Assessment of embryo viability based on morphokinetics in cattle and horses

ABSTRACT In both bovine and equine reproduction, fertility is a lowly heritable trait that has an exceedingly large financial impact and may be a limiting factor on the ability to grow genetic lines. In order to increase pregnancy rates using Assisted Reproductive Techniques (ART) such as Embryo Transfer (ET), in vitro fertilization (IVF), and cryopreservation, producers must have access to accurate decision-making tools. Traditionally, the embryo grading system accepted by industry standards is based on morphological evaluation, and is a method that has remained relatively unchanged for over four decades that uses subjective evaluation that varies with exposure of technicians to training and experience. Inconsistency in grading among embryologists can ultimately lead to decreased conception rates through increased potential in discarding viable embryos. Seventeen In Vivo derived (IVD) morula stage fertilized equine embryos, 5 cloned equine embryos, 19 IVD blastocyst stage fertilized equine embryos, and 73 IVD, fertilized bovine embryos were collected from donor animals to be evaluated before transfer into recipient mares and cows. Embryos were graded according to the International Embryo Transfer Society (IETS) manual guidelines (IETS, 2009), that is the accepted industry standard of grading. Of the embryos graded, those that were given a quality score of Grade 1 or 2 were chosen for morphokinetic evaluation. The objective of this study was to discern whether IETS quality Grade 1 or 2 embryos could be measured based on their morphokinetic activity to determine if this activity was indicative of embryo viability, enforce embryos that would establish pregnancies, and that would fail to establish pregnancies Both equine and bovine embryos were filmed within 3 h of transfer with the complete embryo in view of the camera. Videos were then amplified using video motion magnification (VMM) resulting in 300x amplification of the videos and revealed previously hidden components of these embryos.. Embryo morphokinetic activity became humanly perceptible and measurable by changes in morphological dimensions following the VMM step. Data measurements were taken on the embryo inner cellular mass area (ICM), complete embryo and ICM area, and on the vertical, diagonal, and horizontal axis of the sub-zonal (perivitelline) space, zona pellucida (ZP), and trophectoderm (TE). Of the 72 bovine embryos transferred, as Grade 1 and 2, 19% failed to form a pregnancy as affected by morphokinetic activity. Of the 41 equine embryos transferred, 37% of the Grade 1 and 2 embryos failed to form a pregnancy. Morula stage equine embryos presented differences on the overall axis (P ≤ 0.01), ZP thickness (P < 0.05), and perivitelline space shift (P < 0.01), as well as tendencies when measuring the area of the ICM (P < 0.09). Once bovine embryos were analyzed and evaluated as range of overall morphokinetic activity per section measured, the effect of pregnancy due to that activity was most significant when measuring the complete embryo area (P < 0.03) and embryo complete change in diameter along the X-axis and Y-axis (P < 0.02) with average changes of 1242.66μm2 and 8.41μm, respectively, in embryos resulting in pregnancies. Both species demonstrate and exemplify that there is more to embryo grading than just morphology based on the observed effect on pregnancy status and morphokinetic values. By evaluating all of these factors, there are a wider range of parameters to rely on for the selection of viable embryos outside of the morphology.