17 Using Photobiomodulation to Improve Bovine Oocyte Maturation

Abstract Oocyte cytoplasmic maturation is critical in determining the success of subsequent embryo development. Bovine oocytes matured in vitro yield less embryonic development rates than in vivo matured oocytes and new strategies are needed to improve the culture system. 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 and has the potential to modify the oocyte metabolome. The objective of this work was to affect the cumulus-oocyte-complex metabolic state using photobiomodulation, aiming to enhance cytoplasmic maturation and subsequent embryonic development. A standardized in vitro fertilization (IVF) 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. Compacted COCs were cultured in in vitro maturation media (n = 50/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 start of maturation (L-20). At hour 22 from start of maturation, COCs were fertilized by co-culture with bull spermatozoa (1x106 motile sperm/mL) for 18 hours, then subjected to cumulus cell removal. Presumptive zygotes were placed in in vitro culture media (IVC) for 48 hours when cleavage rate was assessed. Embryos with ≥ 8 cells were selected and placed in fresh IVC until evaluation of blastocyst rate at 168 and 192 hours from start of fertilization. The experiment was replicated 6 times. Blastocysts present at 168 hours from start of fertilization were fixed in 5% formalin, stained with Hoechst 33342, imaged with Cytation 1 (Agilent Technologies Inc.), and blastocyst cell number was counted using ImageJ software (ImageJ2 2.9.0) in 4 replicates. For statistical analysis, each well was considered as experimental unit in a model of repeated measures, main effects of time and treatment, blocked by replicate (Mixed procedure, SAS Institute Inc.). One-way ANOVA test was used to analyze the average cell number per well by group, blocked by replicate (JMP Pro 16.1.0). There was a significant (P < 0.05) effect of treatment, replicate, and time for embryo development and there was a tendency (P = 0.6) for treatment effect on blastocyst cell number. Cleavage rates were 81.3 ± 2.5; 88.5 ± 2.5; 87.3 ± 2.5 % (LSM ± SEM), blastocyst rates were 49.1± 2.5; 49.3 ± 2.5; 51.6 ± 2.5 % and cell numbers were 159.9 ± 3.5; 149.3 ± 3.5; 164.0 ±3.5 for groups control, L-16, and L-20 respectively. The positive effect of photobiomodulation treatment on cleavage rate and blastocyst cell number suggests there is a window of sensitivity to 660-665nm light between 16 and 20 hours from start of maturation.