6 Relationships of phototextural characteristics of ovine presumptive zygotes to their developmental potential in vitro

In vitro embryo production (IVP) has widespread implications for infertility treatments, animal husbandry, and conservation programs. The goal of achieving pregnancy with IVP systems calls for noninvasive and accurate methods of embryo screening and selection. Several factors associated with the developmental competence of preimplantation embryos such as chemical composition (e.g. lipid content) of cytoplasm and intracellular architecture could influence their opacity/transparency properties. The present study set out to examine whether phototextural attributes of ovine presumptive zygotes (t0) were reliable markers of their ensuing developmental ability in vitro. Cumulus-oophorus complexes were obtained by ovarian scarification from nine cycling Polish Longwool ewes after slaughter. Following IVM of oocytes and their fertilization with fresh ram semen, the development of embryos (n = 37) to the blastocyst stage was monitored using time-lapse imaging technology. Primo Vision time-lapse system (EAVO+; Vitrolife) was used to capture digital images of developing ovine embryos every 10 min for 8 days post-insemination without disrupting the embryo culture environment. Digital (*.tiff) images of the presumptive zygotes were saved and used for computerized image analyses. The embryos were retrospectively classified as non-arresting (attaining the blastocyst stage; n = 6) or arresting (non-dividing presumptive zygotes or embryo arrest before the ninth mitotic division; n = 31). Commercially available image analytical software (ImageProPlus®) was used to generate bitmaps of the rectangular regions of interest comprising zygotic cytoplasm. Subsequently, a proprietary computer algorithm developed in house (r-Algo) was used to determine if there were clusters of pixels for which first-order phototextural characteristics of presumptive zygotes’ cytoplasm (i.e. mean pixel intensity [MPI], heterogeneity [MPH], and concentration [MPC]) differed significantly between the two subsets of embryos. Algorithmic analysis of the microphotographs of presumptive zygotes revealed the existence of specific pixel ranges for which MPI and MPC, but not MPH values, were statistically distinctive (P < 0.001) between future arresting and non-arresting ovine embryos (pixel range: MPI, 73–75; MPC, 105–106). Within the algorithmically identified pixel clusters, normally developing embryos at t0 significantly exceeded their arresting counterparts in MPI (74.06 ± 0.01 vs. 73.98 ± 0.008) and MPC (1.25 ± 0.22% vs. 0.56 ± 0.07%). Statistical differences were verified using Student t-test (SigmaPlot®; ver. 11.0, Systat Software Inc.). Computerized segmentation of the microscopic images of presumptive zygotes is a promising method of predicting the developmental potential of in vitro-derived mammalian embryos. Therefore, this study introduces a novel, objective, and fully noninvasive (using phototextural analysis) method to enhance embryo assessment.