Selection of viable in vitro-fertilized bovine embryos using time-lapse monitoring in microwell culture dishes

Abstract

Conventionally, in vitro-fertilized (IVF) bovine embryos for transfer are morphologically evaluated at day 7-8 of embryo culture. This method is, however, subjective and results in unreliable selection. We previously described a novel selection system for IVF bovine blastocysts for transfer that traces the development of individual embryos with time-lapse monitoring in our specially developed microwell culture dishes (LinKID micro25). The system can noninvasively identify prognostic factors that reflect viability after transfer. By assessing a combination of identified prognostic factors -timing of the first cleavage; number of blastomeres at the end of the first cleavage; and number of blastomeres at the onset of lag-phase, which results in temporary developmental arrest during the fourth or fifth cell cycle- the pregnancy rate was improved over using conventional morphological evaluation. Time-lapse monitoring with LinKID micro25 could facilitate objective and reliable selection of healthy IVF bovine embryos. Here, we review the novel bovine embryo selection system that allows for prediction of viability after transfer.

Keywords: Bovine embryo; Culture dish; Pregnancy; Selection; Time-lapse monitoring.

Fig. 1.

Effect of conventional morphological code on percentage of pregnancy success at day 30 post-transfer. An in vitro-derived blastocyst, which was selected by conventional morphological code at day 7, was transferred to each synchronized recipient. Code 1: irregularities should be relatively minor and at least 85% of the cellular material should be an intact, viable embryonic mass; code 2: at least 50% of the cellular material should be an intact, viable embryonic mass; and code 3: at least 25% of the cellular material should be an intact, viable embryonic mass.

Fig. 2.

Overview of the novel selection system. In vitro development of bovine IVF embryos in the specially developed microwell culture dish (LinKID micro25) was tracked with time-lapse monitoring (A and B). The timing of the first cleavage, the number of blastomeres at the end of the first cleavage, and the number of blastomeres at lag-phase were examined as prognostic factors (C).

Fig. 3.

The relationship between the (A) timing of the first cleavage in embryos resulting in successful or failed pregnancies after transfer, (B) percentage of pregnant cows after transfer of embryos with different timings for the first cleavage, or (C) different numbers of blastomeres after the first cleavage. The boxes indicate the 25th and 75th percentiles, and the middle horizontal line indicates the median. Whiskers indicate the maximum and minimum values within the acceptable range defined by the two quartiles. Circles denote outliers. Data were analyzed with Student’s t-test. Different letters represent significant differences (^a,b P < 0.05) (A). The percentage of pregnant cows that received embryos with specific first cleavage patterns, based on the timing of cleavage (B) and number of blastomeres (C). Blastocysts were divided into 2 groups, fast and slow, based on the timing of the first cleavage with a cut-off of 27 hpi (B), and 2 or 2–3 blastomeres at the end of the first cleavage (C).