Eventos fisiológicos e bioquímicos relacionados a capacitação espermática de garanhões em condições in vitro

Only two foals have ever been born from conventional equine IVF (in vitro fertilization), both in the early 1990’s. The main reason for the conventional IVF failure is thought to be the stallion spermatozoa, which are not able to penetrate the zona pellucida, most likely due to incomplete activation (capacitation) under in vitro conditions. Capacitation is considered as a consecutive activation of different signaling pathways inducing physiological and biochemical modifications which primes the sperm for fertilization in vitro. To have the capacitation occur, the sperm must be under an environment (in vivo or in vitro) that contains bicarbonate (HCO 3- ), calcium (Ca 2+ ) and albumin. These elements will besides changes in membrane potential, provide changes in cyclic adenosine monophosphate (cAMP) levels, intracellular pH and intracellular Ca 2+ causing plasma membrane reorganization and cholesterol depletion leading the sperm to undergo the acrosome reaction and fertilize the oocyte. The aim of this work was to show by flow cytometry, fluorescence microscopy, cryo-electron microscopy and immunohistochemistry some capacitation steps in stallion spermatozoa in the presence of molecules that can trigger this process and also understand which molecules may be involve in some capacitation steps. The use of the probe Annexin-V in capacitating conditions showed a significant increase in the live, Annexin-V positive sperm population indicating a phosphatidylserine (PS) exposure in stallion sperm during this process (p≤0.05), but it was only in a small percentage of viable sperm. Stimulation of the sAC/cAMP/PKA pathway by caffeine and dibutyryl-cAMP indicated the relevance of this pathway for PS exposure. The observation of three different staining patterns for Annexin-V in live stallion sperm may warrant further investigation with respect to whether these represent sequential steps in membrane remodeling. Regarding the effects in membrane fluidity and induced acrosome reaction different concentrations of calcium did not interfere in the population live merocyanine and PNA-positive spermatozoa (p>0.05). Also, different concentrations of progesterone (P4) and bovine serum albumin (BSA) were not able to increase membrane fluidity or induced acrosome reaction (p≤0.05). However, calcium should be present in the medium since EGTA (ethylene glycol-bis (β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid; 2 mM) was able to block all this capacitation steps, but there is no need for higher concentrations (2 mM) to start capacitation in stallion sperm as is needed in other species. Regarding the adenilato cyclases (ACs), the presence of sAC (soluble) in the stallion sperm was shown to be responsible for the increase in membrane fluidity in the presence of bicarbonate. This finding was possible to demonstrate with the use of LRE1, a sAC specific inhibitor that showed no effect in other sperm physiological events (p>0.05). The immunostaining of sAC in stallion sperm indicated different localization compared to boar. In stallions, the enzyme was present as a dotted line (diadem-like pattern) distal from the acrosomal area, while in boars, the sAC was present in the acrosomal area and in the neck. The action of forskolin indicated that tmAC (transmembrane) may be present in the stallion sperm and may play a role in the acrosome reaction. However, the fact that a relatively high concentration (500 µM) was needed to show this effect compared to other reports, indicates that more studies like use of specific inhibitors of tmAC (as ddAdo) or immunostaining of the AC1 to 9 must be performed in stallion sperm to confirm the findings. In conclusion, the findings in this work elucidate several questions in stallion capacitation as PS exposure, role of different concentrations of calcium, progesterone and BSA in this process and the role of adenylyl cyclases in some stallion capacitation events showing that capacitation is a complex process that differs among species. Keywords: Adenylyl cyclases. Capacitation. Membrane reorganization. Stallion spermApenas dois potros foram nascidos de FIV (fertilização in vitro) convencional em equinos, ambos no início dos anos 90. O principal motivo do insucesso da técnica é direcionado ao espermatozoide do garanhão, que não é capaz de penetrar a zona pelúcida e fertilizar o oócito provavelmente devido a sua incompleta ativação (capacitação) em condições in vitro. Capacitação pode ser definida como o conjunto de mudanças físicas e biológicas que acontecem no espermatozoide o tornando apto a fertilizar o oócito em condições in vivo ou in vitro. Para que esse processo ocorra, o espermatozoide deve estar submetido a um ambiente que contenha bicarbonato (HCO 3- ), cálcio (Ca 2+ ) e albumina. Essas substâncias causarão mudanças nas concentrações de adenosina monofosfato cíclico (AMPc), pH e Ca 2+ intracelular, além alteração no potencial de membrana causando reorganização da membrana plasmática e depleção do colesterol, levando ao espermatozoide à reação acrossomal e fertilização do oócito. Infelizmente, nos equinos várias etapas da capacitação ainda não estão bem elucidadas. O objetivo desse trabalho foi demonstrar, por meio do uso de citometria de fluxo, microscopia de fluorescência, crio-eletromicroscopia e imunohistoquímica, algumas etapas da capacitação espermática de garanhões na presença de moléculas que podem estimular esse processo, além de compreender qual adenilato ciclase (solúvel ou transmembrana) está envolvida em algumas das etapas desse evento. O uso da probe Annexina-V em condições capacitantes demonstrou a ocorrência da exposição da fosfatidilserina (PS) em espermatozoides de garanhão durante o processo de capacitação, porém em uma pequena porcentagem da população (p≤0,05). Além disso, o uso de cafeína e dibutiril-AMPc indicam a relevância da via sAC/pkA para esse processo (p≤0,05). A observação de três diferentes padrões de fluorescência da Annexina-V no espermatozoide viável de garanhão sugere etapas sequenciais na remodelação da membrana plasmática. Em relação aos efeitos na fluidez de membrana e reação acrossomal induzida, diferentes concentrações de Ca 2+ não interferiram na população espermática viva, merocianina e FITC-PNA 647 positiva (p≥0,05). O cálcio deve estar presente no meio capacitante uma vez que, na presença de seu quelante EGTA, essas etapas da capacitação foram bloqueadas. Porém o cálcio não deve estar obrigatoriamente presente em elevadas concentrações para iniciar a capacitação em espermatozoides de garanhão como é necessário em outras espécies. Diferentes concentrações de progesterona (P4) e albumina sérica bovina (BSA) também não foram capazes de aumentar a fluidez de membrana ou induzir reação acrossomal nos espermatozoides de garanhão (p≥0,05). Em relação as adenilato ciclases, a adenilato ciclase solúvel (sAC) no espermatozoide de garanhão demonstrou ser responsável pelo aumento da fluidez de membrana na presença de bicarbonato. Esse resultado foi demonstrado pelo uso do LRE1, um inibidor específico da sAC que demonstrou não causar efeitos colaterais em outros eventos fisiológicos espermáticos como alteração do potencial mitocondrial (p≥0,05). A imunohistoquímica da sAC em espermatozoide de garanhões demonstrou diferente localização comparada à sAC de suínos. No espermatozoide de garanhão essa enzima está presente formando uma linha pontilhada como um cordão na parte distal do acrosoma enquanto no suíno essa enzima está presente na parte apical da região acrossomal e na porção inicial da peça intermediária. A ação da forskolina indicou que a adenilato ciclase transmembrana (tmAC) possivelmente está presente no espermatozoide de garanhão e pode desempenhar um papel na reação acrossomal. Contudo, o fato da necessidade de uma alta concentração (500 µM) necessária para demonstrar seu efeito comparada a outras espécies indica que mais estudos como o uso de inibidores específicos (como o ddAdo) ou imunohistoquímica da tmAC (AC1-9) devem ser realizados no intuito de corroborar e validar os resultados encontrados. Em conclusão os resultados obtidos no presente trabalho elucidam diversos questionamentos na capacitação espermática de garanhões como exposição da PS, papel de diferentes concentrações Ca 2+ , P4 e BSA nesse evento além do papel de diferentes adenilato ciclases em alguns estágios da capacitação demonstrando que a capacitação é um processo complexo que varia entre as espécies. Palavras-chave: Adenilato ciclases. Capacitação. Espermatozoide de garanhão. Reorganização de membrana.Conselho Nacional de Desenvolvimento Científico e TecnológicoCoordenação de Aperfeiçoamento de Pessoal de Nível Superio

Use of Equicoll-SmallTM for filtering cryopreserved semen with different extenders of stallions of Mangalarga Marchador breed

O aumento do uso da inseminação artificial (IA) em equinos com sêmen congelado necessita de estudos que possibilitem a melhora da qualidade dos espermatozoides criopreservados e diminua a variabilidade individual entre os garanhões quanto à congelabilidade dos ejaculados. Isto porque as taxas de fertilidade obtidas com o sêmen equino criopreservado são inferiores àquelas obtidas com o sêmen fresco e refrigerado dessa espécie e ao sêmen bovino criopreservado. Este estudo visou verificar a ação de diferentes crioprotetores adicionados ao diluente além da eficácia da solução coloidal Equicoll-SmallTM na filtragem do sêmen equino pós-descongelamento no intuito de se obter uma população espermática com menor concentração de patologias e com maior número de características favoráveis à fecundação. Foram utilizados quatro garanhões da raça Mangalarga Marchador, hígidos e com históricos reprodutivos normais. Após o descongelamento, as amostras de sêmen foram submetidas ou não à centrifugação com o Equicoll-SmallTM, e avaliadas por meio de sondas fluorescentes através da citometria de fluxo. A maioria dos parâmetros espermáticos mostrou melhora após a centrifugação em camada única (SLC) com o Equicoll-SmallTM (P<0,05). No pós-descongelamento as amostras de sêmen criopreservadas em diluentes com o crioprotetor glicerol mostraram piores resultados comparadas às amostras de sêmen criopreservadas em diluentes com outros crioprotetores (DMFA e DG) em relação à integridade de membrana plasmática e organização da bicamada lipídica (P<0,05). O uso do coloide propiciou a recuperação de uma população espermática com melhor integridade de membrana, maior organização da bicamada lipídica, menor potencial mitocondrial e menor integridade acrossomal (P<0,05). O rendimento espermático médio foi de 6,95 % (±0,91 %). Diante dos resultados obtidos, concluiu-se que o uso de diluentes com os crioprotetores DMFA e DG mostraram ser mais eficazes em manter a viabilidade espermática in vitro em comparação ao diluente com o crioprotetor glicerol no pós-descongelamento. Além disso, o uso do Equicoll-SmallTM se mostrou eficaz na obtenção de uma população espermática viável, principalmente nas amostras de sêmen com maior porcentagem de crioinjúrias, porém seu rendimento espermático (concentração espermática final inseminante) pode ser um fator limitante para o uso desta técnica como rotina.The use of artificial insemination (AI) in horses with frozen semen requires efforts to increase the quality of cryopreserved spermatozoa and decrease individual variability among stallions as theirs ejaculates freezeability. This is because of fertility rates obtained from cryopreserved stallion semen are lower than those obtained with fresh and cooled semen of this species and of cryopreserved bovine semen. This study aimed to verify the action of different cryoprotectants added to the diluent in addition to the effectiveness of the colloidal solution Equicoll-SmallTM, in filtering the stallion semen post-thawing in order to obtain a sperm population with lower concentrations of pathologies and with more favorable fertilization characteristics. Four healthy stallions of Mangalarga Marchador breed with normal reproductive historic were used. After thawing the semen samples were subjected or not to centrifugation with Equicoll-SmallTM and evaluated by fluorescent dyies via flow cytometry. Most sperm parameters showed improvement after Single Layer Centrifugation (SLC) with Equicoll-SmallTM (p<0.05). After thawing the treatment with glycerol showed worse results compared to others cryoprotectants (dimethylformamide and dimethylformamide+glycerol) in relation to the integrity of the plasma membraneand organization of the lipid bilayer (P<0.05). SLC recovered a sperm population with better membrane integrity, better organization of the lipid bilayer, lower mitochondrial potential and lower acrosomal integrity (P<0.05). The average of the sperm yield was 6.95% (±0.91%). Based on these results, it is concluded that the treatments with dimethylformamide and dimethylformamide + glycerol are shown to be more effective in maintaining the sperm viability in vitro when compared to the glycerol after thawing. In addition, it was concluded that the SLC with Equicoll-SmallTM is effective in obtaining a viable sperm population, especially in those samples that show more cryoinjuries, but its sperm yield can be a limiting factor for the technique.Coordenação de Aperfeiçoamento de Pessoal de Nível Superio

Bicarbonate-Stimulated Membrane Reorganization in Stallion Spermatozoa

Classical in vitro fertilization (IVF) is still poorly successful in horses. This lack of success is thought to be due primarily to inadequate capacitation of stallion spermatozoa under in vitro conditions. In species in which IVF is successful, bicarbonate, calcium, and albumin are considered the key components that enable a gradual reorganization of the sperm plasma membrane that allows the spermatozoa to undergo an acrosome reaction and fertilize the oocyte. The aim of this work was to comprehensively examine contributors to stallion sperm capacitation by investigating bicarbonate-induced membrane remodelling steps, and elucidating the contribution of cAMP signalling to these events. In the presence of capacitating media containing bicarbonate, a significant increase in plasma membrane fluidity was readily detected using merocyanine 540 staining in the majority of viable spermatozoa within 15 min of bicarbonate exposure. Specific inhibition of soluble adenylyl cyclase (sAC) in the presence of bicarbonate by LRE1 significantly reduced the number of viable sperm with high membrane fluidity. This suggests a vital role for sAC-mediated cAMP production in the regulation of membrane fluidity. Cryo-electron tomography of viable cells with high membrane fluidity revealed a range of membrane remodelling intermediates, including destabilized membranes and zones with close apposition of the plasma membrane and the outer acrosomal membrane. However, lipidomic analysis of equivalent viable spermatozoa with high membrane fluidity demonstrated that this phenomenon was neither accompanied by a gross change in the phospholipid composition of stallion sperm membranes nor detectable sterol efflux (p > 0.05). After an early increase in membrane fluidity, a significant and cAMP-dependent increase in viable sperm with phosphatidylserine (PS), but not phosphatidylethanolamine (PE) exposure was noted. While the events observed partly resemble findings from the in vitro capacitation of sperm from other mammalian species, the lack of cholesterol removal appears to be an equine-specific phenomenon. This research will assist in the development of a defined medium for the capacitation of stallion sperm and will facilitate progress toward a functional IVF protocol for horse gametes

Seasonal variation in the reproductive activity of male goats raised under tropical climate conditions

This study analyzed seasonal variations in the testes, the concentration of sex hormones, the parameters of fresh and thawed semen, and the sexual behavior of male Alpine goats from a temperate region in a tropical climate and possible interference with fertility. The maximum and minimum temperature and luminosity were recorded daily, while seminal, hormonal, and behavioral assessments were carried out every fortnight. The maximum and minimum temperature (°C) and luminosity (h) were recorded daily always at 17.00 h. The scrotal circumference (cm), testicular volume (mL), volume (mL), appearance (creamy, milky, aqueous) and coloration (white, white-yellowish, and yellowish) seminal, turbulence or mass movement (0 to 5), progressive spermatic motility (0 to 100%), spermatic force (0 to 5), concentration (spermatozoids/mL), spermatic pathologies, hypoosmotic test (%), serum levels of FSH (mUI/mL), LH (mUI/mL), testosterone (ng/mL), and sexual behaviors were carried out every fortnight. There was a difference between the scrotal circumference evaluated monthly, testicular volume, volume and concentration of fresh semen, sperm vigor of the thawed semen, serum levels of testosterone, FSH and LH, and some sexual behaviors. Thus, the changes that occur in the quantity and quality of sperm, in the hormonal profile, and in sexual behaviors should not be regarded as an impediment to the use of male Alpine goats in tropical climates throughout the year. These variations do not lead to changes in the semen that may compromise the fertility of these animals

Relationship of testicular biometry with semen variables in breeding soundness evaluation of Nellore bulls

This study aimed to evaluate the correlation between testicular biometry and semen variables, as well as, to relate testicular variables to the probability of selecting Nellore bulls with desirable sperm morphology when conducting breeding soundness evaluations (BSE). A total of 2055 BSEs from 506 bulls comprised the dataset. Biometric variables evaluated were: scrotal circumference, testicular volume, width, length, ratio and eccentricity; and semen variables were sperm motility, major sperm defects, minor sperm defects and normal sperm. Data of testicular biometry were correlated with data for semen variables using the Pearson’s correlation assessment. Effects of testicular variables in selecting for sperm morphology of bulls in the BSE were evaluated by logistic regression. Scrotal circumference, testicular volume, length and width were positively correlated to sperm motility (0.18 to 0.19) and normal sperm (0.24 to 0.27) and negatively correlated with values for major defects (−0.24 to −0.27), but for testicular ratio and eccentricity there were coefficients near zero for all semen traits. Testicular ratio and eccentricity were not suitable for predicting the probability of selecting a bull based on semen variables using the BSE, but scrotal circumference, testicular volume, length and width were highly significant (P <  0.0001) with moderate values of area under ROC (Receiver Operating Characteristics) curve (0.608 to 0.620)

Seasonal variation in the reproductive activity of male goats raised under tropical climate conditions

ABSTRACT This study analyzed seasonal variations in the testes, the concentration of sex hormones, the parameters of fresh and thawed semen, and the sexual behavior of male Alpine goats from a temperate region in a tropical climate and possible interference with fertility. The maximum and minimum temperature and luminosity were recorded daily, while seminal, hormonal, and behavioral assessments were carried out every fortnight. The maximum and minimum temperature (°C) and luminosity (h) were recorded daily always at 17.00 h. The scrotal circumference (cm), testicular volume (mL), volume (mL), appearance (creamy, milky, aqueous) and coloration (white, white-yellowish, and yellowish) seminal, turbulence or mass movement (0 to 5), progressive spermatic motility (0 to 100%), spermatic force (0 to 5), concentration (spermatozoids/mL), spermatic pathologies, hypoosmotic test (%), serum levels of FSH (mUI/mL), LH (mUI/mL), testosterone (ng/mL), and sexual behaviors were carried out every fortnight. There was a difference between the scrotal circumference evaluated monthly, testicular volume, volume and concentration of fresh semen, sperm vigor of the thawed semen, serum levels of testosterone, FSH and LH, and some sexual behaviors. Thus, the changes that occur in the quantity and quality of sperm, in the hormonal profile, and in sexual behaviors should not be regarded as an impediment to the use of male Alpine goats in tropical climates throughout the year. These variations do not lead to changes in the semen that may compromise the fertility of these animals

Bicarbonate-Stimulated Membrane Reorganization in Stallion Spermatozoa

Classical in vitro fertilization (IVF) is still poorly successful in horses. This lack of success is thought to be due primarily to inadequate capacitation of stallion spermatozoa under in vitro conditions. In species in which IVF is successful, bicarbonate, calcium, and albumin are considered the key components that enable a gradual reorganization of the sperm plasma membrane that allows the spermatozoa to undergo an acrosome reaction and fertilize the oocyte. The aim of this work was to comprehensively examine contributors to stallion sperm capacitation by investigating bicarbonate-induced membrane remodelling steps, and elucidating the contribution of cAMP signalling to these events. In the presence of capacitating media containing bicarbonate, a significant increase in plasma membrane fluidity was readily detected using merocyanine 540 staining in the majority of viable spermatozoa within 15 min of bicarbonate exposure. Specific inhibition of soluble adenylyl cyclase (sAC) in the presence of bicarbonate by LRE1 significantly reduced the number of viable sperm with high membrane fluidity. This suggests a vital role for sAC-mediated cAMP production in the regulation of membrane fluidity. Cryo-electron tomography of viable cells with high membrane fluidity revealed a range of membrane remodelling intermediates, including destabilized membranes and zones with close apposition of the plasma membrane and the outer acrosomal membrane. However, lipidomic analysis of equivalent viable spermatozoa with high membrane fluidity demonstrated that this phenomenon was neither accompanied by a gross change in the phospholipid composition of stallion sperm membranes nor detectable sterol efflux (p > 0.05). After an early increase in membrane fluidity, a significant and cAMP-dependent increase in viable sperm with phosphatidylserine (PS), but not phosphatidylethanolamine (PE) exposure was noted. While the events observed partly resemble findings from the in vitro capacitation of sperm from other mammalian species, the lack of cholesterol removal appears to be an equine-specific phenomenon. This research will assist in the development of a defined medium for the capacitation of stallion sperm and will facilitate progress toward a functional IVF protocol for horse gametes