram realizados: Percoll tradicional e quatro técnicas de mini-Percoll (I- 5000 x g, 5min; II- 2500 x g, 5min; III- 1250 x g, 5min; IV- 700 x g, 10min). Após o descongelamento e a seleção espermática em cada técnica utilizada (0h), amostras foram avaliadas quanto à taxa de recuperação espermática, motilidade, integridade de membrana plasmática, capacitação e morfologia. Ao final, foram incubadas a 37 ºC por uma, duas e três horas. A taxa de recuperação média (9,1±1,4%) e a maioria dos parâmetros de motilidade foram similares (P>0,05) entre os tratamentos. VCL foi maior (P<0,05) após MP-II, III e IV (66,1±4,5) quando comparados ao Percoll tradicional (46,3±4,9). O status da capacitação e a integridade de membrana foram similares (P>0,05) entre os tratamentos. Pela primeira vez, foi demonstrado que a redução do volume do gradiente utilizado e do tempo de centrifugação, associada com o aumento da força de centrifugação nos protocolos de Percoll, pode ser usada com sucesso na seleção espermática de sêmen congelado de ovinos. O mini-Percoll pode ser utilizado em alternativa à técnica de Percoll tradicional, diminuindo custos e tempo de manipulação do sêmen durante a técnica.(AU)

Roles of sialic acids in sperm maturation and capacitation and sperm-egg recognition / 中华男科学杂志

Sialic acids are a subset of nine-carbon alpha-keto aldonic acids involved in various biological functions. Sialic acid on the sperm surface is closely related to sperm maturation and capacitation and sperm-egg recognition, which makes sperm negatively charged to avoid accumulation and covers some antigenic determinants there to increase the survival rate of sperm in the female reproductive tract. The loss of sialic acids is an important factor mediating sperm capacitation. Moreover, the sialic acid at the extremity of the protein polymer is involved in signal identification in sperm-egg recognition. Here, we review the current understanding of sialic acids in sperm maturation and capacitation and sperm-egg recognition.

Influence of L-arginine during bovine in vitro fertilization.

The objective of this work was to evaluate the effect of using L-arginine during in vitro fertilization (IVF) on in vitro embryonic development using Bos taurus and Bos indicus semen. Effect of different concentrations (0, 1, 10 and 50 mM) of L-arginine, added to the IVF medium, was evaluated on the fertilization rate at 18 h post-fertilization (hpf), NO3-/NO2- production during IVF by the Griess colorimetric method (30 hpf), cleavage and blastocyst rates (on Day 2 and Day 7 of culture, respectively) and total blastocyst cell number (Day 7 of culture). The results reveal that the addition of 50 mM L-arginine to IVF medium, with either Bos taurus or Bos indicus spermatozoa, decreased the cleavage rate and blastocyst rate compared to the control group. Other concentrations did not affect embryo production. However, 1 mM L-arginine with Bos indicus semen increased the proportion of hatched blastocysts. These results indicate that high L-arginine concentrations may exhibit toxic effects on bovine gametes during in vitro fertilization.

Concentración espermática mínima requerida para inseminación intrauterina mediante capacitación por migración ascendente / Low sperm concentration required to IUI through training by upward migration

Objetivo: Determinar cuál es la cantidad mínima necesaria de espermatozoides móviles que se requiere para realizar la inseminación intrauterina y evaluar la morfología estricta de Kruger y la movilidad espermática antes y después de la capacitación por migración ascendente. Métodos: Estudio prospectivo de 35 muestras de semen de hombres infértiles, se lavaron alícuotas de 1 mL de semen fresco, se centrifugaron y sobre el centrifugado se colocó una capa de medio de capacitación para lograr una migración ascendente. Resultados: Los valores de movilidad y formas normales espermáticas se observaron significativamente aumentados en las muestras después de la capacitación. Fue posible recuperar ≥ 2 x 106 espermatozoides móviles aun en muestras aparentemente inapropiadas caracterizadas por hipospermia u oligozoospermia severa, pero contenían en el total del eyaculado al menos 5 millones de espermatozoides móviles que permitieron un elevado porcentaje de recuperación espermática. Conclusiones: La posibilidad de obtener altos porcentajes de recuperación de espermatozoides móviles en el total del eyaculado permite la inseminación intrauterina como técnica de reproducción asistida en pacientes oligozoospérmicos antes de elegir fertilización in vitro o inyección citoplasmática del espermatozoide cuando el factor masculino es la causa de infertilidad.

Objective: To determine what is the minimum necessary amount of motile sperm required for intrauterine insemination and to evaluate the Kruger strict morphology test and sperm motility before and after training sperm by swim up. Methods: Prospective study of 35 semen samples from infertile men, aliquots of 1 mL of fresh semen was washed, centrifuged and over the pellet was placed a layer of capacitation medium to achieve an upward migration. Results: The values of motility and normal sperm forms were observed in the samples significantly increased after the training. It was possible to recover ≥2 x 106 motile sperm even in seemingly inappropriate samples with hypospermia or severe oligozoospermia, but these contained in the total ejaculate at least 5 million of motile spermatozoa that allowed a high percentage of retrieval. Conclusions: The possibility of obtaining high recoveries of motile sperm in the total ejaculate allows IUI as assisted reproduction technique in oligozoospermic patients before choosing IVF or cytoplasmic sperm injection when the male factor is cause of infertility.

Effect of exogenous nitric oxide on sperm motility in vitro

BACKGROUND: Nitric oxide (NO) has been shown to be important in sperm function, and the concentration of NO appears to determine these effects. Studies have demonstrated both positive and negative effects of NO on sperm function, but have not been able to provide a clear link between NO concentration and the extent of exposure to NO. To study the relationship between nitric oxide and sperm capacitationin vitro, and to provide a theoretical basis for the use of NO-related preparations in improving sperm motility for in vitro fertilization, we investigated the effects of NO concentration and time duration at these concentrations on in vitro sperm capacitation in both normal and abnormal sperm groups. We manipulated NO concentrations and the time duration of these concentrations using sodium nitroprusside (an NO donor) and NG-monomethyl-L-argenine (an NO synthase inhibitor). RESULTS: Compared to the normal sperm group, the abnormal sperm group had a longer basal time to reach the appropriate concentration of NO (p < 0.001), and the duration of time at this concentration was longer for the abnormal sperm group (p < 0.001). Both the basal time and the duration of time were significantly correlated with sperm viability and percentage of progressive sperm (p < 0.001). The experimental group had a significantly higher percentage of progressive sperm than the control group (p < 0.001). CONCLUSIONS: We hypothesize that there is a certain regularity to both NO concentration and its duration of time in regards to sperm capacitation, and that an adequate duration of time at the appropriate NO concentration is beneficial to sperm motility.

Effects of different concentrations of parecoxib sodium on rat sperm motility, capacitation and acrosome reaction in vitro / 中华麻醉学杂志

Objective To evaluate the effects of different concentrations of parecoxib sodium on the rat sperm motility,capacitation and acrosome reaction in vitro.Methods The sperm samples from Sprague-Dawley rat epididymis were collected by Klinefelter diffusion method and randomly divided into 4 groups (n =18 each) using a random number table:control group (group C),and parecoxib sodium 100,500,1 000 μmol/L groups (P1-3 groups).Parecoxib sodium with the final concentrations of 100,500 and 1 000 μmol/L was added to the culture medium.The samples were then incubated for 5 h in an airtight container filled with 5 ％ CO2 at 37 ℃.Then sperm motility was examined in vitro at 37 ℃ and analyzed by the computer-assisted sperm analysis,including the sperm motility ((a + b)％),average path velocity (VAP),straight line velocity (VSL),curvilinear velocity (VCL) and amplitude of lateral head displacement (ALH).The capacitation effect was assessed by using the chlortetracycline staining and phase-contract microscopy.The acrosome reaction was evaluated by coomassie brilliant blue staining.Results The VAP,VSL,VCL and capacitation ability of the sperm were gradually decreased in C and P1-3 groups (P ＜ 0.05).Compared with group C,(a + b)％ in P2,3 groups and ALH in P2 group were significantly decreased (P ＜ 0.05).There was no significant difference in the acrosome reaction between groups (P ＞ 0.05).Conclusion Parecoxib sodium has significant inhibitory effects on the rat sperm motility and capacitation in a dose-dependent manner,while has no effect on the acrosome reaction in vitro.

Actividad espermicida y citotóxica del extracto de Sapindus saponaria L. (jaboncillo) / Evaluation of the spermicidal and cytotoxic activity of extracts of Sapindus saponaria L. (jaboncillo)

Introducción: los espermicidas están entre los métodos anticonceptivos que pueden inmovilizar o matar los espermatozoides. Objetivo: evaluar la actividad espermicida y citotóxica de los extractos de Sapindus saponaria L., conocida como jaboncillo, sobre espermatozoides humanos y la línea celular HeLa, respectivamente. Métodos: las muestras de semen donadas por individuos sanos se incubaron con los extractos de Sapindus saponaria L. y sus respectivas fracciones. La movilidad y la viabilidad espermática se evaluó antes y después de cada tratamiento. Adicionalmente, el efecto citotóxico del extracto se valoró sobre la línea celular HeLa mediante el ensayo 3-(4,5 dimetiltiazol-2-il)-5-(3-carboximetoxifenil)-2-(4-sulfofenil)-2H-tetrazolio (MTS). Resultados: el máximo efecto espermicida se observó cuando las muestras de semen se incubaron con la fracción polar del extracto de hojas de Sapindus saponaria L., luego de 5 min de tratamiento (p< 0,05). No se encontró efecto citotóxico en la línea celular HeLa luego de 6 y 12 h de tratamiento con la fracción polar del extracto de hojas. Conclusión: el extracto de Sapindus saponaria L. puede ser una nueva opción como espermicida con menos efectos adversos.

Introduction: spermicides are contraceptive methods aimed at either immobilizing or killing spermatozoa. Objective: evaluate the spermicidal and cytotoxic activity of extracts of Sapindus saponaria L. (jaboncillo) on human spermatozoa and the HeLa cell line, respectively. Methods: semen samples from healthy individuals were incubated with extracts of Sapindus saponaria L. and their fractions. Sperm motility and viability were measured before and after each treatment. Additionally, the cytotoxic effect of the extract on the HeLa cell line was assessed by the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxy methoxy phenyl)-2-(4-sulfophenyl)-2H-tetrazolium MTS assay. Results: maximum spermicidal effect was observed when semen samples were incubated with the polar fraction of Sapindus saponaria L. leaf extract after 5 minutes of treatment (p< 0.05). No cytotoxic effect on the HeLa cell line was found after 6 and 12 hours of treatment with the polar fraction of the leaf extract. Conclusion: the extract of Sapindus saponaria L. may be a new spermicidal option with fewer adverse effects.

Las proteínas del plasma seminal incrementan la viabilidad espermática post-descongelación del semen de toros Sanmartinero / Seminal plasma proteins increase the post-thaw sperm viability of Sanmartinero bull’s semen

Objetivo. El objetivo de este trabajo fue evaluar el efecto de la adición de proteínas del plasma seminal sobre el porcentaje de espermatozoides bovinos viables post-descongelación. Materiales y métodos. Los espermatozoides se congelaron usando dos medios (citrato-fructosa-yema y Bioxcell®) y la obtención de proteínas de plasma seminal de bajo peso molecular se realizó por medio de cromatografía líquida de baja presión. Las proteínas de interés eluyeron en las fracciones 21-25 y se sometieron a electroforésis en una y dos dimensiones. Los espermatozoides se incubaron a 37°C durante una hora, con 0.5, 1.0, 1.5 y 2.0 mg de la fracción 21-25. Se incluyeron dos tratamientos adicionales: uno con proteínas totales del plasma seminal y otro sin proteína. Resultados. La electroforésis bidimensional de las fracciones confirmó la presencia de siete puntos de proteína de bajo peso molecular (14-16 kDa y punto Isoeléctrico de 5.0 - 5.5). La adición de estas proteínas aumentó 20% (p<0.05), el porcentaje de espermatozoides viables post-descongelación en muestras congeladas en medio citrato-fructosa-yema (con dosis de 1 ó 1.5 mg de proteína/106 espermatozoides), y 25% (p<0.05) en muestras congeladas en medio Bioxcell® (con dosis de 0.5 mg de proteína/106 espermatozoides). Conclusiones. Los resultados de esta investigación sugieren el posible uso de proteínas de bajo peso molecular del plasma seminal, para disminuir el efecto deletéreo de la criopreservación en los espermatozoides.

Objective.This study was performed to evaluate the effect of the addition of proteins on the post-thawing viability of spermatozoa. Materials and methods. Spermatozoa were frozen with two different media: Citrate-fructose and Bioxcell®. The isolation of seminal plasma proteins of low molecular weight was performed through low pressure liquid chromatography. It was determined that the proteins of interest eluted in fractions 21-25, and two dimensional electrophoresis was performed. Thawed sperm was incubated at 37°C for one hour with 0.5, 1, 1.5 and 2.0 mg of 21-25 fraction protein. Two additional treatments were included: one with seminal plasma total protein, and another one without protein. Results. Two dimensional electrophoresis of protein confirmed the presence of two bands of 14 and 16 kDa and seven spots with iso-electric points between 5.0 - 5.5 respectively. Incubation of the spermatozoa with the 21-25 fraction showed that sperm viability increases by 20% with doses of 1 and 1.5 mg of protein/106 spermatozoa in the citrate-fructose medium, and 25% with 0.5 mg of protein/106 spermatozoa in Bioxcell® medium. A positive effect in sperm viability was demonstrated although it depends on the doses of protein and the cryopreservation medium used. Conclusions. This investigation suggests that the use of seminal plasma proteins can be useful for reducing the harmful effect on sperm cryopreservation.

Efecto de cinco extractos de plantas colombianas sobre espermatozoides humanos / Effect of five Colombian plant extracts on human spermatozoids

Introducción: las pruebas de capacitación espermática y la actividad espermicida o inmovilizante usando extractos de plantas permiten incrementar los conocimientos actuales en el área reproductiva. Objetivos: evaluar el efecto de 5 extractos de las plantas colombianas Bocconia frutescens, Bomarea setaceas, Muehlenbeckia platyclada, Zanthoxylum lenticulare y Piper subpedale sobre los espermatozoides humanos. Métodos: los espermatozoides humanos se incubaron con los extractos de las plantas, se valoró su movilidad y viabilidad. Adicionalmente la capacitación espermática se evaluó utilizando albúmina sérica bovina. Resultados: después de una evaluación inicial del efecto de los extractos de las plantas sobre la movilidad o viabilidad de los espermatozoides, se seleccionaron los extractos de B. frutescens y B. setaceas para realizar los ensayos de capacitación espermática, porque no alteraron ni la movilidad ni la viabilidad espermática, y se seleccionaron los extractos de M. platyclada, Z. lenticulare y P. subpedale para los ensayos de actividad espermicida debido a que afectaron la movilidad progresiva espermática. Conclusiones: este trabajo permitió proponer 3 plantas con promisoria actividad espermicida, y se logró estandarizar un sistema biológico en el cual se pudo evaluar el efecto capacitante de algunos extractos sobre los espermatozoides humanos.

Introduction: the sperm capacitation test and the spermicidal activity or immobilization using plant extracts, allows for an increase in the current knowledge on the reproductive area. Objectives: to evaluate the effect of five extracts from Colombian plants Bocconia frutescens, Bomarea setaceas, Muehlenbeckia platyclada, Zanthoxylum lenticulare and Piper subpedale on human spermatozoa. Methods: human spermatozoa were incubated with each extract and their motility and viability were evaluated. Additionally, sperm capacitation was evaluated using bovine serum albumin. Results: after an initial assessment of the effect of plant extracts on human spermatozoa, the extracts from B. frutescens and B. setaceas were selected for capacitating tests since they did not change the sperm motility and viability. Additionally, M. platyclada, Z. lenticulare and P. subpedale extracts were selected for immobilization or spermicidal activities tests because they had had an effect on sperm progressive motility. Conclusions: this study suggested three plants with promising spermicidal effect; in addition to the standardization of a biological system, allowing the assessment of the capacitation effect of some extracts over human spermatozoa.

In vitro assessment of reproductive toxicity of cigarette smoke and deleterious consequences of maternal exposure to its constituents

Cigarette smoke is known to be a serious health risk factor and considered reproductively toxic. In the current study, we investigated whether constituents of cigarette smoke, pyrazine, 2-ethylpyridine, and 3-ethylpyridine, adversely affect reproductive functioning such as oocyte maturation and sperm capacitation. Our findings indicated that three smoke components were involved in retardation of oocyte maturation in a dose-dependent manner and the lowest-observed-adverse-effect level (LOAEL) was determined to be 10-10M. However, individual smoke components administrated at the LOAEL did not attenuate oocyte maturation, demonstrating that all three toxicants were equally required for the observed growth impairment. When exposed to all three components at 10-10M during in vitro capacitation, murine sperm lost forward progression and were unable to show adequate hyperactivation, which is indicative of the incompletion of the capacitation process. Only sperm administrated with 3-ethylpyridine alone showed significant reduction in capacitation status, suggesting the chemical is the one responsible for disrupting sperm capacitation. Taken together, this is the first report that documents the effect of cigarette smoke components on oocyte maturation and sperm capacitation. The present findings demonstrate the adverse effects of smoke constituents of mammalian reproduction and the differences in sensitivity to smoke components between male and female gametes. Since both processes take place in the female reproductive system, our data provide new insights into deleterious consequences of maternal exposure to cigarette smoke.

Effects of inhalation anesthetics on human sperm motility and capacitation in vitro / 中华麻醉学杂志

Objective To investigate the effects of inhalation anesthetics on human sperm motility and capacitation in vitro. Methods Sperm samples were obtained from normal adults and prepared with discontinuous percoll gradient centrifugation technique. The samples were incubated for 5 h in an airtight glass container filledwith 5% CO2-95% air at 37 ℃ with or without sevoflurane (SEV 2%, 4% ) or isoflurane (ISO 1.1%, 2.2% ).Then human sperm motility was examined in vitro at 37℃ and analyzed by the computer-assisted sperm analysis (CASA), including sperm motility (a + b)%, curvilinear velocity (VCL), straight line velocity (VSL), averagepath velocity (VAP) and amplitude of lateral head displacement (ALH). The capacitation effect was assessed by using the chlortetracycline (CTC) staining and phase-contract microscopy. Results 2% and 4% SEV significantly reduced (a + b)% , VCL, VSL and VAP in a dose-dependent manner, while only 4% SEV significantly decreased ALH and the capacitation ability of the sperm compared with control group. 2.2% ISO significantly decreased ( a + b)%, VCL, VSL and VAP compared with control and 1.1% ISO group. The capacitation ability of the sperm was significantly decreased by 1.1% and 2.2% ISO as compared with control group. Conclusion Sevoflurane and isoflurane have significant inhibitory effects on human sperm motility and capacitation in a dose-dependent manner. Sevoflurane has stronger inhibitory effect than isoflurane.

Inducción de la reacción acrosómica en espermatozoides de ratón mediante solubilizados de zona pelucida de alpaca (Lama pacos L.)

La acrosina, es uno de los componentes principales del acrosoma, presenta actividad similar a la tripsina, y es liberado luego de la reacción acrosómica (RA). El objetivo del presente trabajo fue estudiar la inducción de la RA en espermatozoides de ratón con zonas pelúcidas homólogas y heterólogas. Espermatozoides de ratón capacitados por 2 horas en medio IVF suplementado con albúmina, heparina y suero sintético a 37 ºC y 5% CO2 fueron incubados en ausencia de Zona Pelucida (ZP) o en presencia de solubilizados de ZP aisladas de ratón (0,78 mg/ml) y alpaca (0,78 mg/ml y 2,35 mg/ml). La RA se evaluó mediante inmunocitoquímica con anticuerpo monoclonal anti-acrosina humana C5F10 a intervalos de 1 hora durante 4 horas. Los resultados obtenidos en espermatozoides de ratón evidencian un incremento significativo (p< 0,05) de la reacción acrosómica inducida con ZP homóloga y heteróloga.

Acrosin is one of the principal components in the acrosome, has trypsin-like activity and is secreted after the Acrosome Reaction (RA). The aim of this study was to study the induction of the acrosomal reaction in mouse sperms with homologous- Pellucid Zone and heterologous- Pellucid Zone. Mouse sperms were capacitated by two hours in IVF medium, supplemented with albumin, heparin and synthetic serum to 37 °C and 5% CO2, and before they were incubated in absence of ZP and in present of solubilized ZP from mouse (0,78 mg/ml) and alpaca (0,78 mg/ml y 2,35 mg/ml). The RA was evaluated through immunocytochemistry with monoclonal antibodies anti-human acrosin C5F10 within intervals of 1 hour during four hours. The results show a significant increase (p<0,05) in acrosome reaction induced with homologous-ZP and heterologous-ZP.

Percutaneous testicular sperm aspiration and intracytoplasmic sperm injection in the treatment of severe male infertility / 中华妇产科杂志

Objective To investigate the efficacy of intracytoplasmic sperm injection (ICSI) combined with percutaneous testicular sperm aspiration (PTSA)in the treatment of severe male infertility. Methods From October 1998 to December 2000, 162 couples were enrolled, and ICSI and PTSA were adopted. Ovarian stimulation was achieved by the short protocol. All metaphase Ⅱ (MⅡ) oocytes were selected for ICSI. Results 1 517 MⅡ oocytes were injected in 185 cycles, 990 fertilized (65.3%), and 152 embryo implanted. 54 couples achieved clinical pregnancy (35.5%). Conclusion PTSA combined with ICSI is a rapid, convenient, painless and effective approach for the treatment of severe male infertility.

PC3 prostasome promotive effects on fresh or cryopreserved human spermatozoa in swim-up medium / 中华泌尿外科杂志

Objective To investigate the motility stimulant effects of prostasome like granules from the PC3 prostate cancer cell line (here called PC3 prostasome). Methods Fresh or cryopreserved human spermatozoa in Earle's balanced salt solution (EBSS) supplemented with human serum albumin (HSA,10 mg/ml) or PC3 prostasome (0.10 mg/ml or 0.25 mg/ml)after swim up to compare the difference of motility or the recovery of motility. Results For the fresh samples within different time of incubation or different parameters in one hour incubation,addition of PC3 prostasomes 0.10 mg/ml was superior to HSA in sperm motility stimulation ( P