Combination of nanoparticle green tea extract in tris-egg yolk extender and 39 °c thawing temperatures improve the sperm quality of post-thawed Kacang goat semen

Kacang goats are small ruminants produced by low-income households in smallholder and farm to reduce poverty and prevent undernutrition. Studies to find a cryopreservation protocol for Kacang goat semen are expected to multiplication of genetically superior animals selected by the paternal lineage. This study evaluated the effect of thawing temperature and supplementation of the green tea extract nanoparticle in skim milk-egg yolk (SM-EY) extender on post-thaw sperm quality of Kacang goat semen. Six ejaculates of Kacang goat were diluted in SM-EY supplemented or not (control group) with 0.001 mg/mL NPs GTE. The diluted semen was packaged with 0.25 mL straws (insemination dose: 60x106 sptz/mL) and cryopreserved. Then, six samples of the control group and NPs GTE groups were thawed at 37°C or 39°C sterile water for 30 s and submitted to sperm quality evaluations. The sperm viability, motility, and intact of the plasma membrane (IPM) were higher (p<0.05) in NPs GTE group than control group. In contrast, the NPs GTE group presented lower (p<0.05) malondialdehyde levels and sperm DNA fragmentation (SDF) compared with the control group. The catalase levels were not significantly different (p > 0.05) between the control and NPs GTE groups. Thawing at 39°C resulted in higher (p<0.05) sperm viability, motility, and IPM than thawing at 37°C. However, thawing at 39°C group presented lower (p<0.05) malondialdehyde levels compared with thawing at 37°C. SDF and catalase levels were similar (p>0.05) between thawing at 37°C and thawing at 37°C. In conclusion, supplementation of 0.001 mg/mL of NPs GTE in SM-EY extender and thawing temperature of 39°C resulted in a better quality of frozen-thawed Kacang goat semen.(AU)

Different approaches for assessing sperm function

Different approaches can be used to assess sperm function in different conditions, i.e. sperm storage, freezing-thawing or activation by induction of capacitation and acrosome reaction. In this review we will focus on the assays routinely performed in our laboratories, giving a literature support to critically analyse different approaches. In fact, researchers usually tend to look for the “one shot” parameter that could explain itself a specific process; it is our conviction that a multiparametric approach is still more valid, as some changes in sperm function are very complex and could be explained only by operating in different ways. Sperm motility, the most evident sperm characteristic, should be assessed by computer-aided sperm analysers that permit an objective evaluation of the motility and its kinematic parameters. Commercial and open source instruments are available and could be profitably used together with specific statistical approaches. The use of microscopy, and particularly fluorescent microscopy, could be a very useful tool to assess different parameters in sperm cells both by fluorophores that give indication of a determined function, and by immunolocalization of proteins, that permits the discover of new features or to explain particular sperm functions. The same substrates could be used also in flow cytometry: the difference is that it permits to study wider sperm populations (and their subpopulation distribution). Flow cytometry is undergoing a very wide use in spermatology and technical and experimental rigor is needed to obtain reliable results. Metabolic assessment of sperm features, particularly energetic supply, ATP formation and other enzyme activities, could represent a very important challenge to acquire new information and complete/integrate those derived from other techniques. Finally, functional assays such as oocyte binding and in vitro fertilization, represent a very strong tool to assess sperm function in vitro, as they could evidence the functional intactness of some pathways.

Different approaches for assessing sperm function

Different approaches can be used to assess sperm function in different conditions, i.e. sperm storage, freezing-thawing or activation by induction of capacitation and acrosome reaction. In this review we will focus on the assays routinely performed in our laboratories, giving a literature support to critically analyse different approaches. In fact, researchers usually tend to look for the “one shot” parameter that could explain itself a specific process; it is our conviction that a multiparametric approach is still more valid, as some changes in sperm function are very complex and could be explained only by operating in different ways. Sperm motility, the most evident sperm characteristic, should be assessed by computer-aided sperm analysers that permit an objective evaluation of the motility and its kinematic parameters. Commercial and open source instruments are available and could be profitably used together with specific statistical approaches. The use of microscopy, and particularly fluorescent microscopy, could be a very useful tool to assess different parameters in sperm cells both by fluorophores that give indication of a determined function, and by immunolocalization of proteins, that permits the discover of new features or to explain particular sperm functions. The same substrates could be used also in flow cytometry: the difference is that it permits to study wider sperm populations (and their subpopulation distribution). Flow cytometry is undergoing a very wide use in spermatology and technical and experimental rigor is needed to obtain reliable results. Metabolic assessment of sperm features, particularly energetic supply, ATP formation and other enzyme activities, could represent a very important challenge to acquire new information and complete/integrate those derived from other techniques. Finally, functional assays such as oocyte binding and in vitro fertilization, represent a very strong tool to assess sperm function in vitro, as they could evidence the functional intactness of some pathways.(AU)

Impacts of oxidative stress on bovine sperm function and subsequent in vitro embryo development

Low levels of reactive oxygen species (ROS) in sperm are essential for various sperm functions such as capacitation, hyperactivation and acrosome reaction. However, increased synthesis of ROS or a disruption of antioxidative status (e.g. in cryopreserved sperm) can induce oxidative stress (OS). Sperm are particularly vulnerable to OS, as their plasma membrane contains large amounts of polyunsaturated fatty acids and they have limited antioxidative capacity (due to low cytoplasmic volume). Oxidative stress disturbs sperm function by damaging sperm proteins, lipids and DNA. Under relatively low OS sperm may retain their fertilizing ability, which might result in transfer of impaired paternal molecules (e.g. damaged DNA) to the fertilized oozyte. Oocytes can repair damaged paternal DNA, but only to a certain extent. Most embryos are either repaired (based on limited DNA damage in blastocysts) or eliminated (based on low percentage of blastocyst formation when sperm with damaged DNA is used for fertilization). However, some blastocysts had increases in both DNA damage and apoptosis, which could have important implications for subsequent development. In several studies, exogenous antioxidants improved quality of sperm exposed to oxidative stress and subsequent embryo development. However, there is still a knowledge gap regarding whether these alterations affect embryonic survival and further development to a live fetus and healthy offspring.

Impacts of oxidative stress on bovine sperm function and subsequent in vitro embryo development

Low levels of reactive oxygen species (ROS) in sperm are essential for various sperm functions such as capacitation, hyperactivation and acrosome reaction. However, increased synthesis of ROS or a disruption of antioxidative status (e.g. in cryopreserved sperm) can induce oxidative stress (OS). Sperm are particularly vulnerable to OS, as their plasma membrane contains large amounts of polyunsaturated fatty acids and they have limited antioxidative capacity (due to low cytoplasmic volume). Oxidative stress disturbs sperm function by damaging sperm proteins, lipids and DNA. Under relatively low OS sperm may retain their fertilizing ability, which might result in transfer of impaired paternal molecules (e.g. damaged DNA) to the fertilized oozyte. Oocytes can repair damaged paternal DNA, but only to a certain extent. Most embryos are either repaired (based on limited DNA damage in blastocysts) or eliminated (based on low percentage of blastocyst formation when sperm with damaged DNA is used for fertilization). However, some blastocysts had increases in both DNA damage and apoptosis, which could have important implications for subsequent development. In several studies, exogenous antioxidants improved quality of sperm exposed to oxidative stress and subsequent embryo development. However, there is still a knowledge gap regarding whether these alterations affect embryonic survival and further development to a live fetus and healthy offspring.(AU)

EFEITO IN VITRO DO FLUIDO E CÉLULAS EPITELIAIS DO OVIDUTO BOVINO NA FASE FOLICULAR OU LUTEAL NA FUNÇÃO E CAPACITAÇÃO DE ESPERMATOZOIDES OVINOS

O fluido do oviduto (FO) desempenha importante função na capacitação espermática e fertilização. Ainda, as células epiteliais do oviduto (CEO) interagem com o espermatozoide (SPZ) prolongando a sua vida útil. Evidências na literatura sugerem que o FO varia de acordo com a fase do ciclo estral, no entanto, pouco se sabe sobre os efeitos dessa variação na função espermática. Assim, o objetivo do estudo foi avaliar o efeito do FO da fase folicular e luteal e a interação do SPZ com CEO na modulação da função espermática e dos processos de capacitação/reação acrossômica (RA). Para alcançar esse objetivo, dois experimentos foram realizados. Para isso, FO na fase folicular e luteal (classificação baseada na morfologia ovariana) e CEO foram obtidas de ovidutos de vacas oriundas de matadouro. Para ambos os experimentos, após a técnica de swim-up (pool de três carneiros), os SPZ foram distribuídos aleatoriamente nos grupos experimentais. No experimento 1, 8 x 106 SPZ/mL foram adicionados ao meio: i) Fert-TALP (controle positivo), ii) Fert-TALP sem indutores da capacitação espermática (Fert-TALP modificado; controle negativo), iii) Fert-TALP modificado, suplementado com 10% de FO da fase folicular ou iv) FertTALP modificado, suplementado com 10% de FO na fase luteal. No experimento 2, para mimetizar as condições do oviduto durante a fase folicular e luteal, as CEO foram cultivadas no meio TCM-199, suplementada com 10% de soro fetal bovino até a confluência. Previamente (24 h) ao cocultivo com SPZ, foi realizada a indução das condições luteal e folicular pela suplementação dos hormônios 17 -estradiol (E2) e progesterona (P4) no meio nas concentrações detectadas no oviduto durante estas fases. Para o cocultivo, 8 x 106 SPZ/mL foram adicionados na presença da monocamada de CEO: i) CEO fase folicular cocultivo de SPZ e CEO no meio FertTALP, com suplementação de 290 pg/mL E2 e 6 ng/mL P4 (concentração similar a fase folicular); ii) CEO fase luteal cocultivo de SPZ e CEO no meio Fert-TALP, com suplementação de 80 pg/mL E2 e 85 ng/mL P4 (concentração similar a fase luteal); iii) cultivo de SPZ no meio Fert-TALP, com suplementação de 290 pg/mL de E2 e 6 ng/mL P4 e iv) cultivo de SPZ no meio Fert-TALP, com suplementação de 80 pg/mL E2 e 85 ng/mL P4. Para o grupo controle positivo, foi utilizado o meio Fert-TALP convencional. Os SPZ foram incubados a 38 °C em 5% de CO2 e os parâmetros da cinemática espermática, integridade da membrana plasmática (MP) e status da capacitação espermática foram avaliados após 0, 2, 4, 6 e 18 e 24 h. As variáveis foram submetidas ao teste two-way ANOVA com medidas repetidas (Bonferroni; P < 0,05). Os resultados do Experimento 1, mostraram que a incubação de até 4 h com FO independente do estágio do ciclo estral promoveu aumento na maioria dos parâmetros da cinemática espermática de forma similar (P 0,05) ao grupo controle positivo, enquanto que no Experimento 2, esses parâmetros foram reduzidos (P < 0,05) pelo co-cultivo entre SPZ e CEO, independente do estágio do ciclo estral. A integridade da MP não foi afetada (P 0,05) pela incubação com FO e CEO na fase folicular ou luteal. Incubação com FO independente do estágio do ciclo estral apresentou maior (P < 0,05) taxa de RA em comparação ao grupo controle negativo após longo tempo de incubação (18-24 h) enquanto que a interação das CEO (folicular ou luteal) reduziu (P < 0,05) a taxa de capacitação espermática em comparação ao grupo controle positivo ao longo da incubação. Conclui-se que utilizando a espécie ovina como modelo, a adição do FO na fase folicular ou luteal promove um aumento na cinemática espermática até 4 h de incubação e na taxa de RA após 18-24 h, enquanto que a interação das CEO com SPZ reduz a cinemática espermática e atrasa a capacitação espermática in vitro.

Oviduct fluid (OF) provides a suitable environment for the sperm capacitation and fertilization. Still, oviductal epithelial cell (OEC) interacts with sperm prolonging sperm lifespan. Evidences on literature suggest that OF varies according to the stage of estrous cycle, however, there are only few reports about the effect of this variation on sperm function. Thus, the objective of the study was to evaluate the effect of OF at follicular and luteal phases and the interaction of sperm with OEC on the modulation of sperm function and capacitation/acrosome react (AR) processes. To achieve this goal, two experiments were performed. For this, OF at follicular and luteal phases (classification based on ovarian morphology) and OEC were obtained from cow oviducts at a slaughterhouse. For both experiments, after swim-up technique (pool of three rams) sperm were randomly distributed in the experimental groups. In Experiment 1, 8 x 106 sperm/mL were added in the medium: i) Fert-TALP (positive control), ii) Fert-TALP without capacitating substance (modified Fert-TALP; negative control), iii) modified Fert-TALP supplemented with 10% of OF at follicular phase or iv) modified Fert-TALP supplemented with 10% of OF at luteal phase. In Experiment 2, to mimic oviductal conditions during the follicular and luteal phases, the OECs were cultured in TCM-199 medium supplemented with 10% of fetal bovine serum until confluence. Previously (24 h) to co-culture with sperm, the follicular and luteal conditions were induced by supplementation of 17beta-estradiol (E2) and progesterone (P4) hormones in the Fert-TALP medium, at concentrations detected in the oviduct during these phases. For co-culture, 8 x 106 sperm/mL were added in the presence of OEC monolayer: i) OEC follicular phase co-culture of sperm and OEC in Fert-TALP medium supplemented with 290 pg/mL E2 and 6 ng/mL P4 (concentration similar to follicular phase); ii) OEC luteal phase co-culture of sperm and OEC in FertTALP medium supplemented with 80 pg/mL E2 and 85 ng/mL P4 (concentration similar to luteal phase); iii) sperm culture in Fert-TALP medium supplemented with 290 pg/mL E2 and 6 ng/mL P4 and iv) sperm culture in Fert-TALP medium supplemented with 80 pg/mL E2 and 85 ng/mL P4. The positive control was the conventional Fert-TALP medium. Sperm were incubated at 38 ºC in 5% CO2 and the parameters of sperm kinematics, plasma membrane (PM) integrity and sperm capacitation status were evaluated after 0, 2, 4, 6, 18 and 24 h. The variables were subjected to a repeated measure two-way ANOVA (Bonferroni; P < 0.05). The results of the Experiment 1 showed that incubation up to four hours with OF regardless the stage of estrous cycle promoted an increase in most of kinematic parameters similar (P 0.05) to the positive control group while in Experiment 2, co-culture between sperm and OEC, regardless the stage of estrous cycle, reduced (P < 0.05) these kinematic parameters. The PM integrity was not affected (P 0.05) by incubation with OF and OEC at follicular or luteal phase. Incubation with OF regardless the phase of estrous cycle presented higher (P < 0.05) AR rate compared to the negative control group after a long-time incubation (18-24 h), while the interaction of OEC (either follicular or luteal stage) reduced (P < 0.05) sperm capacitation rate compare to positive control group throughout incubation. It is concluded that the supplementation with OF either at follicular or luteal stage into the medium, promotes an increase in the sperm kinematic for up to 4 h of incubation and AR rate after 18-24 h. In contrast, interaction between sperm and OEC reduce sperm kinematics and delay sperm capacitation in vitro using the ovine specie as a model.

AVALIAÇÕES FUNCIONAIS E BIOQUÍMICAS PARA TOUROS DE ALTA FERTILIDADE

A predição da fertilidade de touros é uma área da pesquisa que tem recebido muita atenção devido a sua importância na indústria de bovinocultura de corte. Um modelo de predição de fertilidade baseado na avaliação precoce dos parâmetros da qualidade de sêmen permitiria identificar e remover indivíduos com fertilidade potencialmente baixa de programas de reprodução. Entretanto, parâmetros convencionais utilizados na avaliação espermática in vitro têm mostrado resultados limitados quanto à capacidade de predizer o potencial de fertilidade do sêmen in vivo. Portanto, o objetivo do presente estudo foi investigar a relação entre a qualidade do sêmen in vitro e a fertilidade in vivo de touros, por meio da correlação de dados de análises bioquímicas e de cinética espermática com a taxa de prenhez. Amostras de sêmen congelado de seis touros Aberdeen Angus foram classificadas como fertilidade alta (n = 3) e baixa (n = 3). O pool de sêmen foi avaliado para a cinética de espermática (sistema computadorizado de análise espermática; CASA), produção de espécies reativas de oxigênio (ROS), peroxidação lipídica, atividade da enzima superóxido dismutase (SOD) e capacidade antioxidante total. As amostras foram avaliadas imediatamente após o descongelamento (PT); após teste de resistência térmica (TRT) a 36ºC por 3 horas; após seleção de espermática (SS) pelo gradiente de densidade de Percoll; e após SS + TRT. Os parâmetros de cinética espermática: motilidade total (MTOT, 51,29±5,60%), motilidade progressiva (MP; 36,12±5,56%) e frequência de batimentos do flagelo (BCF; 8,11±0,77 hz) foram significativamente maiores em touros de baixa fertilidade quando comparados aos touros de alta fertilidade. A velocidade curvilinear (VCL; 40,15±7,92 m/s) foi maior em touros de alta fertilidade após SS + TRT. A velocidade linear (VSL; r = -0,99), a velocidade média do percurso (VAP; r=-0,99), linearidade (LIN; r = -0,98) e BCF (r = -0,99) foram correlacionadas com alta fertilidade após SS + TRT. ROS demonstrou correlação (r=-0,98) com fertilidade após SS. Houve uma diminuição significativa dos níveis de FRAP e ROS após SS em touros de baixa e alta fertilidade (P0,05), enquanto que uma diminuição da peroxidação lipídica foi observada apenas em touros de alta fertilidade. Os resultados não mostraram diferença significativa entre touros de fertilidade baixa e alta na peroxidação lipídica. No entanto, houve uma correlação (r=-0,99) entre a peroxidação lipídica e alta fertilidade após PT e SS + TRT. Esses resultados sugerem que espermatozoides de touros de alta fertilidade são capazes de prevenir o dano induzido pelo estresse oxidativo. Os dados de cinética espermática obtidos pelo CASA demonstram que a avaliação das combinações dos parâmetros poderia predizer a fertilidade in vivo de forma mais precisa do que a avaliação individual desses parâmetros. Além disso, a separação espermática em gradiente de Percoll reduziu os níveis de estresse oxidativo, consequentemente, melhorando a qualidade espermática. O presente estudo compartilha informações uteis para o desenvolvimento futuro de modelos de predição de fertilidade em touros.

Prediction of bull fertility is an area of research that has received much attention due to its importance in the beef cattle industry. A prediction model of fertility based on early assess of semen quality parameters would allow identify and remove sires with potentially low fertility from breeding programs. However, conventional parameters used on in vitro sperm assessments have shown limited results in the ability to predict the fertility potential of semen in vivo. Therefore, the aim of the present study was to investigate the relationship between in vitro semen quality and in vivo fertility of bulls by correlating biochemical and sperm kinetics data with pregnancy rate. Frozen semen samples from six Aberdeen Angus bulls were classified as high (n=3) and low (n=3) fertility. Semen pool was evaluated to sperm kinetics (computer assisted sperm analysis; CASA), reactive oxygen species (ROS) production, lipid peroxidation, superoxide dismutase enzyme (SOD) activity and total antioxidant capacity. Samples were evaluated immediately after thawing (PT); after thermal-resistance test (TRT) at 36ºC for 3 hours; after sperm selection (SS) by Percoll density gradient; and after SS + TRT. Sperm kinetic parameters: total motility (MTOT; 51.29±5.60%), progressive motile (MP; 36.12±5.56%) and beat cross-frequency (BCF; 8.11±0.77 hz) were significantly higher in low-fertility bulls when compared to high fertility ones. Curvilinear velocity (VCL; 40.15±7.92 µm/s) was greater in high fertility bulls after SS + TRT. Straight-line velocity (VSL; r=-0.99), average path velocity (VAP; r=-0.99), linearity (LIN; r=-0.98), and BCF (r=- 0.99) were correlated to high fertility after SS + TRT. ROS showed correlation (r=-0.98) with fertility after SS. There was a significant decrease of FRAP and ROS levels following SS in both low and high fertility bulls (P0.05), whereas a decrease of lipid peroxidation was observed only in high fertility bulls. The results showed no significant difference between low and high fertility bulls in lipid peroxidation. However, there was a correlation (r=-0.99) between lipid peroxidation and high fertility after PT and SS + TRT. Sperm kinetic data obtained by CASA showed that the assessment of a combination parameters could predict in vivo fertility more accurately than the assess of an individual one. In addition, spermatic separation by the Percoll gradient reduced oxidative stress levels, hence, improving sperm quality. The present study shares useful information for the future development of fertility prediction models in bulls. Keywords: . . .

State of the art of assisted human reproduction

Background: Infertility is a disease observed in approximately 10% of the reproductive age population (20-44 years old), and is defined as the failure to conceive after twelve months of regular sexual intercourse, without contraception; in women older than 35 years old, this period is reduced to 6 months. The main causes of infertility are tubal, ovarian and uterine and sperm abnormalities, endometriosis, and those with undetermined causes. Over the past 30 years, several techniques were developed to overcome these factors including gamete cryopreservation, controlled ovarian stimulation, intra-uterine insemination, in vitro fertilization, intracytoplasmatic sperm injection). Review: Despite advances in assisted reproductive technologies (ART), treatment success is still strongly dependent on oocyte and sperm quality, and resulting embryo viability. The most promising advance on oocyte quality assessment is the evaluation of the ovarian reserve by the quantification of the anti-müllerian hormone (AMH). Since ovarian reserve is closely related to oocyte quality, AMH levels could be an indicator of both oocyte production capacity and the potential of these oocytes to generate a viable embryo. On the other hand, despite the development of techniques to overcome male factor infertility, attention has been paid on the semen evaluation, since routine sperm evaluation techniques are known to be ineffective, especially in those cases of unexplained infertility. Therefore, techniques were developed to assess acrosome and membrane integrity, mitochondrial potential, DNA integrity, and fertilizing capacity of sperm. However, further studies are necessary to evaluate sperm DNA integrity without damaging the cell, allowing the injection of a spermatozoon with an intact DNA when using ICSI. Regarding embryo quality, even with a good quality oocyte (as assessed by the current techniques) and an apparently normal sperm, there are still chances of generating an embryo with genetic abnormalities. In such cases, and in cases of recurrent failures, women over 35 years of age, and couples with a pre-existing genetic risk, the preimplantation genetic diagnosis (PGD) appears to be an important tool to improve the odds of pregnancy and avoid abortions or the conception of fetuses with genetic abnormalities. The technique of PGD, usually performed with PCR or FISH, has gained a powerful tool with the development of the Comparative Genomic Hybridization (CGH). However, recent studies aiming to identify markers of oocyte and sperm quality and embryo viability are in course using mass spectroscopy. With this sensitive technique applied to body fluids (i.e., blood, follicular fluid, seminal plasma), granulosa cells, sperm, and culture media, researches are being conducted to non-invasively identify biomarkers that will help understand reproductive mechanisms and to efficiently predict the outcome of ARTs. Conclusion: Significant advances in ART have been observed in the last few years, yet, failures still occur with high frequency. This review will focus on techniques to assess oocyte quality, sperm function and embryo viability, aiming to provide tools for a precise prognosis when treating infertile couples.

Efeito paradoxal de baixas doses de nanopartículas de prata na expressão testicular de genes relacionados com a espermatogênese, hormônios sexuais e funcionalidade espermática

Nanopartículas de prata (AgNPs) destacam-se nas áreas da medicina e da indústria por suas propriedades antibacterianas, antifúngicas e antivirais. O alto grau de comercialização aumenta a preocupação quanto a sua potencial toxicidade sobre os diversos sistemas incluindo o reprodutivo. Este trabalho teve como objetivo avaliar a toxicidade reprodutiva das AgNPs sobre os parâmetros selecionados numa curva de dose-resposta, onde a dose mais alta correspondeu à LOAEL (menor dose com efeito adverso observado) previamente estabelecida (15 g/kg) em ratos Wistar pré-púberes como modelo experimental. Avaliou-se a produção espermática, as reservas espermáticas, o tempo de trânsito espermático, a funcionalidade espermática (integridades acrossômica e da membrana plasmática, atividade mitocondrial e morfologia espermática). A expressão de mRNA nos testículos foi avaliada por PCR quantitativo em tempo real a partir da transcrição reversa (RT-qPCR) para os genes do receptor de andrógenos (Ar), receptor de estradiol (Esr1), receptor de estradiol (Esr2), receptor de estrógeno 1 acoplado a proteína G (Gper) e aromatase (Cyp19a1). Foram dosadas as concentrações séricas de testosterona e estradiol. Os resultados foram analisados estatisticamente pela análise de variância seguida do pós-teste de Dunnet, considerando-se diferenças estatísticas quando p<0,05. A exposição às AgNPs durante a fase pré-púbere resultou na alteração da expressão de genes Ar, Gper e Esr1 relacionados ao controle da espermatogênese, bem como nos níveis de hormônios, reserva, tempo de trânsito e funcionalidade espermáticas. Pode-se concluir que as AgNPs possuem efeitos dose-resposta não lineares agindo como um potencial desregulador endócrino químico e causando decréscimo na fertilidade masculina.

Silver nanoparticles (AgNPs) stand out in the medicine and industry areas by their antibacterial, antifungical and antiviral properties. The high degree of commercialization increases the concerns about potential toxicity in various systems including the reproductive tract. This study aimed to evaluate the reproductive toxicity of AgNPs in the selected parameters in a dose response curve on which the higher dose corresponded to LOAEL (lowest observed adverse effect level) previously established (15 g/kg) in prepubertal Wistar rats as experimental model. It was evaluated the sperm production, the sperm reserves, the sperm transit time, the sperm functionality (acrosome and plasma membrane integrities, mitochondrial activity and sperm morphology). The mRNA expression in the testis was evaluated by reverse transcription followed by real-time polymerase chain reaction (RTqPCR) for androgen receptor (Ar), estradiol receptor (Esr1), estradiol receptor (Esr2), estrogen receptor 1 coupled to the G-protein (Gper) and aromatase (Cyp19a1) genes. It was measured the serum concentrations of testosterone and estradiol. The results were statistically analyzed by analysis of variance followed by Dunnets post-hoc test considering statistical differences when p<0,05. The exposure to AgNPs during the prepubertal period resulted in altered expression of the genes Ar, Gper and Esr1 related to the spermatogenesis control, and in the hormones levels. The sperm parameters were also altered. It is possible to concluded that AgNPs has non-linear dose-response effects acting as a potential endocrine disrupting chemical and causing reduction in male fertility.