208 Photobiomodulation during oocyte maturation enhances blastocyst rates but not blastocyst adenosine triphosphate content

Mitochondria play a key role in the physiology of mammalian oocyte and pre-implantation embryo development. In previous studies, photobiomodulation of oocytes during IVM affected ATP concentrations in mature oocytes and enhanced subsequent embryo development. The objective of this study was to examine ATP concentration and cell numbers in blastocysts derived from oocytes exposed to photobiomodulation treatment during IVM. A standardized IVF protocol and commercial media (IVF Bioscience Inc.) were used. Briefly, bovine ovaries were obtained at a local slaughterhouse and cumulus–oocyte complexes (COCs) were collected by aspiration. Compacted COCs were cultured in IVM medium (50/well) and treated with red light (3 super-bright LED, 660–665 nm; NTE Electronics Inc.) for 10 minutes at 16 h from the start of maturation (L-16), or red light at 20 h (L-20), or no light (control). At 22 h from start of maturation, COCs were fertilized by co-culture with bull spermatozoa (1 × 10⁶ motile sperm mL⁻¹) for 18 h, then subjected to cumulus cell removal by vortexing, and placed in in vitro culture medium (IVC). Developmental rates were assessed as a ratio of embryos over zygotes placed in IVC. Cleavage rate was assessed at 72 h from start of fertilization (hsf) and blastocyst rate at 168 and 188 hsf. Blastocysts present at 188 hsf were stained with BioTracker ATP-Red Live Cell Dye (Sigma-Aldrich) and imaged on a Cytation-1 (Agilent Technologies Inc.) to determine ATP content, then fixed and stained with Hoechst 33342 (Invitrogen) for cell number count. Two bulls were used, one per replicate, for a total of 6 replicates. For embryonic development comparison, each maturation well was considered an experimental unit in a model of repeated measures, main effects of treatment, time, and bull, blocked by replicate. For ATP and cell number, main effects of treatment and bull, blocked by replicate, were used (Mixed procedure, SAS Institute Inc.). Both photobiomodulation treatments had higher development rates than control; there were significant (P < 0.01) effects of treatment, time, and bull. There was no treatment by time interaction (P > 0.05) and the treatment by bull interaction approached significance (P = 0.07). Cleavage rates were 88.5 ± 2.4, 89.0 ± 2.5, and 85.0 ± 1.1% (mean ± s.e.m.) and blastocyst rates were 44.0 ± 5.5, 44.3 ± 5.5, and 35.3 ± 4.6% for groups L-16, L-20, and control, respectively. The photobiomodulation treatment did not affect (P > 0.05) ATP concentrations at the blastocyst stage, but blastocysts from L-20 had higher (P < 0.05) average cell number than control: 145.8^ab ± 10.7; 153.9^a ± 12.4; 142.5 ± 12.0^b (mean ± s.e.m.) for groups L-16, L-20, and control, respectively. In conclusion, this study supports the previous report on enhanced embryo development rates and blastocyst cell number after photobiomodulation treatment of oocytes during IVM. However, the reported difference in ATP concentrations in mature oocytes does not persist at the blastocyst stage.