64 Impact of cleavage morphokinetics on blastocyst development

In vitro embryo production has revolutionized the cattle industry. However, there are still critical limitations in the understanding of embryo competence. The use of time-lapse imaging has significantly enhanced the ability to evaluate embryo development by assessing cleavage patterns, abnormal events, and morphology. Here, we aimed to determine cleavage patterns and morphological features associated with blastocyst development during early embryonic development in cattle. Putative zygotes (n = 1218) were individually cultured in two experimental sites using synthetic oviduct fluid medium (5% CO₂, 5% O₂, 38.5°C). All embryos were produced using semen from one sire. Time-lapse imaging allowed the recording of cleavage timing and patterns, such as ruffling/blebbing, asynchronous cleavage, reversal, or direct cleavage patterns. Overall, 21.3%, 34.3%, and 18.9% embryos halted development at 2–4 cells, 5–8 cells, or morula, respectively, whereas 8.1% developed to blastocysts. Categorical variables were analyzed using logit models that included ruffling, direct, synchronous reverse, and symmetric cleavage as fixed effects and date and plate as random variables, while development was a dependent variable. Quantitative variables were analyzed using linear mixed-effect models that included developmental stage and location as fixed effects and date and plate as random variables. Each tested variable was included as a dependent variable. Cell ruffling increased developmental arrest at 2–4 cells (P < 0.01). Reverse cleavage increased developmental arrest at 2–4 cells and morula stage (P < 0.05). Direct and asynchronous cleavage increased developmental arrest at 2–4 cells, 5–8 cells, and at the morula stage (P < 0.05). Most blastocysts developed out of synchronous (P < 0.01) or symmetric (P < 0.1) cleavages, with no reversed cleavages (P < 0.01). Diameter of putative zygotes and time of first cleavage were associated with developmental arrest at the 2- to 4-cell stage (P < 0.01). Time to reach 4-cell and 8-cell stages was associated with blastocyst development (P < 0.01). Next, we combined all variables into a machine learning model, which predicted 94% (256/272) and 24% (7/29) of the embryos that failed or successfully developed to blastocyst, respectively. In parallel, image analysis of 8-cell embryos using a deep convolutional neural network predicted 73.3% (11/15) and 83.3% (10/12) of the embryos that failed or successfully developed to blastocyst, respectively. Furthermore, we cryopreserved 107 blastocysts followed by thawing and individual culture. Thirty-five blastocysts survived cryopreservation as evaluated by the expansion of the blastocoele. None of the categorical or quantitative variables were significantly associated with competence to survive cryopreservation. We conclude that morphokinetic features were correlated with embryonic competence and are useful for identification of early developmental arrest.

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