Blastocoel fluid removal and melatonin supplementation in the culture medium improve the viability of vitrified bovine embryos

Marques, Thaisa Campos; Santos, Elisa Caroline da Silva; Diesel, T. O.; Martins, Claudia Miranda; Cumpa, H. C. B.; Leme, L. O.; Dode, M. A. N.; Alves, Bênner Geraldo; Costa, Francine Pereira Higino da; Oliveira, E. B. de; Gambarini, M. L.

doi:10.1016/j.theriogenology.2020.10.028

Cited by 8 publications

(8 citation statements)

References 56 publications

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“…We used the tolerance to cold temperatures as a criterion of blastocyst quality [ 87 , 93 ], and we did not observe differences in post-warming survival and TCN between melatonin-treated and untreated embryos during IVC. These results contrast those from previous studies in bovines showing that melatonin supplementation during IVC positively affected the tolerance to cold temperatures of in vitro-produced blastocyst [ 60 , 87 ]. In addition to the previous exposure of in vivo-derived embryos to endogenous melatonin mentioned above, this discrepancy could be explained by the notable differences between species in IVC protocols, which profoundly impact the embryo resistance to damage from low temperatures [ 94 ], and by the greater sensitivity to cold of IVP embryos compared with those derived in vivo [ 32 , 95 ].…”

Section: Discussioncontrasting

confidence: 99%

“…In the present study, we used 1 nM melatonin because that concentration was reported to be the optimum concentration in the maturation [ 22 , 27 , 52 ] and embryo culture [ 22 , 23 , 29 , 30 ] media for porcine embryos obtained via IVF, parthenogenesis, and SCNT. A melatonin concentration of 1 nM has also been found to be effective for bovine embryos fertilized in vitro [ 53 , 54 , 55 , 56 , 57 , 58 , 59 , 60 ], and for the IVC of in vivo-derived mouse embryos [ 61 ]. Furthermore, the concentration of melatonin was effective at protecting porcine oocytes from heat stress [ 62 ] and improving human oocyte tolerance to cold temperatures [ 63 ].…”

Section: Discussionmentioning

confidence: 99%

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Exogenous Melatonin in the Culture Medium Does Not Affect the Development of In Vivo-Derived Pig Embryos but Substantially Improves the Quality of In Vitro-Produced Embryos

et al. 2022

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Cloned and transgenic pigs are relevant human disease models and serve as potential donors for regenerative medicine and xenotransplantation. These technologies demand oocytes and embryos of good quality. However, the current protocols for in vitro production (IVP) of pig embryos give reduced blastocyst efficiency and embryo quality compared to in vivo controls. This is likely due to culture conditions jeopardizing embryonic homeostasis including the effect of reactive oxygen species (ROS) influence. In this study, the antioxidant melatonin (1 nM) in the maturation medium, fertilization medium, or both media was ineffective in enhancing fertilization or embryonic development parameters of in vitro fertilized oocytes. Supplementation of melatonin in the fertilization medium also had no effect on sperm function. In contrast, the addition of melatonin to the embryo culture medium accelerated the timing of embryonic development and increased the percentages of cleaved embryos and presumed zygotes that developed to the blastocyst stage. Furthermore, it increased the number of inner mass cells and the inner mass cell/total cell number ratio per blastocyst while increasing intracellular glutathione and reducing ROS and DNA damage levels in embryos. Contrarily, the addition of melatonin to the embryo culture medium had no evident effect on in vivo-derived embryos, including the developmental capacity and the quality of in vivo-derived 4-cell embryos or the percentage of genome-edited in vivo-derived zygotes achieving the blastocyst stage. In conclusion, exogenous melatonin in the embryo culture medium enhances the development and quality of in vitro-derived embryos but not in in vivo-derived embryos. Exogenous melatonin is thus recommended during embryo culture of oocytes matured and fertilized in vitro for improving porcine IVP efficiency.

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Section: Discussioncontrasting

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Exogenous Melatonin in the Culture Medium Does Not Affect the Development of In Vivo-Derived Pig Embryos but Substantially Improves the Quality of In Vitro-Produced Embryos

et al. 2022

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“…Sabe-se que o aumento da sensibilidade dos embriões PIV aos processos de criopreservação, é decorrente de mudanças na estrutura embrionária, como o menor número de células no embrioblasto, da maior proporção de gotas lipídicas intracelulares e do aumento da permeabilidade da zona pelúcida. Além disso, embriões PIV apresentam menor taxa de compactação do embrioblasto, elevando sua susceptibilidade as crioinjúrias, resultando no maior estresse oxidativo das células, o que leva a diminuição do número de gestações e nascimentos em relação a utilização de embriões frescos (Ramos et al, 2006;Marques et al, 2021).…”

Section: Taxa De Gestação (%)unclassified

Taxa de gestação após transferência de embriões bovinos produzidos in vitro

Trenkel

Neto²,

Cichoski³

et al. 2022

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Objetivou-se com esse estudo avaliar a influência do horário da transferência de embriões, do estádio de desenvolvimento e da qualidade dos embriões transferidos, do processamento dos embriões, do ovário ipsilateral ao corpo lúteo (CL) e do tamanho do CL, sobre a taxa de gestação de receptoras embriões bovinos produzidos in vitro (PIV) oriundos de raças de corte e leite. Para tanto, utilizou-se dados retrospectivos de 419 transferências de embriões bovinos PIV, sendo 245 oriundas de embriões de raças de corte e 174 de raças de leite, entre os meses de fevereiro a novembro de 2021. A taxa de gestação foi calculada considerando-se os efeitos do horário de realização da transferência dos embriões a receptoras, da fase de desenvolvimento embrionário, da qualidade e processamento do embrião, ovário ipsilateral ao CL e do tamanho do CL. As porcentagens médias foram comparadas pelo Qui-quadrado, mediante pacote estatístico SAS versão 9.2, considerando 5% de significância (p<0,05). As 419 transferências de embriões produzidos in vitro resultaram em 29,04% de gestação. Não se observou influência do horário da transferência de embriões às receptoras, do estádio de desenvolvimento dos embriões, do ovário ipsilateral ao CL e do tamanho do CL sobre taxa de gestação de receptoras após transferência de embriões PIV oriundos de raças de corte e leite (p>0,05). Contudo, a qualidade e o processamento dos embriões produzidos in vitro exerceram efeitos sobre a taxa de gestação, após transferência de embriões de raças de corte e leite (p<0,05).

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“…All cells labeled and visualized at both wavelengths were counted to determine the mean TCN and ACN. Intracellular ROS and GSH content were measured using dichlorohydrofluorescein diacetate (C400, Molecular Probes), and 4-chloromethyl-6,8-difluoro-7-hydroxycoumarin (CellTracker Blue CMF2HC, Molecular Probes), respectively, as previously reported (Marques et al, 2021). ROS was detected as green (460 nm wavelength) and GSH as blue (370 nm wavelength) fluorescence, and to evaluate ROS levels and GSH content, the relative fluorescence intensity was considered directly proportional to intracellular ROS levels and GSH content.…”

Section: Detection Of Cellular Apoptosis Ros Generation and Gsh Levelsmentioning

confidence: 99%

Resveratrol-supplemented holding or re-culture media improves viability of fresh or vitrified-warmed in vitro-derived bovine embryos

Silva

Marques

Santos

et al. 2021

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The effect of resveratrol supplementation on fresh (E1) or vitrified/warmed (E2) in vitro produced bovine embryos was investigated by evaluating the time-dependent response. After in vitro production, resveratrol (0.5 µM) was added to the incubation media and after two incubation periods with or without resveratrol, blastocysts were re-cultured for 24h. The rates of re-expansion, hatching, total cell number (TCN), apoptotic cells (ACN), reactive oxygen species (ROS) and intracellular glutathione (GSH) content were evaluated. For E1, the re-expansion rate differed at 6 and 10h within and between treatments (P<0.05), as did the re-expansion rate after 24h (P<0.01). The hatching rate increased after 10h with resveratrol (P<0.01) with differences within (P<0.05), but not between treatments after 24h of re-cultivation. At E2, hatching rate differed between treatments at 24h (P<0.01), with higher TCN in resveratrol-treated blastocysts after 10h (P<0.01). Resveratrol supplementation reduced ROS generation in E1 and E2 after 10h of incubation and increased GSH content (P<0.01). These results indicate that supplementation of holding re-cultivation medium with resveratrol for treatment of fresh or vitrified/warmed in vitro produced bovine embryos has a positive and time-dependent effect. The reduction of ROS content, the increase of GSH and the anti-apoptotic ability of resveratrol are responsible for its protective effects, allowing an extension of embryo storage time before transfer to recipients.

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Blastocoel fluid removal and melatonin supplementation in the culture medium improve the viability of vitrified bovine embryos

Cited by 8 publications

References 56 publications

Exogenous Melatonin in the Culture Medium Does Not Affect the Development of In Vivo-Derived Pig Embryos but Substantially Improves the Quality of In Vitro-Produced Embryos

Exogenous Melatonin in the Culture Medium Does Not Affect the Development of In Vivo-Derived Pig Embryos but Substantially Improves the Quality of In Vitro-Produced Embryos

Taxa de gestação após transferência de embriões bovinos produzidos in vitro

Resveratrol-supplemented holding or re-culture media improves viability of fresh or vitrified-warmed in vitro-derived bovine embryos

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