91 Photobiomodulation of bovine zygotes at 21 hours postfertilization

In vitro production (IVP) of bovine embryos has substantially improved in recent years and, consequently, it is widely used in the bovine industry. However, a significant attrition rate from the 2-cell embryo to the blastocyst stage is still a critical weak point of current IVP systems. Photobiomodulation has been shown to increase ATP and improve embryo developmental rates when applied during late oocyte maturation. We hypothesise that photobiomodulation at the zygote stage will influence the metabolic activity of the early embryo and contribute to higher blastocyst rates compared to untreated control. A standard IVF protocol and commercial media (IVF Bioscience Inc.) were used. Briefly, bovine ovaries were obtained at a local slaughterhouse, cumulus–oocyte complexes (COC) were collected by aspiration. Compacted COCs were cultured in in vitro-maturation medium (50/well) and incubated at 38.5°C in 5% CO₂ in air with high humidity. At 22 h from start of maturation, COCs were fertilized by co-culture with bull spermatozoa (1 × 10⁶ motile sperm/mL) in IVF medium and incubated at 38.5°C in 5% CO₂ in air with high humidity. At 18 h from start of fertilization, presumptive zygotes were subjected to cumulus cell removal by vortexing, and then placed in in vitro culture conditions (IVC) at 38.5°C in 5% CO₂ and 5% O₂ with high humidity. At 21 h from start of fertilization, presumptive zygotes were treated with red light (Light source: 3 super-bright LED; wavelength 660–665 nm; NTE30041; NTE Electronics INC.) for 10 min (light group), or no light (control group). Developmental rates were assessed as a ratio of embryos over zygotes placed in IVC. Cleavage rate was assessed at 72 h postfertilization and embryos with 4 cells or more were moved to fresh IVC medium. Blastocyst rates were assessed at 168 and 192 h postfertilization. For statistical analysis, each well was considered an experimental unit in a model of repeated-measures, main effects of treatment and time, blocked by replicate (Mixed procedure, SAS Institute Inc.). One of three bulls was used for each replicate for a total of eight replicates. The photobiomodulation treatment had no significant (P > 0.05) effect in embryo development rates compared to control. There was a significant (P < 0.05) effect of time, overall cleavage rate was higher than overall blastocyst rate. Cleavage rates were 78.3 ± 4.6 and 79.6 ± 3.6% (mean ± standard error of the mean) and blastocyst rates were 34.3 ± 4.6 and 32.4 ± 4.6% for Light and Control groups, respectively. There was no treatment by time interaction. In conclusion, the photobiomodulation treatment used in this experiment did not enhance blastocyst production rates. Further research is needed to determine whether there is a specific window of sensitivity to red light at the zygote stage or if a different photobiomodulation protocol could be used to modify the metabolic stage of the early bovine embryo.