Centro de Investigación en Tecnología de Embriones (CIETE), Animal Improvement Program, Universidad Nacional Agraria La Molina, Lima, Perú.
Department of Animal Science, Faculty of Veterinary Sciences, Universidad de Concepción, Chillán, Concepción, Chile.
PLoS One. 2023 Aug 10;18(8):e0289751. doi: 10.1371/journal.pone.0289751. eCollection 2023.
The objective of this study was to evaluate the time of blastulation monitored by time-lapse technology to predict in vitro viability of bovine blastocysts. This technology can be a powerful tool for bovine embryos selection with higher implantation capacity and competence. Also, in humans an early blastulation is associated with higher quality and pregnancy rate. Cumulus oocyte complexes (COCs) were matured for 20 to 22 h and then fertilized by co-incubation of COCs and spermatozoa (10,000 sperm per oocyte) for 18 h. Presumptive zygotes were placed individually in microwells, in droplets of commercial culture medium. The Primo Vision TL system (EVO+; Vitrolife) captured digital images of developing embryos every 15 minutes. The time frame from IVF to the start of blastulation (tSB) and to blastocyst development (tB) was recorded. After day 7.5, the blastocysts were in vitro culture for 48 h until day 9.5 after IVF to evaluate post hatching development. In vitro viability was evaluated at day 9.5: those with a diameter greater than 200 μm and a total cell count greater than 180 were classified as viable (value 1), while the rest were classified as non in vitro viable (value 0). The area under the ROC curve (AUC) was estimated to determine the predictive power of in vitro viability through blastulation time. In addition, binary logistic regression analysis was used to generate a mathematical model with morphokinetic variables that allow the best prediction of in vitro viability. In 13 sessions, the blastocyst production rate was 46.2% (96/208). The cut-off time to discriminate early or late blastulation was 149.8 h. The post-hatching development of the embryos with early blastulation was 63.3% (31/49), being statistically superior (p = 0.001) than the late blastulation group 14.9% (7/47). Likewise, the time of blastulation showed an accuracy of 90.8% (p < 0.001) in predicting in vitro viability of bovine blastocysts. In conclusion, the selection of blastocysts based on blastulation time (< 155 h) and blastocyst diameter measured on day 7.5 after IVF (> 180 μm) maximizes the in vitro viability.
本研究旨在评估通过时间延迟技术监测囊胚期以预测牛囊胚体外活力的时间。这项技术可以成为选择具有更高着床能力和竞争力的牛胚胎的有力工具。此外,在人类中,早期囊胚期与更高的质量和妊娠率相关。卵丘卵母细胞复合物(COC)成熟 20-22 小时,然后通过 COC 和精子(每个卵子 10,000 个精子)共孵育 18 小时进行受精。假定的受精卵被单独放置在微井中,在商业培养基的液滴中。Primo Vision TL 系统(EVO+;Vitrolife)每 15 分钟拍摄一次胚胎发育的数字图像。记录从体外受精到囊胚期开始(tSB)和囊胚发育(tB)的时间框架。第 7.5 天后,将囊胚在体外培养 48 小时,直到体外受精后第 9.5 天,以评估孵化后发育情况。第 9.5 天评估体外活力:直径大于 200μm且总细胞计数大于 180 的被归类为有活力(值 1),其余的被归类为无体外活力(值 0)。通过囊胚形成时间评估 ROC 曲线下面积(AUC),以确定通过囊胚形成时间预测体外活力的能力。此外,使用二元逻辑回归分析生成具有形态动力学变量的数学模型,该模型可以最好地预测体外活力。在 13 次实验中,囊胚生成率为 46.2%(96/208)。区分早期或晚期囊胚的截止时间为 149.8 小时。早期囊胚胚胎的孵化后发育为 63.3%(31/49),明显优于晚期囊胚组的 14.9%(7/47)(p = 0.001)。同样,囊胚形成时间在预测牛囊胚体外活力方面的准确率为 90.8%(p < 0.001)。总之,基于囊胚形成时间(<155 小时)和体外受精后第 7.5 天测量的囊胚直径(>180μm)选择囊胚可以最大限度地提高体外活力。