Morphokinetic Profiling Suggests That Rapid First Cleavage Division Accurately Predicts the Chances of Blastulation in Pig In Vitro Produced Embryos

Abstract

The study of pig preimplantation embryo development has several potential uses: from agriculture to the production of medically relevant genetically modified organisms and from rare breed conservation to acting as a physiologically relevant model for progressing human and other (e.g., endangered) species' in vitro fertilisation technology. Despite this, barriers to the widespread adoption of pig embryo in vitro production include lipid-laden cells that are hard to visualise, slow adoption of contemporary technologies such as the use of time-lapse incubators or artificial intelligence, poor blastulation and high polyspermy rates. Here, we employ a commercially available time-lapse incubator to provide a comprehensive overview of the morphokinetics of pig preimplantation development for the first time. We tested the hypotheses that (a) there are differences in developmental timings between blastulating and non-blastulating embryos and (b) embryo developmental morphokinetic features can be used to predict the likelihood of blastulation. The abattoir-derived oocytes fertilised by commercial extended semen produced presumptive zygotes were split into two groups: cavitating/blastulating 144 h post gamete co-incubation and those that were not. The blastulating group reached the 2-cell and morula stages significantly earlier, and the time taken to reach the 2-cell stage was identified to be a predictive marker for blastocyst formation. Reverse cleavage was also associated with poor blastulation. These data demonstrate the potential of morphokinetic analysis in automating and upscaling pig in vitro production through effective embryo selection.

Keywords: blastocyst; morphokinetics; pig; predictive parameters; time-lapse.

Figure 1

Key developmental events during early pig embryogenesis. Representative examples of nine key embryonic developmental events: (A): 2-cells; (B): 3-cells; (C): 4-cells; (D): 5+ cells; (E): compaction; (F): morula; (G): cavitation/early blastocyst; (H): expanding blastocyst; (I): hatching blastocyst. Images taken from a Geri^® Assess platform. The time taken to reach each of these events was recorded for each embryo. Magnification: 10×.

Figure 2

Comparison of the developmental kinetics of blastulating and non-blastulating in IVP pig embryos. ** denotes p < 0.01. (A): A boxplot showing the number of hours post-insemination (hpi) taken to reach each developmental stage for both blastulating (n = 47) and non-blastulating (n = 64) cleaved embryos. Analysis with a Mann U Whitney test with Bonferroni adjusted p-values showed a significant difference between the two groups for the time taken to the 2-cell (W = 370.5, p = 0.0015) and morula stage (W = 89, p = 0.0017). There was no significant difference found for the 3-cell (W = 460.5, p = 0.2), 4-cell (W = 381, p = 0.16) and 5+ cell stage (W = 453.5, p = 0.56). (B). Visualisation of the development of individual blastulating and non-blastulating IVP pig embryos. A line graph to show the time (hpi) taken to reach each developmental stage during early embryogenesis for both blastulating and non-blastulating (n = 111) cleaved IVP pig embryos.

Figure 3

ROC curve and AUC score for the time taken to the 2-cell stage as a predictor of further development to the blastocyst stage. ROC = receiver operating characteristics curve. AUC = Area under the curve. AUC scores are interpreted as no discrimination = 0.5, acceptable discrimination = 0.7–0.8, excellent discrimination = 0.8–0.9 and outstanding discrimination = >0.9.