PSIII-4 - Effect of photobiomodulation treatment on ATP concentrations in bovine oocytes during maturation

Abstract: Oocyte cytoplasmic maturation is critical in determining the success of subsequent embryo development. It has been proposed that light in the red-to-near infrared optical region (~600–1000 nm) enhances cellular metabolic activity through activation of the mitochondrial respiratory chain, increasing intracellular ATP concentrations, and has the potential to modify the oocyte metabolome. Previous research indicated a higher cleavage rate and similar blastocyst rates for bovine oocytes treated with photobiomodulation at hours 16 and 20 from the start of maturation compared to control. For the present study, two separate experiments were conducted to determine ATP concentration in oocytes during maturation after photobiomodulation treatment. A standardized in vitro maturation (IVM) protocol and commercial media (IVF Bioscience) were used. Briefly, bovine ovaries were obtained at a local slaughterhouse, cumulus oocyte complexes (COCs) were collected by aspiration of small (2-6 mm) follicles. For experiment 1, compact COCs were cultured in in vitro maturation media (n=30/well) and treated with no light (control); light (660-665nm) for 10 minutes at hour 16 from start of maturation (L-16) and light for 10 minutes at hour 20 from the start of maturation (L-20). At hour 24 of in vitro maturation, COCs were vortexed to remove cumulus, stained with BioTracker ATP-Red Live Cell Dye (Sigma-Aldrich SCT045), imaged with Cytation-1 (Agilent Technologies Inc.) in 6 replicates. For experiment 2, COCs were cultured in in vitro maturation media (n=40/well) and treated with no light (control) and light (660-665nm) for 10 minutes at hour 16 from start of maturation (L-16). At hours 18, 20, 22 and 24 of in vitro maturation, COCs were vortexed to remove cumulus, stained with BioTracker ATP-Red Live Cell Dye, and imaged with Cytation-1 in 4 replicates. Statistical analysis: for experiment 1, OneWay ANOVA and Student’s T-Test were performed (JMP software 16.0); in experiment 2, each well was considered as an experimental unit in a model of repeated measures, main effects of time and treatment, blocked by replicate (Mixed procedure, SAS Institute Inc.). In experiment 1, there was a significant (P < 0.05) effect of treatment; ATP concentrations were higher in L-16 than control, and L-20 was intermediate. ATP concentrations were 19579.3^a ± 596.5, 21652.0^b ± 602.7 and 19710.9^ab ± 570.6 relative fluorescence units (RFU) for control, L-16, and L-20 respectively. In experiment 2, there was a significant (P < 0.05) effect of treatment and no significant effect of time. Overall ATP concentrations were 25668 ± 894.6 and 28332 ± 894.6 RFU for control and L-16 respectively. In conclusion, Photobiomodulation treatment with LED 660-665nm for 10 minutes at hour 16 from start of oocyte maturation increases intracellular ATP concentrations from hour 18 to 24 of maturation.