14 Multidimensional imaging of in vitro-produced bovine embryos: preliminary data

Embryo selection is mainly done via morphological evaluation. Owing to the subjectivity of the embryologist’s view, this visual method is limited. Sharing knowledge with colleagues using embryo pictures taken via a camera attached to a light microscope still lacks information as the images are only two-dimensional. The aim of this study was to compare images of in vitro-produced (IVP) bovine embryos via normal light microscopy pictures, using an extended focus imaging (EFI) technique on the same embryos that allowed pictures of objects defined at more levels. Embryos were produced using cumulus–oocyte complexes of slaughtered cattle and a standard IVP protocol (Abele et al. 2014 Zygote 22, 41–49). At Day 7 of in vitro culture, embryos of the following developmental stages were selected and placed in TCM-199 with HEPES: morula, blastocyst, and expanded and hatching blastocyst. The light microscope used was an Olympus IX73 (Eppendorf) connected via a camera system (Olympus SC180) with a PC together with the corresponding imaging software cellSens Dimension (version 1.18). Two pictures of every embryo were taken, a normal two-dimensional image first and a multidimensional one second, applying the manual acquisition process “instant extended focal imaging” (InstantEFI) of the software. The focus was set at the lowest point of the embryo, and the recording process was started and manually focused through the whole embryo to its top. The software automatically took as many pictures as possible, and after the process ended, a combined image was delivered. This process was performed for 35 embryos and yielded 70 pictures, including normal images (control) and EFI ones, of every embryo. The pictures were collected in one file in a random order and four experienced practitioners were asked to determine stage and quality of every embryo according to the IETS guidelines (IETS manual, 5th edition; study part A). At the end of the document, five embryos were presented as examples for the stages above with both imaging techniques in direct comparison (study part B). The practitioners were also asked to choose the image of the embryo that allowed easier staging. The returned files were analyzed anonymously. Average values of stages and grades from the four participants were calculated. Owing to the low number of participants, no statistical analysis was performed. For 17 out of 35 embryos, stage and grade were the same between controls and the EFI pictures (part A). Eighteen embryos differed in stage or grade; however, in most cases the difference was less than one point and one at most. Providing the more developed stage or a better grade was equally distributed between both techniques. Comparing them directly to each other (part B) revealed that in most cases, the EFI pictures were preferred over control pictures for staging of the embryos. In summary, the EFI technique seems to be comparable to the already known pictures via light microscopy and could provide the same and eventually more precise results for staging and grading. Further surveys with more participants have to be conducted to allow generalizing conclusions; in particular, the direct comparison of the techniques for different grades should be added.