115 Video data reveals bovine embryo stress in response to fluctuations in ambient room temperature

Temperature is known to affect pre-implantation embryo development and should be controlled. However, practical considerations of the conventional embryo transfer procedure subject the embryos to fluctuations in temperature as the embryos are collected from the in vivo uterine environment, to the in vitro laboratory. This transition typically involves the embryo culture environment decreasing from 38°C (body temperature) to ~20°C (room temperature), possibly inducing damage to the embryo. The objective of this study was to use graphic image processing (GIP) techniques to measure real-time bovine embryo morphokinetic activity as the embryo transitions from 38°C to room temperature. Ninety-five embryos were collected from donor Holstein females during routine field practice and placed into a holding media. The temperature of the holding media was recorded, and a 30-s video of the embryos were recorded on a View4k camera at 30 frames per second (fps). Environmental room temperature ranged from 33.3°C to 22.8°C. Embryos were immediately transferred into eligible recipients and pregnancy outcomes were confirmed with ultrasound. The GIP methods to quantify embryo activity were applied to videos. By subtracting the digital numeric value of each pixel from the value of the corresponding pixel in the previous frame, an output that is the absolute difference between pixel values per frame was determined. This measurement was recorded for each subsequent frame for the entire duration of the video (30 s × 30 fps = 900 total frames) for each embryo. Mean pixel change (MPC) per embryo was calculated and compared to media temperature. Pearson correlation coefficient and Student’s t-test was performed in R. It was found MPC is positively correlated with a decrease in temperature, showing embryos have a higher MPC (thus more morphokinetic activity) at lower temperature than higher temperatures (r = 0.7). Morphokinetic activity is highly correlated at 30°C to 33°C and 22.8°C to 23.8°C (r > 0.8). Morphokinetic activity demonstrated a greater range and more variability per embryo at 25.5°C to 28.8°C (r < 0.4). Forty-six of 95 (48.4%) embryos produced a pregnancy. The MPC ranged from 0.7 to 1.75. This data shows an increase in embryo morphokinetic activity as media temperature decreases, suggesting cooler temperatures induces embryo stress. Increased activity is likely due to increased embryo metabolism as the embryo attempts to maintain homeostasis in the changing environment. Interestingly, there was more variability of morphokinetic activity in embryos in media temperatures 25.5°C to 28.8°C, as some embryos maintained a more quiescent state and others demonstrated a sharp spike in increased activity, suggesting this temperature range is an inflection point that triggers a response in some embryos. When pregnancy outcomes were compared, embryos which maintained a more quiescent state during the inflection point established more pregnancies (P < 0.05). Data suggest these techniques can be used to detect subclinical signs of embryonic stress and select hearty embryos robust to temperature fluctuations. Further studies are ongoing.