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.

Quality control systems for assays and assay components are crucial to achieving optimum standards in healthcare. In human IVF, the mouse embryo assay is commonly used to assess the quality of media and instruments. Despite its popularity, there are some well-known issues with the assay, including insensitivity to sub-optimal conditions, ethical issues due to animal use and variation in end-point interpretation between operators. Here, we detail the development of a P19 embryonic carcinoma cell-based assay with the potential to be an alternative method for quality control testing of human IVF media, and we use this assay to test varying levels of the critical energy sources pyruvate, glutamine and calcium lactate in human blastocyst media. Trademark features of the mammalian cold stress response were also incorporated as potential tools for increasing assay sensitivity. Assessed by resorufin fluorescence, P19 cells responded to changes in pyruvate and glutamine in the presence of cold stress and the mTOR inhibitor rapamycin but demonstrated natural sensitivity to calcium lactate concentrations. In addition to increasing cell sensitivity, cold stress enhanced cell growth, whilst rapamycin treatment further amplified the effects of cold stress. The assay described is robust, suitable for detecting sub-optimal culture conditions and indicates the extent to which different energy substrates affect cell growth and proliferation.