41 Effect of photobiomodulation in blastocysts post-vitrification

Cellular cryopreservation affects mitochondria function and subsequent cell survival. In previous studies, photobiomodulation of oocytes increased ATP levels in mature oocytes and enhanced subsequent embryo development and blastocyst cell numbers. The objective of this study was to examine the effect of photobiomodulation treatment in blastocysts post-vitrification. A standardized IVF protocol and commercial media (Stroebech Media) 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 (three wells at 50 oocytes/well). At 22 h from the start of maturation, COCs were fertilized by co-culture with bull spermatozoa (2 × 10⁶ motile sperm mL⁻¹) for 18 h, then subjected to cumulus cell removal by vortexing. A different bull was used for each replicate. Presumptive zygotes were placed in in vitro culture (IVC) conditions. Forty grade-1 blastocysts were used in each of six replicates, 10 per group, in four groups of a 2 × 2 factorial design (total 240 blastocysts) as follows: at 168 h post-fertilization, blastocysts were removed from IVC, split into two groups and placed in holding medium. One group was vitrified on Cryolock® (Biotech, Inc.) using the Stroebech vitrification kit, following manufacturer protocol, with two blastocysts per Cryolock. After vitrification, the nonvitrification group was split into two wells and returned to IVC, where they received no treatment (Control) or were treated with a red light (660–665nm) for 10 min (Light). The vitrified group was then warmed using the Stroebech warming kit. The embryos from each Cryolock were separated, randomly placed in one of two wells, and returned to IVC, where they received either no treatment (Vit Control [VC]) or were treated at 30 min postwarming with a red light for 10 min (Vit Light [VL]). Re-expanded blastocysts at 48 h post-vitrification were fixed and stained with Hoechst 33342 (Invitrogen H21492) for cell number count. Survival rate was assessed as a ratio of re-expanded blastocysts post-vitrification over total blastocysts per group. Data were analyzed as a 2 × 2 factorial using a Mixed model, with main effects of light, cryopreservation, and their interaction blocked by replicate. Significance was set at P < 0.05. There was a main effect of cryopreservation on survival rates, 75.6 ± 4.4% and 47.0 ± 3.0% (mean ± SEM) for non-vitrified and vitrified groups, respectively. However, there was no interaction between groups, as survival rates were 71.3 ± 7.3%, 79.8 ± 4.8%, 42.5 ± 4.4%, and 51.5 ± 3.6% for groups Control, Light, VC, and VL, respectively. There was a main effect of cryopreservation on average cell numbers and a significant interaction. Average cell numbers were 168.9 ± 5.3; 201.2 ± 9.0; 158.4 ± 7.4; and 175.0 ± 9.1 for groups Control, Light, VC, and VL, respectively. In summary, photobiomodulation did not affect the survival rate or average cell number in the cryopreserved blastocysts, but it did increase cell numbers in the nonvitrified blastocysts. Further research is needed to characterize the effects of red light on the bioenergetic capacity of blastocysts.