Efeitos da suplementação com anetol durante a miv e civ na maturação oocitária e na produção e qualidade de embriões bovinos
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Data
2021-11-03
Autores
Janini, Ludimila Cardoso Zoccal
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Universidade Estadual Paulista (Unesp)
Resumo
A produção in vitro de embriões (PIV) vem apresentando um crescimento significativo, representando 73,7% dos embriões produzidos mundialmente em 2019. Apesar de sua viabilidade comercial, a PIV necessita de aprimoramento para expandir ainda mais seu potencial. Desta forma, o aumento nas taxas de produção e qualidade embrionária proporcionaria ganhos substanciais na pecuária. Objetivou-se avaliar o efeito do anetol na MIV, nas doses de 300 e 3000µg/mL (Experimento 1) e avaliar o efeito do anetol na MIV e/ou CIV, na dose de 300µg/mL, na produção e qualidade de embriões bovinos (Experimento 2). Foram coletados ovários provenientes de frigoríficos utilizados nos dois experimentos. Folículos de 2 a 8mm foram puncionados e submetidos à maturação com meio maturação in vitro (MIV) suplementado com SFB 10%. No experimento 1, foram divididos em 3 grupos experimentais, sendo o Grupo Controle composto por (meio base); Grupo M300 (meio base suplementado com 300 µg/ml de anetol) e Grupo M3000 (3000 µg/ml). Após a MIV, a maturação nuclear foi avaliada por meio da técnica de análise da configuração da cromatina para obtenção dos resultados de metáfase II. No experimento 2, a MIV foi realizada com meio base (Grupo Controle) ou suplementado com (300 µg/ml de anetol). Posteriormente, os oócitos foram submetidos à fecundação in vitro (FIV) e cultivo in vitro (CIV), sendo divididos em 04 grupos: Grupo Controle M0-C0 maturados e cultivados em meio sem adição de anetol, Grupo M0-C300 maturados sem anetol e cultivados em meio com 300 µg/ml, Grupo M300-C0 µg/ml maturados com 300 µg/ml de anetol e cultivados sem anetol e o Grupo M300-C300 com meio contendo 300 µg/ml de anetol na maturação e no cultivo. No 3º dia do cultivo (D3), após a FIV foi avaliado a clivagem, no 7º dia após a FIV (D7), foi avaliado a produção de blastocistos e no 8º dia (D8) foi avaliado e classificado os blastocistos por meio de analise morfológica em estereomicroscópico. Como análise estatística foi utilizado a análise de variância, com on way ANOVA, utilizando o software GraphPad Prism versão 6.0 (GraphPad Software Inc, La Jolla, CA, USA), e testes de Tukey (dados paramétricos). Observou-se que 300 µg/mL de anetol no meio MIV não promoveu diferença nas taxas de MII quando comparado ao grupo controle. Entretanto, a adição de 3000 µg/mL de anetol reduziu significativamente a porcentagem de CCOs que chegaram à metáfase II em relação aos demais grupos (P≤0,05). Em relação ao índice de clivagem, observamos que a adição de 300 µg/ml de anetol durante a MIV e a CIV aumentou a porcentagem de embriões clivados quando comparado aos demais grupos experimentais (P≤ 0,05). Entretanto, nenhuma diferença foi encontrada entre os grupos quanto a produção embrionária, avaliada pela taxa de formação de blastocistos no D8 (P>0,05), nem sobre a qualidade embrionária, obtida por meio da contagem do número total de células embrionárias (P>0,05). Apesar das evidencias dos efeitos antioxidantes do anetol, encontradas na literatura, neste estudo o teste de TBARS realizado no meio de produção de embriões não apresentou significância estatística. Os genes SOD 1, CAT e GST relacionados a resistência ao estresse oxidativo, apresentaram aumento em sua abundancia nas células da granulosa quando foi utilizado o anetol. Alem disso, os genes COX2 e HAS também se apresentaram elevados, ressaltando o efeito benéfico do anetol sobre o metabolismo das CCs. Os embriões produzidos com ócitos maturados em presença de anetol também apresentaram elevação de transcritos do gene GPX1 que está envolvido na metabolização do peróxido de hidrogenio. A elevação da abundancia destes transcritos demonstra a contribuição do anetol para a neutralização do efeito das EROS. Por outro lado, apesar do alto número de embriões produzido no grupo maturado e cultivado com anetol, os genes IFNT2 e HASPA1A se encontravam diminuídos. Conclui se que a adição de 300 µg/mL de anetol durante a MIV e a CIV melhora a clivagem embrionária sem comprometer o desenvolvimento posterior do blastocisto.
INTRODUCTION: In vitro embryo production (IVP) has shown significant rise, reaching 68.7% of the embryos produced worldwide in 2018. Despite its commercial viability, IVP needs improvement to further expand its potential. Thus, livestock would benefit with substantial gains from increased production rates and embryo quality. OBJECTIVE: The objective was to evaluate the effect of anethole on in vitro maturation (IVM) at doses of 300 µg/mL and 3,000 µg/mL (Experiment 1) and to evaluate the effect of anethole on IVM and/or in vitro culture (IVC) at a dose of 300 µg/mL on the production and quality of bovine embryos (Experiment 2). MATERIAL AND METHODS: A total of 700 oocytes from ovaries from slaughterhouses were collected to be used in the two experiments. The 2 to 8 mm follicles were punctured and submitted to maturation with IVM medium supplemented with 10 % fetal bovine serum (FBS). In Experiment 1, a total of 300 oocytes were used and divided into 3 experimental groups: the Control Group, composed of basal medium; Group M300 (basal medium supplemented with 300 µg/ml anethole); and Group M3000 (basal medium supplemented with 3,000 µg/ml). After IVM, nuclear maturation was evaluated using chromatin configuration analysis technique to obtain the results of metaphase II (MII) with the best concentration, which was used in Experiment 2. In Experiment 2, IVM was performed either with basal medium only (Control Group) or with supplementation (Group M300 – 300 µg/ml anethole). Subsequently, 400 oocytes were subjected to in vitro fertilization (IVF) and IVC, and then divided into four groups: Control Group M0-C0: matured and cultivated in medium without addition of anethole; Group M0-C300: matured without anethole and cultivated in medium with 300 µg/ml anethole; Group M300-C0: matured with 300 µg/ml anethole and cultivated without anethole; and Group M300-C300: matured and cultivated in medium containing 300 µg/ml anethole. On the 3rd day of culture (D3), cleavage was evaluated after IVF; on the 7th day after IVF (D7), blastocyst production was evaluated; and on the 8th day (D8), blastocysts were evaluated and classified 5 through morphological analysis in a stereomicroscope. Statistical analysis was carried out by using the one-way analysis of variance (one-way ANOVA), as well as Tukey tests for parametric data. RESULTS: When 300 µg/mL anethole were added to the MIV medium, there was no difference in MII rates in comparison to the control group. However, the addition of 3,000 µg/mL anethole significantly reduced the percentage of cumulus-oocyte complex (COC) that reached MII in relation to the other groups (P ≤ 0.05). Regarding the cleavage index, the addition of 300 µg/ml anethole during IVM and IVC was observed to increase the percentage of cleaved embryos when compared to the other experimental groups (P ≤ 0.05). Nevertheless, no difference was found between the groups regarding embryo production, which was assessed by blastocyst formation rate on D8 (P > 0.05), nor related to embryo quality, which was obtained by counting the total number of embryonic cells (P > 0.05). In spite of the evidence of antioxidant effects of anethole pointed out in the literature, the TBARS test performed in this study in the embryo production medium did not show any statistical significance. The SOD1, CAT, and GST genes related to resistance to oxidative stress presented increased abundance in granulosa cells when anethole was used. In addition, the COX2 and HAS genes were also elevated, highlighting the beneficial effect of anethole on the metabolism of COCs. Similar to in embryos produced with cells matured in the presence of anethole, a rise was detected in transcripts of the GPX1 gene, which is involved in the metabolization of hydrogen peroxide. The increased abundance of these transcripts demonstrates the role of anethole in neutralizing the effect of ROS. On the other hand, regardless of the large number of embryos produced in the group matured and cultivated with anethole, the IFNT2 and HASPA1A genes were decreased. CONCLUSION: The addition of 300 µg/mL anethole during IVM and IVC improves embryonic cleavage without compromising blastocyst development.
INTRODUCTION: In vitro embryo production (IVP) has shown significant rise, reaching 68.7% of the embryos produced worldwide in 2018. Despite its commercial viability, IVP needs improvement to further expand its potential. Thus, livestock would benefit with substantial gains from increased production rates and embryo quality. OBJECTIVE: The objective was to evaluate the effect of anethole on in vitro maturation (IVM) at doses of 300 µg/mL and 3,000 µg/mL (Experiment 1) and to evaluate the effect of anethole on IVM and/or in vitro culture (IVC) at a dose of 300 µg/mL on the production and quality of bovine embryos (Experiment 2). MATERIAL AND METHODS: A total of 700 oocytes from ovaries from slaughterhouses were collected to be used in the two experiments. The 2 to 8 mm follicles were punctured and submitted to maturation with IVM medium supplemented with 10 % fetal bovine serum (FBS). In Experiment 1, a total of 300 oocytes were used and divided into 3 experimental groups: the Control Group, composed of basal medium; Group M300 (basal medium supplemented with 300 µg/ml anethole); and Group M3000 (basal medium supplemented with 3,000 µg/ml). After IVM, nuclear maturation was evaluated using chromatin configuration analysis technique to obtain the results of metaphase II (MII) with the best concentration, which was used in Experiment 2. In Experiment 2, IVM was performed either with basal medium only (Control Group) or with supplementation (Group M300 – 300 µg/ml anethole). Subsequently, 400 oocytes were subjected to in vitro fertilization (IVF) and IVC, and then divided into four groups: Control Group M0-C0: matured and cultivated in medium without addition of anethole; Group M0-C300: matured without anethole and cultivated in medium with 300 µg/ml anethole; Group M300-C0: matured with 300 µg/ml anethole and cultivated without anethole; and Group M300-C300: matured and cultivated in medium containing 300 µg/ml anethole. On the 3rd day of culture (D3), cleavage was evaluated after IVF; on the 7th day after IVF (D7), blastocyst production was evaluated; and on the 8th day (D8), blastocysts were evaluated and classified 5 through morphological analysis in a stereomicroscope. Statistical analysis was carried out by using the one-way analysis of variance (one-way ANOVA), as well as Tukey tests for parametric data. RESULTS: When 300 µg/mL anethole were added to the MIV medium, there was no difference in MII rates in comparison to the control group. However, the addition of 3,000 µg/mL anethole significantly reduced the percentage of cumulus-oocyte complex (COC) that reached MII in relation to the other groups (P ≤ 0.05). Regarding the cleavage index, the addition of 300 µg/ml anethole during IVM and IVC was observed to increase the percentage of cleaved embryos when compared to the other experimental groups (P ≤ 0.05). Nevertheless, no difference was found between the groups regarding embryo production, which was assessed by blastocyst formation rate on D8 (P > 0.05), nor related to embryo quality, which was obtained by counting the total number of embryonic cells (P > 0.05). In spite of the evidence of antioxidant effects of anethole pointed out in the literature, the TBARS test performed in this study in the embryo production medium did not show any statistical significance. The SOD1, CAT, and GST genes related to resistance to oxidative stress presented increased abundance in granulosa cells when anethole was used. In addition, the COX2 and HAS genes were also elevated, highlighting the beneficial effect of anethole on the metabolism of COCs. Similar to in embryos produced with cells matured in the presence of anethole, a rise was detected in transcripts of the GPX1 gene, which is involved in the metabolization of hydrogen peroxide. The increased abundance of these transcripts demonstrates the role of anethole in neutralizing the effect of ROS. On the other hand, regardless of the large number of embryos produced in the group matured and cultivated with anethole, the IFNT2 and HASPA1A genes were decreased. CONCLUSION: The addition of 300 µg/mL anethole during IVM and IVC improves embryonic cleavage without compromising blastocyst development.
Descrição
Palavras-chave
Estresse oxidativo, Espécies reativas de oxigênio (EROS), Anetol, PIV, Desenvolvimento embrionário, Bovino, Oxidative stress, Bovine