197 The use of photostimulation to enhance oocyte cytoplasmic maturation

Abstract

Phototherapy uses monochromatic light from low-power lasers and light-emitting diodes (LEDs) to modulate biological processes. It has been proposed that the red-to-near infrared optical region (~600-1000 nm) enhances cellular metabolic activity by activation of the mitochondrial respiratory chain. However, photostimulation induces the generation of oxide free radicals and could create oxidative stress in exposed cells. The main objective was to use photostimulation to affect the cumulus-oocyte complex metabolic state, aiming to enhance cytoplasmic maturation rates and subsequent embryonic development. A secondary objective was to determine the toxicity of the proposed photostimulation protocol. Abattoir-derived ovaries were used. All media was from IVF Biosciences (Falmouth, Cornwall, UK). Follicles 2 to 6 mm in diameter were aspirated. Oocytes with compact cumulus and homogeneous cytoplasm were selected, and 50 oocytes/well were placed in in vitro maturation medium (0 h) and incubated at 38.5°C in 5% CO₂ in air with high humidity in the presence (treatment = exposure for 2 min to super-bright LED 1 and 2h after the beginning of maturation; LED wavelength of 660-665 nm; NTE30041; NTE Electronics Inc.) or absence (Control) of light. After maturation (22 h), oocytes were split into two wells (25 oocytes/well) and subjected to IVF with semen from two different bulls for 18 to 20 h. Cumulus cells were separated by vortexing, zygotes were placed in in vitro culture medium, and incubated at 38.5°C in 5% CO₂ in air with high humidity. Culture medium was renewed every 48 h. Cleavage, morula, and blastocyst rates were recorded as a percentage of the number of oocytes subjected to IVF per treatment. The experiment was replicated 4 times. Statistical analysis was conducted using the Mixed procedure (SAS 9.4, SAS Institute Inc.) with repeated-measures and autoregressive covariance. The model's random effect was well within treatment. Fixed effects were bull, stage of development, and treatment. There was no difference (P = 0.8) between treatments for any stage of development measured (cleavage: 76.4 ± 2.7 vs. 74.8 ± 4.1; morula: 36.1 ± 4.8 vs. 35.9 ± 5.8; blastocyst: 20.8 ± 3.2 vs. 20.6 ± 4.4 for control and treatment respectively; mean ± s.e.). Sire affected development: bull 1 had a greater percentage (P < 0.05) cleavage (82.9 ± 0.02 vs. 68.3 ± 0.02), morula (42.6 ± 0.05 vs. 29.4 ± 0.04), and blastocyst (27.8 ± 0.04 vs. 13.6 ± 0.01) development than bull 2. There was no treatment × bull interaction (P = 0.9). In conclusion, there were no stimulatory or toxic effects of this preliminary photostimulation protocol. Further research is needed to develop an optimal protocol that shows a metabolic effect and, potentially, an enhancement of in vitro cytoplasmic maturation rates and subsequent embryonic development.