Lectin spatial immunolocalization during in vitro capacitation in Tursiops truncatus spermatozoa

Spermatozoa interactions with the female reproductive tract and oocyte are regulated by surface molecules such as glycocalyx. The capacitation process comprises molecular and structural modifications which increase zona pellucida binding affinity. Lectins allowed us to describe glycocalyx changes during maturation, capacitation and acrosome reaction. This study had as its aim to identify lectin binding patterns using four lectins with different carbohydrate affinity in bottlenose dolphin (Tursiops truncatus) spermatozoa both before and after in vitro capacitation. Two semen samples from the same dolphin obtained on consecutive days were used, with four different lectin binding patterns becoming visible in both samples before and after capacitation. A highly stained equatorial segment with prolongations at the edges appeared as the most frequent pattern with Wheat germ agglutinin (WGA) in uncapacitated spermatozoa. However, it was homogeneously distributed over the acrosomal region after capacitation. Instead, the use of Peanut agglutinin (PNA) resulted in most spermatozoa showing high labelling in the acrosomal periphery region before capacitation and a homogeneous staining in the acrosomal region within the population of capacitated spermatozoa. Nevertheless, the most representative patterns with Concavalin A (ConA) and Aleuria aurantia agglutinin (AAA) lectins did not change before and after capacitation, labelling the acrosomal region periphery. These findings could contribute to the understanding of the reproductive biology of cetaceans and the improvement of sperm selection techniques.(AU)

Lectin spatial immunolocalization during in vitro capacitation in Tursiops truncatus spermatozoa

Spermatozoa interactions with the female reproductive tract and oocyte are regulated by surface molecules such as glycocalyx. The capacitation process comprises molecular and structural modifications which increase zona pellucida binding affinity. Lectins allowed us to describe glycocalyx changes during maturation, capacitation and acrosome reaction. This study had as its aim to identify lectin binding patterns using four lectins with different carbohydrate affinity in bottlenose dolphin (Tursiops truncatus) spermatozoa both before and after in vitro capacitation. Two semen samples from the same dolphin obtained on consecutive days were used, with four different lectin binding patterns becoming visible in both samples before and after capacitation. A highly stained equatorial segment with prolongations at the edges appeared as the most frequent pattern with Wheat germ agglutinin (WGA) in uncapacitated spermatozoa. However, it was homogeneously distributed over the acrosomal region after capacitation. Instead, the use of Peanut agglutinin (PNA) resulted in most spermatozoa showing high labelling in the acrosomal periphery region before capacitation and a homogeneous staining in the acrosomal region within the population of capacitated spermatozoa. Nevertheless, the most representative patterns with Concavalin A (ConA) and Aleuria aurantia agglutinin (AAA) lectins did not change before and after capacitation, labelling the acrosomal region periphery. These findings could contribute to the understanding of the reproductive biology of cetaceans and the improvement of sperm selection techniques.

Mini-Percoll technique induces similar capacitation features in domestic ruminant frozen-thawed spermatozoa regardless of the presence of heparin

Background: Sperm capacitation is a process consists of a series of functional, biochemical, and biophysical modificationsthat render the ejaculated sperm competent for oocyte fertilization. Secreted by the female reproductive tract epithelium,heparin promotes capacitation by binding to and removing seminal plasma proteins, which are adsorbed to the sperm PMand would inhibit capacitation. There is substantial evidence that cryopreservation promotes capacitation-like changes inbull, ram and buck sperm. Our general hypotheses were: (a) cryopreserved ram sperm suffer capacitation more quicklythan buck and bull sperm under the same conditions; (b) the capacitation status of ruminant cryopreserved sperm is similarwhether or not heparin is present after the mini-Percoll technique; and (c) ruminant frozen-thawed sperm selected by miniPercoll and incubated within media without heparin supplementation is not impaired in terms of capacitation status andsperm agglutination. This study aimed to compare sperm parameters of ovine, caprine, and bovine frozen-thawed spermafter mini-Percoll processing followed by incubation with or without heparin supplementation.Materials, Methods & Results: Commercial semen of all species were used. Sperm samples were selected by mini-Percolland supplemented (or not) with heparin within an incubation medium for 18 h. Sperm kinematics (CASA system analyzes),capacitation status (CTC staining) and sperm agglutination were evaluated after thawing, mini-Percoll, 1.5 h, 3 h, 6 h and18 h. In comparison with post-thawing analysis, ovine species demonstrated a reduction (P < 0.05) in most of the spermmotility parameters after mini-Percoll. Conversely, ovine samples presented...

Mini-Percoll technique induces similar capacitation features in domestic ruminant frozen-thawed spermatozoa regardless of the presence of heparin

Background: Sperm capacitation is a process consists of a series of functional, biochemical, and biophysical modificationsthat render the ejaculated sperm competent for oocyte fertilization. Secreted by the female reproductive tract epithelium,heparin promotes capacitation by binding to and removing seminal plasma proteins, which are adsorbed to the sperm PMand would inhibit capacitation. There is substantial evidence that cryopreservation promotes capacitation-like changes inbull, ram and buck sperm. Our general hypotheses were: (a) cryopreserved ram sperm suffer capacitation more quicklythan buck and bull sperm under the same conditions; (b) the capacitation status of ruminant cryopreserved sperm is similarwhether or not heparin is present after the mini-Percoll technique; and (c) ruminant frozen-thawed sperm selected by miniPercoll and incubated within media without heparin supplementation is not impaired in terms of capacitation status andsperm agglutination. This study aimed to compare sperm parameters of ovine, caprine, and bovine frozen-thawed spermafter mini-Percoll processing followed by incubation with or without heparin supplementation.Materials, Methods & Results: Commercial semen of all species were used. Sperm samples were selected by mini-Percolland supplemented (or not) with heparin within an incubation medium for 18 h. Sperm kinematics (CASA system analyzes),capacitation status (CTC staining) and sperm agglutination were evaluated after thawing, mini-Percoll, 1.5 h, 3 h, 6 h and18 h. In comparison with post-thawing analysis, ovine species demonstrated a reduction (P < 0.05) in most of the spermmotility parameters after mini-Percoll. Conversely, ovine samples presented...(AU)

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)

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)

Comparison of different sperm selection techniques in ram frozen-thawed sperm

Background: The success of fertilization is directly associated with semen quality and the sperm preparation. Considering the common use of cryopreserved spermatozoa, there is a need to develop strategies for sperm preparation in order to achieve a sperm sample of high quality through a rigorous selection of sperm. Thus, sperm cells are being more extensively investigate. This study aimed evaluating the influence of different sperm selection techniques on ram sperm parameters in semen preparation.Materials, Methods & Results: Frozen-thawed commercial semen from 10 Santa Inês rams was subjected to either: swim-up, Percoll, mini-Percoll, sperm washing by centrifugation or a control group. After each technique, samples were incubated at 37o.C for 1 h, 2 h and 3 h. At post-selection moment (0 h) and at each interval, sperm recovery rate, motility, capacitation and plasma membrane (PM) integrity were analyzed. The lowest (P 0.05). Most part of motility parameters were not affected (P > 0.05) by the technique at 0 h; just swim-up obtained higher (P 0.05) capacitated [...]

Comparison of Different Sperm Selection Techniques in Ram Frozen-Thawed Sperm

Background: The success of fertilization is directly associated with semen quality and the sperm preparation. Considering the common use of cryopreserved spermatozoa, there is a need to develop strategies for sperm preparation in order to achieve a sperm sample of high quality through a rigorous selection of sperm. Thus, sperm cells are being more extensively investigate. This study aimed evaluating the influence of different sperm selection techniques on ram sperm parameters in semen preparation.Materials, Methods & Results: Frozen-thawed commercial semen from 10 Santa Inês rams was subjected to either: swim-up, Percoll, mini-Percoll, sperm washing by centrifugation or a control group. After each technique, samples were incubated at 37C for 1 h, 2 h and 3 h. At post-selection moment (0 h) and at each interval, sperm recovery rate, motility, capacitation and plasma membrane (PM) integrity were analyzed. The lowest (P 0.05) recovery rate was recorded after swim-up (1.0 ± 0.3%), whilst the others were similar (P > 0.05). Most part of motility parameters were not affected (P > 0.05) by the technique at 0 h; just swim-up obtained higher (P 0.05) values for VSL (41.8 ± 11.1) and VAP (46.9 ± 11.2). Overall, swim-up presented higher (P 0.05) values for most of motility parameters over time of incubation. The control group led to more (P 0.05) capacitated

Comparison of different sperm selection techniques in ram frozen-thawed sperm

Background: The success of fertilization is directly associated with semen quality and the sperm preparation. Considering the common use of cryopreserved spermatozoa, there is a need to develop strategies for sperm preparation in order to achieve a sperm sample of high quality through a rigorous selection of sperm. Thus, sperm cells are being more extensively investigate. This study aimed evaluating the influence of different sperm selection techniques on ram sperm parameters in semen preparation.Materials, Methods & Results: Frozen-thawed commercial semen from 10 Santa Inês rams was subjected to either: swim-up, Percoll, mini-Percoll, sperm washing by centrifugation or a control group. After each technique, samples were incubated at 37o.C for 1 h, 2 h and 3 h. At post-selection moment (0 h) and at each interval, sperm recovery rate, motility, capacitation and plasma membrane (PM) integrity were analyzed. The lowest (P < 0.05) recovery rate was recorded after swim-up (1.0 ± 0.3%), whilst the others were similar (P > 0.05). Most part of motility parameters were not affected (P > 0.05) by the technique at 0 h; just swim-up obtained higher (P < 0.05) values for VSL (41.8 ± 11.1) and VAP (46.9 ± 11.2). Overall, swim-up presented higher (P < 0.05) values for most of motility parameters over time of incubation. The control group led to more (P > 0.05) capacitated [...](AU)

Sobrevivência in vitro de sêmen criopreservado equino e de ruminantes após indução à capacitação espermática e da reação acrossômica para aplicação na produção in vitro de embriões

O objetivo deste estudo foi determinar a eficiência de um protocolo de indução de capacitação espermática e de reação acrossômica in vitro em sêmen criopreservado de espécies mamíferas domésticas para uso na fecundação in vitro (FIV), utilizando a espécie bovina como modelo e controle. No Experimento I, comparou-se o efeito de diferentes concentrações de heparina (5, 10 e 15 UI/mL) na indução da capacitação espermática (CE) in vitro do sêmen criopreservado das espécies bovina, bubalina, ovina, caprina e equina. Também se avaliou a resposta do sêmen bovino capacitado in vitro ao cálcio ionóforo (A23187) para a indução da reação acrossômica (RA) in vitro e, por fim, este foi utilizado para a FIV de oócitos bovinos sem zona pelúcida (ZP) para avaliar as taxas de fecundação monospérmica e polispérmica. No Experimento II, avaliou-se a resposta do sêmen equino criopreservado frente a concentrações maiores de heparina (50 e 100 UI/mL) e de cálcio ionóforo (A23187) para a indução da CE e da RA in vitro. A sobrevivência dos espermatozoides após o descongelamento, a incubação, a CE e a RA, foram avaliadas pelas colorações com azul de tripano-Giemsa, e com clortetraciclina (CTC), respectivamente. No Experimento I, a espécie bovina apresentou baixa mortalidade espermática após incubação (7 a 16%) e foi responsiva ao tratamento com heparina (59 a 84%), havendo diferença nas respostas dos touros frente à heparina (p<0,05), conforme esperado. As espécies bubalina, equina e ovina não responderam aos tratamentos com heparina (<20%) e apresentaram elevada mortalidade após a incubação com heparina (18 a 77%), havendo uma correlação negativa entra as taxas de mortalidade e a proporção de espermatozoides capacitados (p<0,05; r= -0,70). Já o sêmen caprino apresentou menor mortalidade (19 a 42%) e resposta moderada à heparina (14 a 31%). A FIV de oócitos bovinos sem ZP com espermatozoides bovinos após CE e RA in vitro resultou em maiores taxas de fecundação monospérmica (40%) e menores de polispermia (2%) em comparação à FIV convencional, com oócitos com ZP (24% e 18%, respectivamente; p<0,05). No Experimento II, dois de cinco garanhões mostraram ser mais sensíveis à heparina (10 a 30%) e ao cálcio ionóforo (A23187) para a indução da RA in vitro (52 a 67%), mas com elevadas taxas de mortalidade após a incubação (49 a 74%). Contudo, o protocolo aplicado no presente trabalho resultou na indução da capacitação espermática e reação acrossômica in vitro do sêmen equino criopreservado.

This study aimed to determine the efficiency of a protocol for the induction of in vitro sperm capacitation and acrosome reaction in frozen semen of domestic species for use in in vitro fertilization (IVF) procedures, using bovine semen as control. In Experiment I, the effect of different heparin concentrations (5, 10 and 15 IU/mL) on the induction of in vitro sperm capacitation (SC) of frozen bovine, bubaline, ovine, caprine and equine semen was compared. The response of capacitated frozen bovine semen to calcium ionophore (A23187) for induction of in vitro acrosome reaction (AR) was also evaluated, being used for the IVF of zona-free bovine oocytes to assess monospermic and polyspermic fertilization rates. In Experiment II, the response of frozen equine semen to heparin (50 e 100 IU/mL) and calcium ionophore (A23187) for induction of in vitro SC and AR was evaluated. Sperm survival rates after thawing, incubation, SC AR were evaluated by trypan blue-Giemsa and by chlortetracycline (CTC) stains, respectively. In Experiment I, bovine semen had low sperm mortality after incubation (7 to 16%) and high response to heparin (59 to 84%), with bulls showing different responses to heparin. Buffalo, equine and ovine semen did not respond to heparin treatments (<20% of capacited cells) and showed high cell mortality after incubation (18 to 77%), with a negative correlation observed between mortality and the proportion of capacitated sperm cells (p<0,05; r= -0.70). Goat semen, in turn, had moderate mortality (19 to 42%) and response to heparin (14 to 31%). The IVF of zona-free bovine oocytes with in vitro acrosome reacted bovine sperm resulted in higher monospermic fertilization rates (40%) and lower polyspermia (2%) than conventional IVF using intact oocytes (24% and 18%, respectively; p<0.05). In Experiment II, two out of five stallions responded to heparin (10 a 30%) and to calcium ionophore to induce AR in vitro (52 a 67%), with high mortality rates after incubation (49 to 74%). However, the protocol used in the present study resulted in the in vitro sperm capacitation and acrosome reaction of cryopreserved equine semen.

EFFECTS OF BOVINE SERUM ALBUMIN (BSA) AND BICARBONATE ON THE ACHIEVEMENT OF IN VITRO CAPACITATION AND SUBSEQUENT PROGESTERONEINDUCED ACROSOME REACTION IN BOAR SPERMATOZOA

Objetivou-se avaliar os efeitos de diferentes concentrações de bicarbonato e albumina sérica bovina (BSA) adicionados ao meio de capacitação in vitro sobre a indução da reação acrossômica no sêmen suíno. Oito diferentes tratamentos com diferentes concentrações de bicarbonato (0, 5, 15 e 38 mM) e BSA (0 e 5 mg/ml) foram testados: 1) controle negativo (No BSA / No Bic), o meio básico sem bicarbonato ou BSA; 2) meio básico suplementado apenas com 5 mg / mL de BSA (BSA / No Bic); 3) meio básico suplementado com 5 mM de NaHCO3 e 5 mg / mL de BSA (BSA + 5 mM Bic); 4) meio básico suplementado apenas com 5 mM de NaHCO3 (sem BSA + 5 mM Bic); 5) meio básico suplementado com 5 mM de NaHCO3 e 5 mg / mL de BSA (BSA + 15 mM Bic); 6) meio básico suplementado apenas com 15 mM de NaHCO3 (sem BSA + 15 mM Bic); 7) meio básico suplementado com NaHCO3 38 mM e 5 mg / mL de BSA (BSA + Bic 38 mM); e 8) meio básico suplementado apenas com NaHCO3 38 mM (sem BSA + Bic 38 mM). As células espermáticas foram submetidas aos meios e incubadas a 38,5 C com 5% de CO2 por 5h. A motilidade espermática, integridade de membrana, distúrbio lipídico de membrana, níveis de cálcio intracelular, atividade mitocondrial e níveis de fosforilação de tirosina de GSK3 e DARPP32 foram determinados após 0, 2 e 4 h de incubação e também após 30 e 60 min da adição de progesterona. Os meios sem BSA não se capacitaram, obtendo resultados semelhantes ao meio não capacitante (sem BSA e sem bicarbonato) (P<0,05). Os meios com BSA e bicarbonato a 15 ou 38 mM apresentaram (P<0,05) altos níveis de cálcio (Fluo-3 e Rhod 5), desordem lipídica (M540) e fosforilação de proteínas (GSK3). O meio com BSA e bicarbonato a 15 mM apresentou (P<0,05) maior motilidade e viabilidade que o meio com BSA e bicarbonato a 38 mM. O meio com BSA e sem bicarbonato ou com bicarbonato a 5 mM se capacitaram, porém com menores indices de capacitação que os meios que contiam BSA e bicarbonato a 15 ou 38 mM. Os resultados mostraram que a capacitação in vitro de sêmen suíno está relacionada com a presença de BSA no meio capacitante e a presença do bicarbonato não é um fator limitante para a capacitação. O meio que obteve melhor resultado de capacitação foi o BSA com bicarbonato a 15 mM.

The objective of the present work was to evaluate the effects of different concentrations of bicarbonate and bovine serum albumin (BSA), added to the in vitro capacitation medium, on the induction of the acrosome reaction in boar semen. Eight different treatments with different concentrations of bicarbonate (0, 5, 15 and 38 mM) and BSA (0 and 5 mg / ml) were tested: 1) negative control (No BSA / No Bic), which was the basic medium without bicarbonate or BSA; 2) basic medium supplemented with 5 mg/mL BSA only (BSA / No Bic); 3) basic medium supplemented with 5 mM NaHCO3 and 5 mg/mL BSA (BSA + 5 mM Bic); 4) basic medium supplemented with 5 mM NaHCO3 only (No BSA + 5 mM Bic); 5) basic medium supplemented with 5 mM NaHCO3 and 5 mg/mL BSA (BSA + 15 mM Bic); 6) basic medium supplemented with 15 mM NaHCO3 only (No BSA + 15 mM Bic); 7) basic medium supplemented with 38 mM NaHCO3 and 5 mg/mL BSA (BSA + 38 mM Bic); and 8) basic medium supplemented with 38 mM NaHCO3 only (No BSA + 38 mM Bic). Sperm cells were submitted to the medium and incubated at 38.5 C with 5% of CO2 for 5 h. Sperm motility, membrane integrity, membrane lipid disorder, intracellular calcium levels, mitochondrial activity and tyrosine phosphorylation levels of GSK3 and DARPP32 were determined after 0, 2 and 4 h of incubation and after 30 and 60 min of the addition of progesterone. The medium without BSA did not capacitated, generating similar results to the non-capacitive medium (without BSA and without bicarbonate) (P <0.05). Mediums with BSA and bicarbonate at 15 or 38 mM showed high (P <0.05) levels of calcium (Fluo-3 and Rhod 5), lipid disorder (M540) and protein phosphorylation (GSK3). The medium with BSA and 15 mM bicarbonate presented (P <0.05) greater motility and viability than the medium with BSA and bicarbonate at 38 mM. The medium with BSA and without bicarbonate or with 5 mM bicarbonate, were capacitated, however with lower indices than medium containing BSA and bicarbonate at 15 or 38 mM. The analyzes showed that the in vitro capacitation of boar semen is related to the presence of BSA in the capacitive medium and the presence of bicarbonate is not a limiting factor for the capacitation. The medium that obtained the best qualification result was BSA with bicarbonate at 15 mM.

COMPARAÇÃO DO PERFIL PROTEICO DE ESPERMATOZOIDES EM DIFERENTES ESPÉCIES

Proteínas presentes no plasma seminal de animais domésticos apresentam diversas funções na fisiologia espermática, atuando em processos de capacitação, reação acrossômica, interação entre gametas e proteção ao espermatozoide. Desse modo, o presente estudo caracterizou proteínas presentes em células espermáticas de ovinos, caprinos, equinos, suínos e cães. Por meio de espectrometria de massas, foram identificadas um total de 128 proteínas no espermatozoide das espécies utilizadas. Desse total, proteínas identificadas foram encontradas em todas as espécies, no entanto seis proteínas estavam presentes em quatro das cinco espécies estudadas, sendo elas a angiotensin-converting enzyme, ATP-synthase subunit a, hexoquinase 1, malate dehydrogenase, pyruvate dehydrogenase e succinyl-CoA. Foi possível identificar 15 proteínas comuns às espécies ruminantes, além de terem sido encontradas ainda 11 proteínas exclusivas em ruminantes e 2 proteínas exclusivas em não ruminantes. Esse estudo foi o primeiro a caracterizar as proteínas presentes no espermatozoide de cães, além de comparar cinco espécies incluindo animais ruminantes e não-ruminantes. Apesar da alta similaridade entre algumas espécies, o espermatozoide apresenta diferenças proteicas na sua composição, o que poderia explicar as diferentes variações no processo de fertilização.

Proteins present in seminal plasma of domestic animals present several functions in sperm physiology, acting in capacitation processes, acrosome reaction, interaction between gametes and sperm protection. Thus, the present study characterized proteins present in sperm cells from sheep, goats, horses, pigs and dogs. By means of mass spectrometry, a total of 128 proteins were identified in the spermatozoa of the species used. From this total, identified proteins were found in all species, however six proteins were present in four of the five species studied: angiotensin-converting enzyme, ATP synthase subunit, hexokinase 1, malate dehydrogenase, pyruvate dehydrogenase and succinyl- CoA. It was possible to identify 15 proteins common to ruminant species, besides 11 exclusive proteins were found in ruminants and 2 proteins exclusive to non-ruminants. This study was the first to characterize the proteins present in the spermatozoon of dogs, in addition to comparing five species including ruminant and non-ruminant animals. Despite the high similarity between some species, the spermatozoid presents protein differences in its composition, which could explain the different variations in the fertilization process.

Participação da via L-arg/NO/GMPc na capacitação in vitro e seu impacto no perfil proteômico de espermatozoides bovinos

A L-arginina (L-arg) tem sido utilizada nos estudos em reprodução animal por ser precursora da síntese de óxido nítrico (NO). O NO por sua vez desempenha um papel importante em espermatozoides, nas vias de sinalização que levam à fosforilação de proteínas, evento necessário à capacitação espermática. Entretanto faltam estudos moleculares que caracterizem as proteínas relacionadas a esse processo e que justifiquem os efeitos positivos do NO observado em estudos anteriores na capacitação espermática in vitro em bovinos. Portanto, esse estudo teve por objetivo principal caracterizar o proteoma dos espermatozoides bovinos após a capacitação in vitro com adição de L-arg. Além disso, objetivou-se elucidar se os efeitos da L-arg durante a capacitação espermática de bovinos são relacionados à ativação da via NO/GMPc/PKG1. Em um primeiro estudo, objetivou-se caracterizar as proteínas, pela espectrometria de massas, de espermatozoides capacitados in vitro com L-arg ou não (controle) e relacionar esses achados com a qualidade espermática, avaliada pela integridade de membrana, atividade mitocondrial, motilidade/vigor espermático e percentual de capacitação. No segundo estudo, objetivou-se elucidar se o tratamento L-arg/NO durante a capacitação espermática in vitro de bovinos, tem ação pela via NO/GMPc/PKG1. Para isso, os espermatozoides foram tratados com L-arg, Rp-8-Br-PET-cGMPS (RP, inibidor seletivo da PKG1) ou o PTIO (quelante do NO), e o GMPc foi avaliado pelo ensaio de ELISA. Em ambos os estudos, os dados foram submetidos à análise de variância e as médias comparadas pelo teste SNK com 5% de probabilidade. No primeiro estudo, todas as características de qualidade avaliadas foram melhores no grupo com L-arg em relação ao controle (P<0,05). Além disso, 367 proteínas foram encontradas, das quais 40 foram diferencialmente expressas entre o grupo com L-arg e o controle, e uma (AKAP3) foi expressa exclusivamente no grupo com L-arg (P<0,05). No segundo estudo, efeitos negativos foram encontrados nos espermatozoides tratados com PTIO e RP em relação ao controle na capacitação espermática (P<0,05), enquanto que na motilidade/vigor, o tratamento com PTIO teve a menor taxa em relação ao controle e demais tratamentos (P<0,05). Na dosagem do GMPc intracelular, observou-se que a concentração foi menor nos tratamentos PTIO e L-arg + PTIO, mas foi mais alto nos tratamentos RP e L-arg + RP em relação ao controle e à L-arg (P<0,05). Com esses resultados, podemos concluir que em bovinos: 1) o sistema L-arg/NO tem um papel importante sobre a qualidade espermática e capacitação espermática, mas independente da via GMPc/PKG1; e 2) foi observado um padrão diferencial de expressão de proteínas entre tratamento e controle, que pode estar diretamente ligado aos mecanismos moleculares envolvidos na ação da L-arg sobre a capacitação in vitro em bovinos.

L-arginine (L-arg) has been used in animal reproduction studies as a precursor of the synthesis of nitric oxide (NO). NO plays an important role in spermatozoa, in the signaling pathways that lead to protein phosphorylation, a necessary event for sperm capacitation. However, molecular studies that characterize the proteins related to this process and that justify the positive effects of NO observed in previous studies on in vitro sperm capacitation in cattle are lacking. Therefore, this study aimed to characterize the bovine sperm proteome after the in vitro capacitation with addition of L-arg. In addition, we aimed to elucidate whether the effects of L-arg during bovine sperm capacitation are related to NO/cGMP/PKG1 pathway activation. In a first study, the objective was to characterize the proteins by mass spectrometry of capacitated sperm in vitro with L-arg (treatment) or not (control) and to relate these findings with the sperm quality, evaluated by membrane integrity, mitochondrial activity, sperm motility/vigor and percentage of capacitation. In the second study, the objective was to elucidate whether the L-arg/NO treatment during the in vitro sperm capacitation of cattle, acts via NO/cGMP/PKG1 signaling pathway. For this, sperm were treated with L-arg, Rp-8-Br-PET-cGMPS (RP, selective inhibitor of PKG1) or PTIO (NO chelator) were used, and cGMP was evaluated by ELISA assay. In both studies, the data were submitted to analysis of variance and the means compared by the SNK test with 5% probability. In the first study, all the quality characteristics evaluated were better in the L-arg group compared to the control (P<0.05). In addition, 367 proteins were found, of which 40 were differentially expressed between L-arg group and control, and one (AKAP3) was expressed exclusively in the L-arg group (P<0.05). In the second study, negative effects were found in the sperm treated with PTIO and RP compared to the control in the sperm capacitation (P<0.05), whereas in the motility/vigor, the treatment with PTIO had the lowest rate compared to the control and other treatments (P<0.05). In the intracellular cGMP measurement, it was observed that the concentration was lower in the PTIO and L-arg + PTIO treatments, but was higher in the RP and L-arg + RP treatments compared to the control and L-arg (P<0.05). With these results, we can conclude that in bovines: 1) the L-arg/NO system plays an important role on sperm quality and sperm capacitation, but independent of the cGMP/PKG1 signaling pathway; and 2) a differential protein expression pattern was observed between treatment and control, which may be directly related to the molecular mechanisms involved in the action of L-arg on in vitro sperm capacitation in cattle.

Membrana plasmática de espermatozoides bovinos: efeito de metabólitos do oxigênio, antioxidantes e criopreservação / Plasmatic membrane of bovine spermatozoa: effect of the species oxygen reactive, antioxidant and cryopreservation

A membrana plasmática é a parte da estrutura do espermatozoide mais susceptível a modificações durante o processo de criopreservação, e sua integridade é fundamental para que os espermatozoides estejam viáveis no momento da fecundação. Sua composição, tipo de fosfolipídeos e quantidade de proteínas são variáveis e influenciam a sensibilidade ao choque térmico. Durante o processo de capacitação, alterações na membrana plasmática são necessárias para que os espermatozoides sejam capazes de efetuar a reação acrossomal e, consequentemente, a fecundação. A presença de metabólitos do oxigênio é necessária para esse processo. No entanto, em excesso, eles são prejudiciais e comprometem a fluidez e a integridade da membrana. Enzimas antioxidantes presentes no plasma seminal e no próprio espermatozoide neutralizam esses metabólitos, evitando estresse oxidativo. Porém, a criopreservação causa desequilíbrio entre produção e neutralização de metabólitos do oxigênio, comprometendo a viabilidade da célula espermática. Na tentativa de amenizar as perdas celulares, diluidores de diferentes composições, inclusive contendo antioxidantes, têm sido testados para a preservação da viabilidade espermática. Esta revisão enfoca a composição lipídica da membrana plasmática do espermatozoide e a produção de metabólitos do oxigênio, relacionando-as com a qualidade espermática de bovinos após o processo de criopreservação e a utilização de diluidores contendo antioxidante.(AU)

The plasmatic membrane is the part of the spermatozoa structure which is the most susceptible to change during cryopreservation and its integrity is fundamental for spermatic viability in the moment of fertilization. Its composition, type of phospholipids and quantity of proteins, is variable and affects the sensibility to thermal shock. During capacitation, alterations occurs in the plasmatic membrane, these alterations are necessary for the spermatozoa to be able to perform the acrosome reaction and hence the fertilization. The presence of free radicals are necessary in this process, however, ROS are harmful in excess and interfere in the fluidity and integrity of the membrane. Antioxidants enzymes in the seminal plasma and in the spermatozoa neutralize these radicals preventing oxidative stress, but the cryopreservation causes imbalance between production and neutralization, compromising the cell´s viability. With the Objective of minimizing cells loss, extenders with different composition, including antioxidants, have been tested for sperm viability preservation This review focuses in the plasmatic membrane composition and species oxygen reactive production, related with quality sperm bovine after cryopreserved process and the use extender containing antioxidant.(AU)

Membrana plasmática de espermatozoides bovinos: efeito de metabólitos do oxigênio, antioxidantes e criopreservação / Plasmatic membrane of bovine spermatozoa: effect of the species oxygen reactive, antioxidant and cryopreservation

A membrana plasmática é a parte da estrutura do espermatozoide mais susceptível a modificações durante o processo de criopreservação, e sua integridade é fundamental para que os espermatozoides estejam viáveis no momento da fecundação. Sua composição, tipo de fosfolipídeos e quantidade de proteínas são variáveis e influenciam a sensibilidade ao choque térmico. Durante o processo de capacitação, alterações na membrana plasmática são necessárias para que os espermatozoides sejam capazes de efetuar a reação acrossomal e, consequentemente, a fecundação. A presença de metabólitos do oxigênio é necessária para esse processo. No entanto, em excesso, eles são prejudiciais e comprometem a fluidez e a integridade da membrana. Enzimas antioxidantes presentes no plasma seminal e no próprio espermatozoide neutralizam esses metabólitos, evitando estresse oxidativo. Porém, a criopreservação causa desequilíbrio entre produção e neutralização de metabólitos do oxigênio, comprometendo a viabilidade da célula espermática. Na tentativa de amenizar as perdas celulares, diluidores de diferentes composições, inclusive contendo antioxidantes, têm sido testados para a preservação da viabilidade espermática. Esta revisão enfoca a composição lipídica da membrana plasmática do espermatozoide e a produção de metabólitos do oxigênio, relacionando-as com a qualidade espermática de bovinos após o processo de criopreservação e a utilização de diluidores contendo antioxidante.

The plasmatic membrane is the part of the spermatozoa structure which is the most susceptible to change during cryopreservation and its integrity is fundamental for spermatic viability in the moment of fertilization. Its composition, type of phospholipids and quantity of proteins, is variable and affects the sensibility to thermal shock. During capacitation, alterations occurs in the plasmatic membrane, these alterations are necessary for the spermatozoa to be able to perform the acrosome reaction and hence the fertilization. The presence of free radicals are necessary in this process, however, ROS are harmful in excess and interfere in the fluidity and integrity of the membrane. Antioxidants enzymes in the seminal plasma and in the spermatozoa neutralize these radicals preventing oxidative stress, but the cryopreservation causes imbalance between production and neutralization, compromising the cell´s viability. With the Objective of minimizing cells loss, extenders with different composition, including antioxidants, have been tested for sperm viability preservation This review focuses in the plasmatic membrane composition and species oxygen reactive production, related with quality sperm bovine after cryopreserved process and the use extender containing antioxidant.

Frozen Jaguar (Panthera onca) sperm capacitation and ability to penetrate zona free hamster oocytes / Capacitação espermática de sêmem congelado de onça pintada (Panthera onca) e ensaio de penetração em oócitos de hamster livres de zona pelúcida

Assisted reproductive technologies in endangered species, such as artificial insemination, in vitro fertilization and embryo transfer, can be viewed as one potential approach for safeguarding species. Toward this aim, the objective of this study was to evaluate the fertility of frozen jaguar (Panthera onca) sperm and Tyrod's Talp PVA capacitation medium using the hamster zona free oocyte penetration assay. Ejaculates were collected from nine animals using electroejaculation and cryopreserved. Sperm capacitation was performed by swim-up technique using Tyrod's Talp PVA medium at room temperature. Penetration was considered when the spermatozoa head decondensation was visualized within the oocyte. This assay showed 15.4 % penetrations (350/2275 oocytes). Results of this study showed high sperm abnormalities, low sperm quality after cryopreservation, and low percentage of penetrations. However, the penetration results showed lhat the cryopreserved jaguar's semen can be used for artificial insemination, in vitro fertilization and intra cytoplasmatic sperm injection, supporting the semen bank creation for this specie.(AU)

Biotecnologias reprodutivas aplicadas a espécies selvagens, como inseminação artificial, fertilização in vitro e transferência de embriões, são vistas como um potencial caminho para proteção das espécies ameaçadas de extinção. Devido a isso, o objetivo deste estudo foi avaliar a fertilidade do sêmen congelado de onças pintadas (Panthera onca) e o meio de capacitação espermática usando o ensaio de penetração em oócitos de hamster livres de zona pelúcida. Ejaculados de nove animais foram coletados por eletroejaculação e criopreservados. Para determinar a capacitação espermática foi utilizada a técnica swin-up com meio Tyrods Talp PVA a temperatura ambiente. No ensaio de penetração em oócitos de hamster livres de zona pelúcida foram considerados como oócitos penetrados aqueles que apresentaram em seu interior a cabeça do espermatozóide descondensada. O ensaio foi realizado em um total de 2275 oócitos, dos quais 350 apresentaram em seu interior a cabeça do espermatozóide descondensada, perfazendo um total de 15.4% de penetração. Os resultados deste estudo demonstraram alto índice de anormalidades espermáticas, baixa qualidade do sêmen e baixa porcentagem de penetrações. Entretanto, os resultados de penetração espermática demonstraram que sêmen congelado de onça pintada poderá ser utilizado para inseminação artificial, fertilização in vitro e injeção intracitoplasmática dando suporte para a criação de um banco de sêmen para esta espécie.(AU)

Comparison of Different Sperm Selection Techniques in Ram Frozen-Thawed Sperm

Background: The success of fertilization is directly associated with semen quality and the sperm preparation. Considering the common use of cryopreserved spermatozoa, there is a need to develop strategies for sperm preparation in order to achieve a sperm sample of high quality through a rigorous selection of sperm. Thus, sperm cells are being more extensively investigate. This study aimed evaluating the influence of different sperm selection techniques on ram sperm parameters in semen preparation.Materials, Methods & Results: Frozen-thawed commercial semen from 10 Santa Inês rams was subjected to either: swim-up, Percoll, mini-Percoll, sperm washing by centrifugation or a control group. After each technique, samples were incubated at 37C for 1 h, 2 h and 3 h. At post-selection moment (0 h) and at each interval, sperm recovery rate, motility, capacitation and plasma membrane (PM) integrity were analyzed. The lowest (P 0.05) recovery rate was recorded after swim-up (1.0 ± 0.3%), whilst the others were similar (P > 0.05). Most part of motility parameters were not affected (P > 0.05) by the technique at 0 h; just swim-up obtained higher (P 0.05) values for VSL (41.8 ± 11.1) and VAP (46.9 ± 11.2). Overall, swim-up presented higher (P 0.05) values for most of motility parameters over time of incubation. The control group led to more (P 0.05) capacitated

Comparison of Different Sperm Selection Techniques in Ram Frozen-Thawed Sperm

Background: The success of fertilization is directly associated with semen quality and the sperm preparation. Considering the common use of cryopreserved spermatozoa, there is a need to develop strategies for sperm preparation in order to achieve a sperm sample of high quality through a rigorous selection of sperm. Thus, sperm cells are being more extensively investigate. This study aimed evaluating the influence of different sperm selection techniques on ram sperm parameters in semen preparation.Materials, Methods & Results: Frozen-thawed commercial semen from 10 Santa Inês rams was subjected to either: swim-up, Percoll, mini-Percoll, sperm washing by centrifugation or a control group. After each technique, samples were incubated at 37C for 1 h, 2 h and 3 h. At post-selection moment (0 h) and at each interval, sperm recovery rate, motility, capacitation and plasma membrane (PM) integrity were analyzed. The lowest (P 0.05) recovery rate was recorded after swim-up (1.0 ± 0.3%), whilst the others were similar (P > 0.05). Most part of motility parameters were not affected (P > 0.05) by the technique at 0 h; just swim-up obtained higher (P 0.05) values for VSL (41.8 ± 11.1) and VAP (46.9 ± 11.2). Overall, swim-up presented higher (P 0.05) values for most of motility parameters over time of incubation. The control group led to more (P 0.05) capacitated

Comparison of Different Sperm Selection Techniques in Ram Frozen-Thawed Sperm

Background: The success of fertilization is directly associated with semen quality and the sperm preparation. Considering the common use of cryopreserved spermatozoa, there is a need to develop strategies for sperm preparation in order to achieve a sperm sample of high quality through a rigorous selection of sperm. Thus, sperm cells are being more extensively investigate. This study aimed evaluating the influence of different sperm selection techniques on ram sperm parameters in semen preparation.Materials, Methods & Results: Frozen-thawed commercial semen from 10 Santa Inês rams was subjected to either: swim-up, Percoll, mini-Percoll, sperm washing by centrifugation or a control group. After each technique, samples were incubated at 37C for 1 h, 2 h and 3 h. At post-selection moment (0 h) and at each interval, sperm recovery rate, motility, capacitation and plasma membrane (PM) integrity were analyzed. The lowest (P 0.05) recovery rate was recorded after swim-up (1.0 ± 0.3%), whilst the others were similar (P > 0.05). Most part of motility parameters were not affected (P > 0.05) by the technique at 0 h; just swim-up obtained higher (P 0.05) values for VSL (41.8 ± 11.1) and VAP (46.9 ± 11.2). Overall, swim-up presented higher (P 0.05) values for most of motility parameters over time of incubation. The control group led to more (P 0.05) capacitated

Comparison of Different Sperm Selection Techniques in Ram Frozen-Thawed Sperm

Background: The success of fertilization is directly associated with semen quality and the sperm preparation. Considering the common use of cryopreserved spermatozoa, there is a need to develop strategies for sperm preparation in order to achieve a sperm sample of high quality through a rigorous selection of sperm. Thus, sperm cells are being more extensively investigate. This study aimed evaluating the influence of different sperm selection techniques on ram sperm parameters in semen preparation.Materials, Methods & Results: Frozen-thawed commercial semen from 10 Santa Inês rams was subjected to either: swim-up, Percoll, mini-Percoll, sperm washing by centrifugation or a control group. After each technique, samples were incubated at 37C for 1 h, 2 h and 3 h. At post-selection moment (0 h) and at each interval, sperm recovery rate, motility, capacitation and plasma membrane (PM) integrity were analyzed. The lowest (P 0.05) recovery rate was recorded after swim-up (1.0 ± 0.3%), whilst the others were similar (P > 0.05). Most part of motility parameters were not affected (P > 0.05) by the technique at 0 h; just swim-up obtained higher (P 0.05) values for VSL (41.8 ± 11.1) and VAP (46.9 ± 11.2). Overall, swim-up presented higher (P 0.05) values for most of motility parameters over time of incubation. The control group led to more (P 0.05) capacitated