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were differentially stained for trophectoderm (TE), inner cell mass (ICM), and apoptotic cells. A qPCR gene expression analysis was performed for embryos in all three systems. Results:The WOW, PM and CG systems developed similar amounts of blastocysts (41, 35 and 36%, respectively; p>0.05). Blastocysts in all three systems showed adequate amounts of ICM and apoptotic cells. Blastocysts in the PM system showed a greater number of TE cells [63.7 versus 58.6% in the CG system (p<0.05)]. Relative mRNA expression of the embryonic genes POUF5F1, GNAS and TP53 did not differ significantly among the systems (p>0.05). ATP5B expression was higher in WOW than in PM (p<0.05), but similar to CG (p>0.05). TJP3 expression was higher in PM than in WOW and CG (p<0.05). Expression of ID2 and CLDN4 was higher in WOW than in PM and CG (p<0.05). The biplot graphic from Principal Component Analysis (PCA) revealed that CG was located near degenerated embryos, whereas PM was located near arrested embryos, larger ICM and TE, and TJP3 expression. WOW was located toward blastocysts, morulae, and expression of CLDN4, ID2 and GNAS. Conclusion: Compared with CG, both the PM and WOW systems are good options for culturing single embryos in the bovine model. Moreover, the PCA results suggest that embryos developed in the WOW system have greater capacity for generating blastocysts with increased ability to form the TE and ICM layers, which might improve their implantation success. Keywords: Bovine embryo; embryo quality; embryo recovery; in vitro culture; in vitro embryo production; polyester mesh culture; single-embryo culture; well-of-the-well culture. ResumenAntecedentes: México está innovando en la industria ganadera a través de la generación in vitro de embriones bovinos con tecnologías de cultivo individual como lo son pozo dentro de pozo (WOW) y malla de poliéster (PM). Estos mantienen los embriones en áreas separadas mientras comparten un mismo medio de cultivo celular. Objetivo: comparar la cantidad y calidad de embriones bovinos producidos en los sistemas de cultivo WOW y PM contra el sistema de cultivo convencional en grupo (CG). Métodos: en total, se evaluaron 345 embriones fertilizados in vitro para determinar la producción de blastocistos generados en los tres sistemas. Se contó el número de células por blastocisto, 69 embriones en cada sistema se tiñeron diferencialmente para trofoectodermo (TE), masa celular interna (ICM) y células ICM, TE e TJP3. WOW foi encontrado para ter blastocistos, mórulas e a expressão de CLDN4, ID2 e GNAS. Conclusão: em comparação com o CG, os sistemas PM e WOW são boas opções para a cultura de embriões individuais no modelo bovino. Além disso, os resultados da PCA sugerem que embriões individuais desenvolvidos no sistema WOW têm maior capacidade de desenvolver blastocistos com maior capacidade de formar as camadas TE e ICM, o que poderia melhorar seu sucesso de implantação.Palavras-chave: cultura em tela de poliéster; cultura individual; cultura in vitro; cultura well-in-well; embriões bovinos; qua...
were differentially stained for trophectoderm (TE), inner cell mass (ICM), and apoptotic cells. A qPCR gene expression analysis was performed for embryos in all three systems. Results:The WOW, PM and CG systems developed similar amounts of blastocysts (41, 35 and 36%, respectively; p>0.05). Blastocysts in all three systems showed adequate amounts of ICM and apoptotic cells. Blastocysts in the PM system showed a greater number of TE cells [63.7 versus 58.6% in the CG system (p<0.05)]. Relative mRNA expression of the embryonic genes POUF5F1, GNAS and TP53 did not differ significantly among the systems (p>0.05). ATP5B expression was higher in WOW than in PM (p<0.05), but similar to CG (p>0.05). TJP3 expression was higher in PM than in WOW and CG (p<0.05). Expression of ID2 and CLDN4 was higher in WOW than in PM and CG (p<0.05). The biplot graphic from Principal Component Analysis (PCA) revealed that CG was located near degenerated embryos, whereas PM was located near arrested embryos, larger ICM and TE, and TJP3 expression. WOW was located toward blastocysts, morulae, and expression of CLDN4, ID2 and GNAS. Conclusion: Compared with CG, both the PM and WOW systems are good options for culturing single embryos in the bovine model. Moreover, the PCA results suggest that embryos developed in the WOW system have greater capacity for generating blastocysts with increased ability to form the TE and ICM layers, which might improve their implantation success. Keywords: Bovine embryo; embryo quality; embryo recovery; in vitro culture; in vitro embryo production; polyester mesh culture; single-embryo culture; well-of-the-well culture. ResumenAntecedentes: México está innovando en la industria ganadera a través de la generación in vitro de embriones bovinos con tecnologías de cultivo individual como lo son pozo dentro de pozo (WOW) y malla de poliéster (PM). Estos mantienen los embriones en áreas separadas mientras comparten un mismo medio de cultivo celular. Objetivo: comparar la cantidad y calidad de embriones bovinos producidos en los sistemas de cultivo WOW y PM contra el sistema de cultivo convencional en grupo (CG). Métodos: en total, se evaluaron 345 embriones fertilizados in vitro para determinar la producción de blastocistos generados en los tres sistemas. Se contó el número de células por blastocisto, 69 embriones en cada sistema se tiñeron diferencialmente para trofoectodermo (TE), masa celular interna (ICM) y células ICM, TE e TJP3. WOW foi encontrado para ter blastocistos, mórulas e a expressão de CLDN4, ID2 e GNAS. Conclusão: em comparação com o CG, os sistemas PM e WOW são boas opções para a cultura de embriões individuais no modelo bovino. Além disso, os resultados da PCA sugerem que embriões individuais desenvolvidos no sistema WOW têm maior capacidade de desenvolver blastocistos com maior capacidade de formar as camadas TE e ICM, o que poderia melhorar seu sucesso de implantação.Palavras-chave: cultura em tela de poliéster; cultura individual; cultura in vitro; cultura well-in-well; embriões bovinos; qua...
The ability of bovine oocytes to reach the blastocyst stage (i.e., embryo with around 150 cells in cattle) in vitro can be affected by technical (e.g., culture medium used) and physiological factors in oocyte donors (e.g., age, breed). As such, the nutritional status of oocyte donors plays a significant role in the efficiency of in vitro embryo production (IVEP), and several nutritional strategies have been investigated in cattle subjected to ovum pick-up (OPU). However, there is no clear consensus on the reliability of nutritional schemes to improve IVEP in cattle. Available evidence suggests that a moderate body condition score (i.e., 3 in a 1–5 scale) in cattle is compatible with a metabolic microenvironment in ovarian follicles that will promote embryo formation in vitro. The usefulness of fatty acid and micronutrient supplementation to improve IVEP in cattle is debatable with the current information available. Overall, the supply of maintenance nutritional requirements according to developmental and productive stage seems to be enough to provide bovine oocyte donors with a good chance of producing embryos in vitro. Future nutrition research in cattle using OPU-IVEP models needs to consider animal well-being aspects (i.e., stress caused by handling and sampling), which could affect the results.
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