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)

Onion peel extract and its constituent, quercetin inhibits human Slo3 in a pH and calcium dependent manner

Sperm function and male fertility are closely related to pH dependent K⁺ current (KSper) in human sperm, which is most likely composed of Slo3 and its auxiliary subunit leucine-rich repeat-containing protein 52 (LRRC52). Onion peel extract (OPE) and its major active ingredient quercetin are widely used as fertility enhancers; however, the effect of OPE and quercetin on Slo3 has not been elucidated. The purpose of this study is to investigate the effect of quercetin on human Slo3 channels. Human Slo3 and LRRC52 were co-transfected into HEK293 cells and pharmacological properties were studied with the whole cell patch clamp technique. We successfully expressed and measured pH sensitive and calcium insensitive Slo3 currents in HEK293 cells. We found that OPE and its key ingredient quercetin inhibit Slo3 currents. Inhibition by quercetin is dose dependent and this degree of inhibition decreases with elevating internal alkalization and internal free calcium concentrations. Functional moieties in the quercetin polyphenolic ring govern the degree of inhibition of Slo3 by quercetin, and the composition of such functional moieties are sensitive to the pH of the medium. These results suggest that quercetin inhibits Slo3 in a pH and calcium dependent manner. Therefore, we surmise that quercetin induced depolarization in spermatozoa may enhance the voltage gated proton channel (Hv1), and activate non-selective cation channels of sperm (CatSper) dependent calcium influx to trigger sperm capacitation and acrosome reaction.

Comportamiento del semen bovino sexado congelado-descongelado en fertilización in vitro (FIV) capacitado mediante BO en dos concentraciones versus percoll / Behavior of sexed frozen-thawed bovine semen on in vitro fertilization (IVF) capacitated with two concentrations of BO versus percoll

RESUMEN: El objetivo fue comparar la tasa de división (TD) y desarrollo embrionario (DE) con semen sexado X (SX) en FIV capacitado con Percoll vs. BO, y evaluar el efecto de dos concentraciones (5x106 vs. 10x106 espermatozoides/ml), capacitado con BO, comparado con el semen no-sexado (NS). Un avance importante es predeterminar el sexo en bovinos, es posible obtener mayor proporción de terneras a partir del citómetro de flujo con la capacidad de seleccionar los espermatozoides X por la diferencia del ADN (4 %, bovinos), con confiabilidad del 90 %. El SX aumenta la eficiencia reproductiva, permite la selección de hembras e incrementa la ganancia genética. Se obtuvieron complejos ovocito cúmulus (COC), de ovarios de matadero, se cultivaron para maduración en gotas de 100 µl de TCM-199 + 5 % de suero fetal bovino + 0.005 U/ml de (FSH-p) + 10 IU hCG/ml + 1µg Estradiol (E2)/ml, 15 COC/gota, cubiertas con aceite mineral, en incubadora (38,5 ºC, 5 % de CO2 y 95 % de humedad), 22 h. Posmaduración se dividieron en 4 grupos (G1, G2, G3, G4) y se realizaron dos experimentos simultáneamente: I) G1: NS a 5x106, G2: SX a 5x106, G3: SX a 10x106 espermatozoides/ml capacitados con BO. II) G2: SX a 4,5x106 espermatozoides/ml capacitados con BO y G4: SX capacitado con Percoll a 5x106 espermatozoides/ ml. Se colocaron 15 COC/gota de semen cubiertas con aceite mineral, en incubadora, 18 h. Para el desarrollo se colocaron en gotas de CR1aa, en incubadora. Se aplicó el test de χ2. La TD a las 48 h entre G1 y G3 no presentó diferencias significativas (p<0,05), sin embargo, en ambos grupos fue significativamente mayor al G2. En el DE al día 7 hubo diferencias significativas (p<0,05) a favor del G4. Se obtuvo mayor DE con el SX capacitado con Percoll respecto al BO y no hubo diferencias entre ambas concentraciones de semen.

SUMMARY: The objective of this study was to evaluate the in vitro fertility of bovine sexed semen (SX) capacitated with Percoll vs. BO. The division rate (DR), embryo development (ED) were evaluated in two concentrations 5x106 vs. 10x106 sperm/ml, capacitated with BO and compared with non-sexed semen (NS). Offspring sexing represents an important advance for livestock production. Flow cytometry separates X and Y spermatozoa by difference in DNA content (4 % greater in X) with 90 % effectiveness. The SX increases the reproductive efficiency, allows the selection of females and increases the genetic gain. Cumulusoocytes complexes (COC) were obtained from slaughterhouse ovaries. They were then cultured 22 hours for maturation in TCM199 + 5 % fetal calf serum + 0.005 IU/ml (FSH-p) + 10 IU hCG/ml + 1 mg Estradiol (E2)/ml, in 100 µl drops with mineral oil, in incubator (38.5 ºC, 5 % CO and 95 % humidity). Post maturation, 4 groups were randomly assigned (G1, G2, G3, G4) and were performed two experiments simultaneously: I) G1 was inseminated with NS at 5x106 sperm/ml, G2: SX at 5x106, G3: SX at 10x106 sperm/ml capacitated with BO. II) G2: SX at 5x106 sperm/ml capacitated with BO and G4: SX capacitated with Percoll at 5x106 sperm/ml. 15 COC/drop of capacitated semen covered with mineral oil and placed in an incubator for 18 hours. For development, they were placed in drops of CR1aa, in an incubator. Results were analyzed with the c square test. At 48 hours, there were no significant differences (p<0.05) in DR between G1 and G3, however, in both groups it was significantly greater than G2. At day 7 there were significant differences (p <0.05) in ED, greater in G4. At 48 hours, there were no significant differences (p<0.05) in DR between G1 and G3, however, in both groups it was significantly greater than G2. At day 7 there were significant differences (p <0.05) in ED, greater in G4. A higher ED was obtained with the SX capacitated with Percoll, with respect to BO and there was no difference between the two semen concentrations.

Palmitoylation of heat shock protein 90 in mouse sperm / 生理学报

Protein palmitoylation, one of post-translation modifications, refers to the addition of saturated 16-carbon palmitic acid to cysteine residues via the thioester bond. It plays key roles in various functional activities, such as the interaction, stability and location of proteins. Heat shock protein 90 (Hsp90), an important molecular chaperone, has been reported to be involved in sperm capacitation. However, it remains unclear whether protein palmitoylation exists in sperm and whether Hsp90 in sperm is palmitoylated under different physiological conditions. In this study, we examined whether the protein palmitoylation is present in mouse cauda epididymis sperm using acyl-biotin exchange method, predicted the potential palmitoylated sites of Hsp90 by the software CSS-Palm 4.0 and detected the palmitoylated Hsp90 in the mouse sperm from caput epididymis and cauda epididymis by immunoprecipitation. We found that some proteins, approximately 50, 65, 72, 85 and 130 kDa, were palmitoylated in mouse cauda epididymis sperm. Five sites in two Hsp90 isoforms were predicted to be palmitoylated. The results also showed that Hsp90 in mouse sperm was palmitoylated and its palmitoylation level was involved in different physiological conditions: the palmitoylation level of cauda epididymis sperm was higher than that of caput epididymis sperm; and the palmitoylation level after capacitation was much higher than that before capacitation. In conclusion, this study reveals that protein palmitoylation is present in mouse sperm and the palmitoylated Hsp90 is associated with different physiological conditions in sperm.

Intracellular calcium-dependent regulation of the sperm-specific calcium-activated potassium channel, hSlo3, by the BK(Ca) activator LDD175

Plasma membrane hyperpolarization associated with activation of Ca²⁺-activated K⁺ channels plays an important role in sperm capacitation during fertilization. Although Slo3 (slowpoke homologue 3), together with the auxiliary γ2-subunit, LRRC52 (leucine-rich-repeat–containing 52), is known to mediate the pH-sensitive, sperm-specific K⁺ current KSper in mice, the molecular identity of this channel in human sperm remains controversial. In this study, we tested the classical BK(Ca) activators, NS1619 and LDD175, on human Slo3, heterologously expressed in HEK293 cells together with its functional interacting γ2 subunit, hLRRC52. As previously reported, Slo3 K⁺ current was unaffected by iberiotoxin or 4-aminopyridine, but was inhibited by ~50% by 20 mM TEA. Extracellular alkalinization potentiated hSlo3 K⁺ current, and internal alkalinization and Ca²⁺ elevation induced a leftward shift its activation voltage. NS1619, which acts intracellularly to modulate hSlo1 gating, attenuated hSlo3 K⁺ currents, whereas LDD175 increased this current and induced membrane potential hyperpolarization. LDD175-induced potentiation was not associated with a change in the half-activation voltage at different intracellular pHs (pH 7.3 and pH 8.0) in the absence of intracellular Ca²⁺. In contrast, elevation of intracellular Ca²⁺ dramatically enhanced the LDD175-induced leftward shift in the half-activation potential of hSlo3. Therefore, the mechanism of action does not involve pH-dependent modulation of hSlo3 gating; instead, LDD175 may modulate Ca²⁺-dependent activation of hSlo3. Thus, LDD175 potentially activates native KSper and may induce membrane hyperpolarization-associated hyperactivation in human sperm.

Avaliação in vitro do sêmen criopreservado de cães naturalmente infectados por Leishmania sp / In vitro evaluation of cryopreserved semen of dogs naturally infected by Leishmania sp

O objetivo deste trabalho foi avaliar a qualidade in vitro do sêmen criopreservado de cães naturalmente infectados por Leishmania sp. Foram coletadas amostras de sêmen de 12 cães, sendo seis positivos (GI) e seis negativos (GII) para leishmaniose visceral (LV), semanalmente, totalizando quatro coletas por animal. O sêmen criopreservado foi avaliado pelo teste de termorresistência rápida (TTR), nos tempos zero, 30 e 60 minutos, pela análise computadorizada (CASA) e por meio de sondas fluorescentes; esta última técnica com o intuito de avaliar a integridade das membranas espermáticas. Houve diferença estatística pela técnica de TTR no parâmetro motilidade progressiva, no tempo 0min (68,33% GI e 72,50% GII), e no vigor espermático (2,67 GI e 3,0 GII), no tempo 30min. Quanto ao CASA, houve diferença estatística apenas na motilidade total (27,50% GI e 57,08% GII), embora os demais parâmetros seminais tenham apresentado valores relativos diminuídos nos cães do GI. Nas análises com sondas fluorescentes, foram observadas diferenças estatísticas entre os grupos quanto à integridade das membranas plasmática e acrossomal e ao potencial mitocondrial das células espermáticas. Concluiu-se que a LV pode comprometer a qualidade do sêmen criopreservado de cães parasitados.(AU)

The aim of this study was to evaluate the in vitro quality of cryopreserved semen of dogs naturally infected by Leishmania sp. 12 dog semen samples were collected, with 06 positive (GI) and 06 negative (GII) for Visceral Leishmaniasis (LV), weekly, totaling 04 collections per animal. The cryopreserved semen was evaluated by the Rapid Termorresistência Test (TTR), at 0, 30 and 60 minutes by computer analysis (CASA) and by means of fluorescent probes, the latter technique in order to assess the integrity of the sperm membrane. There was no statistical difference for the TTR technique in the progressive motility parameter, at time 0min (68.33% GI and GII 72.50%), and sperm vigor (2.67 GI and GII 3.0), time 30min. As for the CASA, there was statistical difference only in total motility (27.50% GI and GII 57.08%), although other semen parameters have submitted lower figures for GI dogs. In the analyzes with fluorescent probes statistical differences were noted between the groups in terms of plasma membrane integrity and acrosomal and mitochondrial potential of sperm cells. It was concluded that LV can compromise the quality of cryopreserved semen of infected dogs.(AU)

Optimizing the transport of porcine semen: a proposal for Brazil / Otimizar o transporte do sêmen suíno: uma proposta para o Brasil

Semen from the first 15mL of the ejaculate (P1) obtained from two boars (30mL) was diluted in glycine-egg yolk extender, cooled at 5°C in a special container and rediluted in standard doses of 3x109 mobile spermatozoa after 12h of storage. Semen was also stored up to 24h after redilution. The physical characteristics of the semen were evaluated at different storage periods (fresh, 0h, 12h, rediluted, 24h, and 36h). The reproductive performance of the boars and their fertility regarding the insemination of primiparous sows were also determined. Two treatments were used: T1-15B sows inseminated with semen originated from hyperconcentrated heterospermic doses (15x109 mobile spermatozoa per dose), rediluted after 12h of storage at 5°C for standard doses of 3x109 mobile spermatozoa per dose and stored at 5°C up to 24h after redilution (n=10); T2-3B sows inseminated with standard heterospermic doses (3x109 mobile spermatozoa per dose), stored at 5°C up to 36h after semen collection (n=10). There was no effect (P>0.05) of treatments on the spermatic motility, even though a pronounced decrease (P>0.05) of their values at 12h of storage was recorded. However, they remained higher than 70% until 36h. There was effect of treatments on spermatic vigour at 0h (P<0.05), when T1-15B vigour was higher. There was also effect of the storage period for both treatments with a progressive decrease throughout 36h of storage, although the differences were not always significant. Pregnancy rates (90%) and the number of total farrowed piglets (15, 11-T1-15B; 13, 44- T2-3B) did not differ (P>0.05) between the treatments. It was concluded that the semen hyperconcentration of 15 billion of mobile spermatozoa per dose, stored at 5°C for 12h, did not result in drawbacks considering the physical characteristics of the semen, maintaining the pregnancy rates and prolificacy of the inseminated sows.

Os primeiros 15mL do ejaculado (P1) de dois varrões foram coletados (30mL) e diluídos em diluidor glicina-gema de ovo, resfriados a 5°C em contêiner especial e rediluídos para doses padrão de 3x109 espermatozoides (sptz) móveis, após 12 horas de armazenamento. Além disso, foram armazenados por até 24 horas após a rediluição, sendo as características físicas avaliadas em diferentes períodos de estocagem (fresco, zero hora, 12h, Red12h, 24h e 36h) e a fertilidade avaliada por meio de fêmeas primíparas inseminadas. Foram realizados dois tratamentos: T1-15B: porcas inseminadas com sêmen de doses heterospérmicas hiperconcentradas (15x109 sptz móveis/dose), rediluídas após 12 horas de armazenamento a 5°C para doses padrão de 3x109 sptz móveis/dose, e armazenadas a 5°C por até 24 horas após a rediluição (n=10); T2-3B: porcas inseminadas com doses heterospérmicas padrão (3x109 sptz móveis/dose), armazenadas a 5°C por até 36 horas após coleta. Não houve efeito (P>0.05) dos tratamentos sobre a motilidade espermática e, embora tenha ocorrido queda (P<0.05) às 12 horas, a motilidade foi superior a 70% durante as 36 horas de armazenamento. Houve efeito (P<0.05) dos tratamentos no tempo zero hora quanto ao vigor espermático, sendo E1T1-15B superior. Além disso, houve efeito do período de estocagem para os dois tratamentos, com queda progressiva do vigor ao longo das 36 horas, embora nem sempre as diferenças tenham sido significativas. As taxas de gestação (90%) e o número total de leitões nascidos (15, 11 - T1-15B; 13, 44 - T2-3B) não diferiram (P>0.05) entre os tratamentos. Concluiu-se que a hiperconcentração do sêmen para 15x109 sptz móveis/dose, armazenado a 5°C por 12 horas não resultou em prejuízos quanto à manutenção das características físicas do sêmen e ao desempenho reprodutivo dos varrões, sendo capaz de manter a taxa de gestação e a prolificidade das fêmeas inseminadas.

Cinética e morfologia de espermatozoides de carpa comum criopreservados em água de coco em pó acp-104 / Kinetics and morphology of common carp sperm cryopreserved in powdered coconut water acp-104

O objetivo deste estudo foi avaliar o efeito de diferentes taxas de diluição seminal sobre a motilidade, velocidade e presença de anormalidades espermáticas no sêmen descongelado de carpa comum (Cyprinus carpio). Utilizaram-se 20 machos sexualmente maduros, pertencentes ao Departamento Nacional de Obras Contra as Secas (DNOCS). A partir do sêmen coletado, foi formado um total de nove pools. O sêmen de cada pool foi avaliado quanto à motilidade, velocidade e morfologia espermática antes e depois da criopreservação. A criopreservação seminal foi realizada em meio ACP-104 + DMSO 10% diluído em 1:1 ou 1:3 (sêmen:diluidor). As amostras foram envasadas em palhetas de 0,25mL, congeladas em vapor de nitrogênio líquido (caixa de poliestireno) e estocadas em nitrogênio líquido (-196 °C). Não houve diferença significativa (P>0,05) entre as taxas de diluições sobre os parâmetros cinéticos e morfológicos de sêmen descongelado de carpa comum. Entretanto, esses parâmetros apresentaram diferença significativa (P<0,05) em relação ao sêmen fresco (controle). Baixos valores de velocidade foram registrados no sêmen descongelado (VCL 42,6-46,5μm/s; VSL 33,2-37,1μm/s; VAP 38,9-43,2μm/s); contudo, observaram-se motilidades acima de 59% (59,8-67,8%) e baixos índices de anormalidades espermáticas (19,3-19,5%), sugerindo que o ACP pode ser usado como um meio favorável à criopreservação do sêmen de carpa comum em qualquer uma das taxas de diluição testadas.

The aim of this study was to determine the effects of various dilution ratios on thaw sperm motility, velocity and presence of spermatic abnormalities in the common carp. 20 males with three years of age, raised at the Departamento Nacional de Obras Contra as Secas (DNOCS) were used. From the collected semen a total of nine pools were formed. The semen from each pool was evaluated for motility, velocity and spermatic morphology before and after cryopreservation. The sperm cryopreservation was performed in medium ACP-104 + DMSO 10% diluted in 1:1 or 1:3 (sperm:extender). The samples were loaded into 0.25mL straws, frozen in liquid nitrogen vapor (Styrofoam) and stored in liquid nitrogen (-196 °C). There was no significant difference (P>0.05) between the ratios of dilution on the kinetic and morphological parameters of thawed semen from common carp. However, these parameters were significantly different (P<0.05) compared to fresh semen (control). Low velocity values were recorded in thawed semen (VCL 42.6-46.5μm/s; VSL 33.2-37.1μm/s; VAP 38.9-43.2μm/s), however, observed motility above 59% (59.8-67.8%), and low levels of spermatic abnormalities (19.3-19.5%) suggested that the ACP-104 can be used as a suitable medium for cryopreservation of common carp semen in any of the tested dilution ratios.

Aloe vera na criopreservação do sêmen de tambaqui (Colossoma macropomum) / Aloe vera in the cryopreservation of tambaqui (Colossoma macropomum) sperm

This study aimed to evaluate the extract of Aloe vera (AV) associated or not with 10% Dimethylsulfoxide (DMSO) in cryopreservation of tambaqui semen. For the formation of the pools (n= 14), 30 males were hormonally induced twice. Each pool had the objective motility, curvilinear velocity, straight-line velocity, average path velocity and morphology analyzed before and after cryopreservation of semen. The means for cryopreservation were constituted of Powder Coconut Water-104 diluent added DMSO and/or AV (5 or 10%). After cryopreservation, motility, velocities and morphology were reduced significantly when compared to fresh semen. For sperm motility the best treatment was that using only DMSO (20,86±8,31) and DMSO + 5% AV (15.71±9.77). For the velocities, the worse treatment was DMSO+10% AV. Treatment with only the addition of DMSO had a significantly higher effect than others on percentage of morphologically normal sperm. The mean correlation found was between motilityand the rate of morphologically normal sperm (r = 0.687). In conclusion, the addition of AV does not provide greater protection for spermatozoa during cryopreservation.

Evaluación del potencial de membrana mitocondrial en espermatozoides humanos capacitados / Mitochondrial membrane potential evaluation in capacitated human sperm

Introducción: los espermatozoides humanos durante su viaje por el tracto reproductivo sufren el procesode capacitación espermática, evento indispensable para la fecundación y que se ha relacionado con cambios en el potencial de membrana mitocondrial. Objetivos: evaluar el efecto de la capacitaciónespermática sobre el potencial de membrana mitocondrial en espermatozoides humanos. Materiales y métodos: se realizó un estudio in vitro en el cual se seleccionaron los espermatozoides puros de nueve voluntarios, los cuales se sometieron a condiciones capacitantes durante seis horas. Posteriormente, se evaluó el efecto de la capacitación sobre el estado del potencial de membrana mitocondrial usando el colorante DiOC6(3) por citometría de flujo. Resultados: de las nueve muestras seminales evaluadas cinco presentaron mayor intensidad media de fluorescencia en el potencial de membrana mitocondrial poscapacitación. Todas las muestras seminales mostraron parámetros normales y no presentaron cambios en la movilidad y la viabilidad espermática durante la selección y la capacitación espermática in vitro. Conclusiones: las modificaciones en el potencial de membrana mitocondrial en respuesta al proceso de capacitación demuestra que existen diferentes poblaciones espermáticas en el eyaculado humano; entender cuál está relacionada con el proceso reproductivo exitoso permitirá aumentar la probabilidad de embarazos.

Introduction: human spermatozoa during their journey through the reproductive tract undergo the capacitation process, an essential event for the fertilization and which has been related with changesin membrane mitochondrial potential. Objective: To evaluate the effect of capacitation process on mitochondrial membrane potential in human sperm. Materials and methods: An in vitro study in which pure sperm from nine semen samples were incubated in capacitated conditions for six hours. Subsequently, the capacitation effect on the state of mitochondrial membrane potential using DiOC6(3) dye through flow cytometry were evaluated. Results: Nine semen samples were evaluated; five had higher mean fluorescence intensity in the mitochondrial membrane potential post capacitation.All semen samples had normal seminal parameters and none of them showed changes in motility and sperm viability during recovery and in vitro capacitation. Conclusions: Changes in the mitochondrial membrane potential in response to the capacitation process shows that there are different sperm populations in the human ejaculated; understand which are related to the successful reproductive process will increase the likelihood of pregnancy.

Morfometria corpórea, características do sêmen, proteínas seminais e testosterona em cervos Cervus unicolor, em cativeiro / Body morphometry, semen characteristics, seminal proteins and testosterone in sambar deer (Cervus unicolor) in captivity

Objetivou-se estudar a morfometria corpórea, as características do sêmen, o perfil proteico do plasma seminal em SDS-PAGE e a concentração sérica de testosterona em cervos-sambar (Cervus unicolor), criados em cativeiro, na estação reprodutiva da primavera. Quatro machos com idades entre 12 e 36 meses foram avaliados em quatro momentos, com intervalos de sete dias, com peso corpóreo (60,5 a 89,0kg), índice de massa corporal (93,07kg/m2 a 126,56kg/m2), volume do ejaculado (0,50±0,35mL a 0,75±0,28mL), motilidade espermática (87,75±4,78% a 90,00±7,07%), defeitos totais (17,25±5,81% a 47,72±17,55%), testosterona sérica (6,43±4,33ng/dL a 166,00±64,48ng/dL) e proteínas do plasma seminal com bandas entre 7,6 e 142kDa. As características dos ejaculados não diferiram (P>0,05) entre as três primeiras colheitas. Houve diferença (P<0,05) para os defeitos espermáticos com elevação na quarta colheita. No plasma seminal de cada cervo, foram identificadas de 16 a 27 bandas de proteínas entre 7,6 e 142kDa. Conclui-se que a qualidade espermática foi satisfatória na primavera. O estresse das contenções sucessivas causou queda da qualidade espermática. A idade influi na concentração sérica de testosterona, a qual foi maior nos cervos aos 36 meses...

The aim of this work was to study the body morphometry, semen characteristics, seminal plasma protein profile in SDS-PAGE and serum testosterone concentration in Sambar Deer (Cervus unicolor), in captivity in the breeding season (spring). Four males aged between 12 and 36 months were assessed in four moments with intervals of seven days with body weight (60.5 to 89.0kg), body mass index (93.07 to 126.56kg/m2), ejaculate volume (0.50±0.35mL to 0.75±0.28mL), sperm motility (87.75±4.78% to 90.00±7.07% ), total defects (17.25±5.81% to 47.72±17.55%), serum testosterone (6.43±4.33 ng/dL to 166.00±64.48ng/dL) and seminal plasma proteins with bands between 7.6 and 142 kDa. The characteristics of ejaculates did not differ (P>0.05) among ejaculates (1st, 2nd and 3rd). There were differences (P<0.05) for sperm defects elevation on the fourth ejaculate. In seminal plasma 16 to 27 protein bands between 7.6 and 142 kDa were identified. In conclusion, sperm quality was satisfactory in the spring and the stress of successive contentions decreased sperm quality. Also, there is influence of age upon serum testosterone concentration which was higher in deer at 36 months...

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.

Características do sêmen de carneiros Dorper, Santa Inês e sem padrão racial definido, pré e pós-congelação, nos períodos chuvoso e seco / Characteristics of the semen of Dorper, Santa Ines and undefined breed sheep, pre-and post-freezing, in the rainy and dry period

Avaliaram-se as características espermáticas de carneiros Dorper, Santa Inês e sem padrão racial definido, nos períodos chuvoso e seco. Após ser colhido por vagina artificial, o sêmen foi avaliado quanto ao volume, ao turbilhonamento, à motilidade, ao vigor, à morfologia e à concentração, congelado e armazenado em botijão criogênico. Depois de descongelado, foram avaliadas a cinemática espermática, a integridade da membrana plasmática, a integridade do acrossoma e a atividade mitocondrial. Vigor espermático, motilidade total, motilidade progressiva, velocidade em linha reta e defeitos maiores não diferiram entre os períodos chuvoso e seco, porém volume, turbilhonamento, linearidade, retilinearidade e frequência de batimentos de cauda foram mais baixos (P<0,05) no período seco; já concentração espermática e defeitos totais apresentaram valores mais baixos no período chuvoso. Valores de integridade do acrossoma e da membrana plasmática, bem como o potencial de membrana mitocondrial, foram mais baixos (P<0,05) no período seco. Conclui-se que os períodos chuvoso e seco influenciam na qualidade espermática de ovinos criados na região Meio-Norte do Brasil e que esses animais têm uma qualidade espermática superior no período chuvoso, quando, portanto, deve ocorrer a criopreservação. Também se observou que, em relação à qualidade espermática, o melhor grupo de carneiros foi o Santa Inês...

The sperm characteristics of Dorper, Santa Ines and undefined breed ram in the rainy and dry seasons was assessed. After collection with an artificial vagina, the semen was evaluated for volume, turbulence, motility, viability, morphology and concentration, frozen and stored in a cryogenic cylinder. After thawing kinematic sperm, plasma membrane integrity, acrosome integrity and mitochondrial activity were evaluated. Sperm vigor, total motility, progressive motility, straight line speed and larger line defects did not differ between the rainy and dry seasons, however, volume, turbulence, linearity, straightness and frequency of tail beats were lower (P<0.05 ) in the dry period, and sperm concentration and total defects showed lower values during the rainy season. Values for acrosome integrity and plasma membrane and mitochondrial membrane potential were lower (P<0.05) in the dry season. It is concluded that the wet and dry period influence sperm quality in ram raised in the Mid - North region of Brazil, with a higher sperm quality in the rainy season, thus suggesting cryopreservation for that period. Also, the better ram group regarding sperm quality, was the Santa Ines...

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.

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.

Qualidade seminal e morfometria testicular de coelhos (Oryctolagus cuniculus) suplementados com geleia real / Seminal quality and testicular morphometry of rabbits (Oryctolagus cuniculus) supplemented with royal jelly

Objetivou-se avaliar o efeito da geleia real na qualidade seminal e na morfometria testicular de coelhos. Quatorze coelhos adultos da raça Nova Zelândia foram distribuídos em três grupos: com administração diária de 1mL de água, via oral (SG); administração diária de 0,5mg (0,5G) de geleia real diluída em 1mL de água, via oral; e administração diária de 1,0mL (1,0G) de geleia real diluída em 1mL de água, via oral. O fornecimento de geleia real foi iniciado 30 dias antes das coletas de sêmen, permanecendo durante todo o período de coleta, totalizando 90 dias. Utilizou-se o método da vagina artificial para coleta de sêmen. Foram avaliados os parâmetros físicos e morfológicos do sêmen e os parâmetros de morfometria testicular. Houve diferença no volume seminal do 0,5G (0,54±0,22) em relação ao SG (0,39±0,13) e ao 1,0G (0,30±0,09) (P<0,05). Para os grupos SG, 0,5G e 1,0G, não houve diferença (P>0,05) para turbilhonamento espermático, concentração espermática, motilidade progressiva e vigor espermático. Os defeitos maiores no grupo 0,5G (8,52±3,26) foram menores do que nos grupos SG (14,09±4,26) e 1,0G (16,1±3,95) (P<0,05). Não houve diferença entre os defeitos menores e os defeitos totais (P>0,05). Os pesos corporal, testicular, epididimário e o índice gonadossomático não diferiram entre os grupos (P>0,05). A ingestão diária de 0,5mg de geleia real apresentou efeitos positivos na morfologia espermática de coelhos.

A trial was carried out to evaluate the effects of royal jelly on the seminal quality and testicular morphometry of rabbits. Fourteen mature rabbits of New Zealand breed were distributed between three groups. The first group was supplied with 1mL of water only (SG), the second group was supplied with 0.5mg of royal jelly diluted in 1mL of water (0.5G), and the third group was supplied with 1mg of royal jelly also diluted in 1mL of water (1.0G). The royal jelly supply started 30 days before semen collection and lasted the entire experimental period. An artificial vagina was used to collect the rabbits' semen. Physical and morphological parameters in the semen and the testicular morphometry were evaluated. Differences were found on the seminal volume in group 0.5G(0,54±0,22) in relation to SG (0,39±0,13) and 1.0G (0,30±0,09) groups (P<0.05). For SG, 0.5G and 1.0G groups, no differences (P>0.05) were found in sperm concentration, gross motility, individual motility and vigor. The total of primary defects in group 0.5G (8,52±3,26) was lower than in groups SG (14,09±4,2) and 1.0G (16,1±3,95) (P<0.05). No significant difference was found between secondary defects and the total defects on the semen (P>0.05). Body, testicular and epididymal weights did not differ between groups, as well to the gonadosomatic index (P>0.05). The ingestion of royal jelly produced positive results on the seminal production of males.

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.

Comparación entre Testsimplets® y Diff-Quik para la evaluación de la morfología espermática / Comparison between Testsimplets® and Diff-Quik to assess sperm morphology

Comparar la evaluación de morfología espermática mediante dos tinciones: Testsimplets® y Diff-Quik y la eficiencia de Testsimplets® y el test de peroxidasa para evaluar leucocitos en semen. Comparamos la morfología espermática de las muestras seminales de 30 pacientes infértiles, evaluada a través de las tinciones Testsimplets® y Diff-Quik. Adicionalmente, en pacientes que presentaron más de 106 de células redondas por mL de semen (n=7), comparamos la evaluación de leucocitos por Testsimplets® y el test de peroxidasa. En el Centro de fertilidad UNIFERTES, en Caracas, Venezuela. No existen diferencias significativas en el porcentaje de espermatozoides morfológicamente normales, siguiendo el criterio estricto de Kruger, entre ambas tinciones. Encontramos diferencias significativas en los porcentajes de los siguientes defectos morfológicos espermáticos, entre tinción: cabeza piriforme, pieza media doblada, irregular o con gota citoplasmática. No se encontraron diferencias significativas en la evaluación de leucocitos en semen mediante Testsimplets® y el test de peroxidasa. Testsimplets® es práctico para ahorrar tiempo y, consecuentemente, mejorar la eficiencia en el tiempo de entrega de resultados. Sin embargo, es considerablemente más costoso que Diff-Quik

To compare sperm morphology by Testsimplets® and Diff-Quik staining. To compare the efficiency of Staining cellular peroxidase and Testsimplets® to assess leukocytes in semen. We compared sperm morphology of 30 infertile patients evaluated by Testsimplets® and Diff-Quik staining. Additionally, in patients with more than 106 round cells per ml semen (n=7), we compared the assessment of leukocytes by Staining cellular peroxidase and Testsimplets®. Fertility clinic UNIFERTES, in Caracas, Venezuela. There was no significant difference in the percentage of morphologically normal sperm, according to Kruger’s strict criteria, between both stains. We found significant differences in the percentages of the following sperm morphological defects between staining: pyriform head, folded middle piece, irregular and presence of cytoplasmic droplets. There was no significant difference in the assessment of leukocytes in semen by Staining cellular peroxidase and Testsimplets®. Testsimplets® is practical for saving time and consequently, improving the efficiency in the delivery time of results. Nonetheless, it is considerably more expensive than Diff-Quik