Establishment of a Low-Level Laser Irradiation Protocol for

Photobiomodulation using low-level laser irradiation has been proposed as a non-thermal approach for stimulating cellular metabolism, but practical protocols for its application to bovine embryos after fertilisation remain insufficiently described. This study aimed to establish a practical low-level laser irradiation procedure at different time points during the in vitro development of bovine embryos. Abattoir-derived oocytes were matured, fertilised with semen from a single bull, and cultured under standard conditions. Entire culture dishes were irradiated using a K-Laser Cube at 800 nm and 0.10 W for 200 s, delivering 20 J, on day one, three or five after fertilisation; an untreated run served as a reference. Embryos were evaluated on day seven according to International Embryo Transfer Society standards. Across four separate production runs, 1345 inseminated oocytes yielded 348 blastocysts meeting IETS stage 6, quality grade 1 criteria (26% overall); blastocyst rates were 27.8%, 22.0%, 25.9% and 28.5% for the reference, day one, day three and day five runs. Because each treatment was tested in a separate run, these values are descriptive and do not permit assessment of treatment efficacy. The procedure was incorporated into routine bovine in vitro embryo production and provides a basis for future validation studies.