Adição de piruvato e coenzima Q10 ao diluente à base de leite desnatado para refrigeração do sêmen equino
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Data
2019-06-14
Autores
Bandeira, Rafael dos Santos
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Editor
Universidade Estadual Paulista (Unesp)
Resumo
O espermatozoide exige um fornecimento constante de energia para a manutenção de suas funções celulares e quando desafiado por processos de criopreservação do sêmen, sofrem danos irreversíveis. Para o desenvolvimento de técnicas que visam o aumento da longevidade espermática, é necessário considerar que mesmo em metabolismo basal, o espermatozoide necessita de substratos para garantir sua motilidade e poder fecundante após a ejaculação. Para atender suas demandas energéticas, estudos recomendam o uso de nutrientes exógenos, como o piruvato de sódio e a coenzima Q10 (CoQ10), substratos fundamentais na bioenergética celular. Visto a importância da refrigeração de sêmen em garanhões e o potencial destas substâncias em melhorar os parâmetros seminais atuando como substrato energético e antioxidante, respectivamente, o presente trabalho tem por objetivo abordar aspectos relacionados ao metabolismo espermático, bem como o papel do piruvato e CoQ10 visando minimizar os efeitos deletérios da refrigeração sobre a qualidade do sêmen equino. Foram adicionadas diferentes concentrações de piruvato de sódio e da CoQ10 ao sêmen de garanhões considerados “good coolers” (GC) e “bad coolers” (BC). Primeiramente, foram estabelecidas as concentrações mais eficazes de piruvato e CoQ10 no diluente de refrigeração BotuSêmen® (Botupharma Botucatu/SP Brasil) para preservar os parâmetros espermáticos na refrigeração a 5° C por até 48 horas. Foi utilizado 1 ejaculado de 25 garanhões das raças Quarto de Milha e Mangalarga Marchador, cada ejaculado foi dividido em 7 frações, sendo uma parte destinada ao grupo controle (CT) e o restante dividido entre o piruvato de sódio e a CoQ10, adicionados nas concentrações de 1 mmol/l (P1), 2 mmol/l (P2), 3 mmol/l de piruvato de sódio (P3), 25 µmol/l (Q25), 50 µmol/l (Q50) e 75 µmol/l de coenzima Q10 (Q75) e submetidos ao processo de refrigeração a 5ºC. Na segunda etapa, foram utilizados 2 ejaculados de 7 garanhões considerados BC, bem como as concentrações mais eficazes de piruvato de sódio e CoQ10 e sua associação. Cada ejaculado foi dividido em 4 frações, sendo P1, Q25, associação de P1 + Q25 e o grupo controle (CT - BotuSêmen®). As amostras foram avaliadas quanto à cinética espermática, integridade de membrana plasmática, produção de espécies reativas ao oxigênio (H2O2 e O2-) e potencial mitocondrial imediatamente após a colheita do sêmen, 24 e 48 horas de armazenamento a 5°C. No primeiro momento, não houve diferença significativa entre os grupos tratados e o controle, para os garanhões GC em nenhum dos tempos analisados. Para os garanhões BC houve diferença significativa no tempo de 48 horas (T48), onde os grupos P1, Q25 e Q75 apresentaram-se superiores ao grupo CT para motilidade total (MT), progressiva (MP) e rápidos (RAP) (p<0,05). Na segunda etapa houve diferença significativa nos grupos tratados, comparados ao controle no T48, apresentando melhores resultados para as avaliações de MT (P1, P1Q25), MP (P1, P1Q25) e RAP (P1, Q25 e P1Q25) (p<0,05). Para a avaliação da concentração de H2O2 intracelular houve diferença entre o grupo P1 em relação ao CT. Conclui-se que o piruvato de sódio na concentração de 1 mmol/l, a coenzima Q10 na concentração 25 µmol/l, ou a sua associação adicionados ao diluidor de refrigeração, melhoram as características de cinética espermática em garanhões “bad coolers” após 48 horas de refrigeração a 5°C.
The spermatozoa require a constant supply of energy for the maintenance of their cellular functions and when challenged by processes of cryopreservation of the semen, they suffer irreversible damages. For the development of techniques that aim to increase sperm longevity, it is necessary to consider that even in basal metabolism, the spermatozoa require substrate to ensure their motility and fertilizing power after ejaculation. To attend their energy demands, studies recommend the use of exogenous nutrients, such as sodium pyruvate and coenzyme Q10 (CoQ10), key substrates in cellular bioenergetics. Considering the importance of semen cooling in stallions and the potential of these substances to improve seminal parameters acting as an energetic and antioxidant substrate, this review aims to address aspects related to cooling, as well as the role of sodium pyruvate and CoQ10 in minimizing the effects of cooling on the quality of equine semen. Different concentrations of sodium pyruvate and CoQ10 have been added to semen from good coolers (GC) and bad coolers (BC). First, the most effective concentrations of sodium pyruvate and CoQ10 in the BotuSêmen® extender (Botupharma Botucatu / SP Brazil) were established to preserve the sperm parameters at 5°C for up to 48 hours. Each ejaculate was split into 7 treatments, with sodium pyruvate and CoQ10 being added at concentrations of 1 mmol/l (P1), 2 mmol/l (P2), 3 mmol/l sodium pyruvate (P3), 25 µmol/l (Q25), 50 µmol/l (Q50) and 75 µmol/l of coenzyme Q10 (Q75) and subjected to the cooling process at 5°C. In the second step, only stallions considered BC were used, as well as the most effective concentrations of sodium pyruvate and CoQ10 and their association. Each ejaculate was split into 4 treatments, being P1, Q20, association of P1 + Q20 and the control group (CT - BotuSêmen®). The samples were evaluated for sperm kinetics, plasma membrane integrity, reactive oxygen species production (H2O2 and O2 - ) and mitochondrial potential immediately after semen collection, 24 and 48 hours of storage at 5°C. At the first moment, there was no significant difference between the treated and control groups for the GC stallions in any of the times analyzed. For the BC stallions, there was a significant difference in the time of 48 hours (T48), where the groups P1, Q25 and Q75 were superior to the CT group for total motility (TM), progressive motility (PM) and rapid (RAP) motile (p <0,05). In the second stage there was a significant difference in the treated groups compared to the control in T48, presenting better results for the evaluations of TM (P1, P1Q25), PM (P1, P1Q25) and RAP (P1, Q25 and P1Q25) (p<0,05). In order to evaluate the intracellular H2O2 concentration there was a difference between the P1 group and the CT. In conclusion, sodium pyruvate at the concentration of 1 mmol/l, coenzyme Q10 at 25 μmol/l or its combination added to cooling extender improved the sperm kinetics characteristics in bad coolers stallions after 48 hours of cooling at 5°C.
The spermatozoa require a constant supply of energy for the maintenance of their cellular functions and when challenged by processes of cryopreservation of the semen, they suffer irreversible damages. For the development of techniques that aim to increase sperm longevity, it is necessary to consider that even in basal metabolism, the spermatozoa require substrate to ensure their motility and fertilizing power after ejaculation. To attend their energy demands, studies recommend the use of exogenous nutrients, such as sodium pyruvate and coenzyme Q10 (CoQ10), key substrates in cellular bioenergetics. Considering the importance of semen cooling in stallions and the potential of these substances to improve seminal parameters acting as an energetic and antioxidant substrate, this review aims to address aspects related to cooling, as well as the role of sodium pyruvate and CoQ10 in minimizing the effects of cooling on the quality of equine semen. Different concentrations of sodium pyruvate and CoQ10 have been added to semen from good coolers (GC) and bad coolers (BC). First, the most effective concentrations of sodium pyruvate and CoQ10 in the BotuSêmen® extender (Botupharma Botucatu / SP Brazil) were established to preserve the sperm parameters at 5°C for up to 48 hours. Each ejaculate was split into 7 treatments, with sodium pyruvate and CoQ10 being added at concentrations of 1 mmol/l (P1), 2 mmol/l (P2), 3 mmol/l sodium pyruvate (P3), 25 µmol/l (Q25), 50 µmol/l (Q50) and 75 µmol/l of coenzyme Q10 (Q75) and subjected to the cooling process at 5°C. In the second step, only stallions considered BC were used, as well as the most effective concentrations of sodium pyruvate and CoQ10 and their association. Each ejaculate was split into 4 treatments, being P1, Q20, association of P1 + Q20 and the control group (CT - BotuSêmen®). The samples were evaluated for sperm kinetics, plasma membrane integrity, reactive oxygen species production (H2O2 and O2 - ) and mitochondrial potential immediately after semen collection, 24 and 48 hours of storage at 5°C. At the first moment, there was no significant difference between the treated and control groups for the GC stallions in any of the times analyzed. For the BC stallions, there was a significant difference in the time of 48 hours (T48), where the groups P1, Q25 and Q75 were superior to the CT group for total motility (TM), progressive motility (PM) and rapid (RAP) motile (p <0,05). In the second stage there was a significant difference in the treated groups compared to the control in T48, presenting better results for the evaluations of TM (P1, P1Q25), PM (P1, P1Q25) and RAP (P1, Q25 and P1Q25) (p<0,05). In order to evaluate the intracellular H2O2 concentration there was a difference between the P1 group and the CT. In conclusion, sodium pyruvate at the concentration of 1 mmol/l, coenzyme Q10 at 25 μmol/l or its combination added to cooling extender improved the sperm kinetics characteristics in bad coolers stallions after 48 hours of cooling at 5°C.
Descrição
Palavras-chave
antioxidante, sêmen, criopreservação, antioxidant, cryopreservation