76 Effects of Reduced Environmental Light on the in Vitro Maturation of Pig Oocytes

Abstract

The effects of light on the steps of embryo manipulation have been described in several species, including humans. There are reports in which exposure of these cells to UV rays from sunlight affects them epigenetically, which could lead to meiotic arrest that would prevent normal maturation from the germinal vesicle (GV) to metaphase II (MII) stage, which would compromise subsequent embryo viability. Development to the blastocyst was not evaluated. The objective of the study was to observe the difference in maturation capacity (GV to MII) of prepubertal gilt oocytes under conditions of reduced ambient light. Thirty Duroc ovaries were recovered at slaughter and immersed in saline (0.9% NaCl) supplemented with penicillin-G (100 IU mL–1) and streptomycin sulfate (100 mg mL–1) at 29 to 34°C for transport to the laboratory where they were punctured with an 18-gauge needle attached to a 10-mL syringe. GV oocytes were selected with at least 3 layers of compact cumulus cells and were incubated with TCM-199 for 42 h in total with replacement with fresh maturation medium at 22 h. Oocytes were subsequently denuded using 0.1% hyaluronidase, fixed with 2% formaldehyde, and stained with Hoechst 33342 (10 min). Thereafter, oocytes were evaluated under a fluorescence microscope for the stage of meiosis: GV, metaphase I (MI), anaphase I/telophase I (ATI), and MII. GV oocytes were divided into two groups of 60 each as follows: Group 1 = handling in ambient light (1 change of culture medium and evaluation at the stereoscope); Group 2 = handling with reduced light (the change of medium was carried out in a dark room with red light on the stereoscope. Chi-square and Student’s t-test with Minitab 16.0 statistical program were used, and differences were considered significant at P < 0.05. There were significant differences in the percentages of maturation stages between Group 1 and Group 2, respectively: GV = 3.33, 3.33% (P = 0.45); MI = 20, 13.3% (P = 0.037); ATI = 33.3, 26.6% (P = 0.03); and MII = 43.3, 56.6% (P = 0.019). In conclusion, rate of maturation to MII was significantly higher with decreased light exposure. There was no difference in the GV groups probably because there was no manipulation at this stage. Further studies are necessary to determine the amount of light needed to optimize oocyte viability and to assess any potential effects on blastocyst production.