Redução do conteúdo lipídico intracitoplasmático de embriões bovinos produzidos in vitro como estratégia para melhorar a crioresistência ao processo de vitrificação
Carregando...
Data
2016-06-06
Autores
Meneghel, Melissa [UNESP]
Título da Revista
ISSN da Revista
Título de Volume
Editor
Universidade Estadual Paulista (Unesp)
Resumo
O presente estudo teve como objetivos avaliar os efeitos de estimuladores da lipólise (Forskolin: Forsk) e inibidores da lipogênese (Ácido Linoléico: LA) durante o cultivo in vitro (CIV) sobre o acúmulo de lipídios intracitoplasmáticos e criotolerância do embrião bovino, bem como avaliar o efeito do tratamento dos embriões com Forsk sobre a taxa de concepção após transferência para receptoras. Oócitos (n=1242) foram maturados in vitro (MIV) durante 22h a 38,5°C e 5% de CO2 em ar em meio TCM-199 bicarbonato com 10% SFB e hormônios. Após fecundação, os prováveis zigotos foram cultivados em meio SOF (grupo Controle) suplementado com: 100 μM LA durante todo o período do CIV (grupo LA); ou 5 μM Forsk a partir do dia 6 do CIV (grupo Forsk); ou com associação de LA e Forsk, como acima descrito (grupo LA+Forsk). O CIV foi conduzido a 38,5°C e 5% de CO2 em ar, por 7 dias. O desenvolvimento embrionário foi avaliado no dia 7 do CIV (D7), quando os blastocistos foram corados com Sudan Black B 1% para determinação do conteúdo lipídico intracitoplasmático. Os embriões foram avaliados em microscopia de luz e as imagens obtidas foram analisadas por Q-Capture Pro Image Software. O grupo Controle foi escolhido como calibrador e a média de cada grupo foi dividida pela média do calibrador para calcular a intensidade relativa de pixels, expressa em unidades arbitrárias. Os blastocistos expandidos foram vitrificados (Vitri Ingá®), aquecidos e cultivados por 24h em SOF para determinação das taxas de re-expansão. Os dados foram analisados por ANOVA seguido por teste de Tukey, e as taxas de re-expansão por qui-quadrado (P<0,05). Não foi observada diferença entre os grupos (P>0,05) na taxa de produção de blastocistos entre os grupos (variação entre 47,2±3,9% e 51,6±5,5%). Observou-se diminuição (P<0,05) da quantidade de lipídeos intracitoplasmáticos nos embriões do grupo Forsk (0,86±0,04) comparado com Controle (0,99±0,02) e LA (1,02±0,02); no entanto, o grupo LA+Forsk (0,95±0,03) não diferiu (P>0,05) dos demais. Não houve diferença (P>0,05) entre os grupos na taxa de re-expansão imediata após o aquecimento (0h: 30,8% a 41,9%). Após cultivo de 24h, a re-expansão foi maior (P<0,05) no grupo Forsk (71,4%) comparado com Controle (46,2%) e LA+Forsk (45,2%); o grupo LA (65,1%) não diferiu dos demais (P>0,05). Baseado nestes resultados, o tratamento com 5 μM Forsk foi escolhido para o desenvolvimento do próximo experimento: oócitos (n=1947) foram aspirados de 22 doadoras Nelore e foram maturados e fecundados in vitro como acima descrito. Os prováveis zigotos foram cultivados de acordo com os tratamentos Controle e Forsk acima descritos e, no D7, foram vitrificados e aquecidos, antes de serem transferidos para receptoras sincronizadas. Os dados da taxa de concepção foram analisados através do modelo linear misto generalizado com medidas repetidas, do pacote Imea4 do software R v3.2.5. A taxa de concepção após transferência dos embriões aquecidos foi semelhante (P>0,05) entre os grupos Controle (25,00±6,53%) e Forsk (30,77±7,39%). De acordo com os resultados obtidos, o tratamento com Forsk foi eficiente para promover a redução da quantidade de lipídeos intracitoplasmáticos e melhorar a criotolerância de embriões bovinos produzidos in vitro. Apesar disso, não aumentou a taxa de concepção.
The objective of the present study was to evaluate the effects of supplementation of in vitro culture (IVC) medium with drugs that stimulates the lipolysis (Forskolin: Forsk) and inhibit the lipogenesis (Linoleic Acid LA) on the intracytoplasmic lipid content and cryotolerance of bovine embryos, as well as to evaluate the effect of treatment of embryos with Forsk on the pregnancy rates after transfer to synchronized recipients. Oocytes (n=1242) were matured in vitro for 22h at 38.5°C and 5% CO2 in air in medium TCM199 with 10% FCS and hormones. After fertilization, presumptive zygotes were cultured in SOF medium (Control group) supplemented with: 100 μM LA throughout the culture period (LA group); or 5 μM Forsk from the 6th day to the end of the culture (Forsk group); or with the association of LA and Forsk, as described above (LA+Forsk group). The IVC was conducted at 38.5°C and 5% CO2 in air, for 7 days. Embryonic development was assessed on day 7 of culture (D7), when blastocysts were stained with 1% Sudan Black B to determine the intracytoplasmic lipid content. The embryos were evaluated by light microscopy and the images were analyzed by Q-Capture Pro Image software. The Control group was chosen as a calibrator and the measured value of each treatment was divided by the mean of the calibrator to generate the relative expression levels of pixels, expressed in arbitrary units. The expanded blastocysts were vitrified (Vitri Ingá®), warmed and cultured for 24h in SOF to evaluate the re-expansion rates. The data were analyzed by ANOVA followed by Tukey's test and rates of re-expansion by qui-square (P<0.05). No differences were observed between treatments (P>0.05) in blastocysts production rates (47.2±3.9% to 51.6±5.5%). Intracytoplasmic lipid content was decrease (P<0.05) in embryos from Forsk group (0.86±0.04) compared to Control (0.99±0.02) and LA (1.02±0.02); however, all these treatments were similar (P>0.05) to LA+Forsk group (0.95±0.03). There were no differences (P>0.05) between treatments in the rates of re-expansion immediately after warming (0h: 30.8% to 41.9%); after 24h of culture post-warming the re-expansion rates were higher (P<0.05) in Forsk group (71.4%) compared with Control (46.2%) and LA+Forsk (45.2%) groups, but there were no diferences between LA group (65,1%) and the other groups (P>0.05). Based on these results, treatment with 5 μM Forsk was chosen for the next experiment: oocytes (n=1947) were aspirated from 22 Nelore donnors and were matured and fertilized in vitro as described above. Presumptive zygotes were cultured according to the treatments Control and Forsk as described above, and in D7 the expanded blastocysts were vitrified and warmed, before being transfered to synchronized recipients. The data of pregnancy rates were analyzed using a generalized linear mixed model framework with repeated measurements, in the lme4 package in R v3.2.5 software. The pregnancy rates after transfer of vitrified-warmed embryos were similar (P>0.05) between Control (25,00±6,53%) and Forsk (30,77±7,39%) groups. According to the results, treatment with Forsk was effective to promote the reduction of intracytoplasmic lipids content in in vitro-produced bovine embryos and improved their cryotolerance. Nevertheless, it did not increase the pregnancy rates.
The objective of the present study was to evaluate the effects of supplementation of in vitro culture (IVC) medium with drugs that stimulates the lipolysis (Forskolin: Forsk) and inhibit the lipogenesis (Linoleic Acid LA) on the intracytoplasmic lipid content and cryotolerance of bovine embryos, as well as to evaluate the effect of treatment of embryos with Forsk on the pregnancy rates after transfer to synchronized recipients. Oocytes (n=1242) were matured in vitro for 22h at 38.5°C and 5% CO2 in air in medium TCM199 with 10% FCS and hormones. After fertilization, presumptive zygotes were cultured in SOF medium (Control group) supplemented with: 100 μM LA throughout the culture period (LA group); or 5 μM Forsk from the 6th day to the end of the culture (Forsk group); or with the association of LA and Forsk, as described above (LA+Forsk group). The IVC was conducted at 38.5°C and 5% CO2 in air, for 7 days. Embryonic development was assessed on day 7 of culture (D7), when blastocysts were stained with 1% Sudan Black B to determine the intracytoplasmic lipid content. The embryos were evaluated by light microscopy and the images were analyzed by Q-Capture Pro Image software. The Control group was chosen as a calibrator and the measured value of each treatment was divided by the mean of the calibrator to generate the relative expression levels of pixels, expressed in arbitrary units. The expanded blastocysts were vitrified (Vitri Ingá®), warmed and cultured for 24h in SOF to evaluate the re-expansion rates. The data were analyzed by ANOVA followed by Tukey's test and rates of re-expansion by qui-square (P<0.05). No differences were observed between treatments (P>0.05) in blastocysts production rates (47.2±3.9% to 51.6±5.5%). Intracytoplasmic lipid content was decrease (P<0.05) in embryos from Forsk group (0.86±0.04) compared to Control (0.99±0.02) and LA (1.02±0.02); however, all these treatments were similar (P>0.05) to LA+Forsk group (0.95±0.03). There were no differences (P>0.05) between treatments in the rates of re-expansion immediately after warming (0h: 30.8% to 41.9%); after 24h of culture post-warming the re-expansion rates were higher (P<0.05) in Forsk group (71.4%) compared with Control (46.2%) and LA+Forsk (45.2%) groups, but there were no diferences between LA group (65,1%) and the other groups (P>0.05). Based on these results, treatment with 5 μM Forsk was chosen for the next experiment: oocytes (n=1947) were aspirated from 22 Nelore donnors and were matured and fertilized in vitro as described above. Presumptive zygotes were cultured according to the treatments Control and Forsk as described above, and in D7 the expanded blastocysts were vitrified and warmed, before being transfered to synchronized recipients. The data of pregnancy rates were analyzed using a generalized linear mixed model framework with repeated measurements, in the lme4 package in R v3.2.5 software. The pregnancy rates after transfer of vitrified-warmed embryos were similar (P>0.05) between Control (25,00±6,53%) and Forsk (30,77±7,39%) groups. According to the results, treatment with Forsk was effective to promote the reduction of intracytoplasmic lipids content in in vitro-produced bovine embryos and improved their cryotolerance. Nevertheless, it did not increase the pregnancy rates.
Descrição
Palavras-chave
Ácido linoleico, Forskolin, Criopreservação, Transferência de embriões PIVE, Concepção, Linoleic acid, Cryopreservation, Pregnancy, IVP embryos transfer