Augmentation of Temperatures, Media and Cryoprotectants on in vitro Fertilization of Pig Oocytes

Abstract

There are numerous challenges to the large-scale production of pig embryos using in vitro procedures. The aim of the present study was to compare the different holding temperatures, in vitro maturation media, and permeating cryoprotectants in the in vitro fertilization of pig oocytes. Ovaries were obtained from the local slaughterhouse, oocytes were retrieved by means of aspiration and slicing techniques and categorized into grades A, B, and C. Good quality oocytes were stored in an Eppendorf tube containing a holding solution at 5, 18, 24, and 38.5°C for 5, 30, 60, and 120 min, respectively. Oocytes were matured for 44 h and then checked for polar body extrusion. Matured oocytes were exposed to cryoprotectant Toxicity Test (TT1) with 7.5% of DMSO + 7.5% of EG and (TT2) with 15% of DMSO + 15% EG. Oocytes were then in vitro fertilized with fresh semen, incubated for 24 h and the pronucleus was checked. The GLM procedure was analyzed using Analysis of Variance (ANOVA) and treatment means were compared with the Least Significance Difference (LSD) test. The results revealed that at 38.5°C, the Cumulus Oocytes Complexes (COCs) expansion rate was significantly (p<0.05) higher (75.8±14.0), no damaged cytoplasm and the polar body was higher (25.5±7.6) compared to all other treatments. Oocytes polar body extrusion were (32.8±13.6, 25.8±9.1 and 11.5±6.9) for NCSU-23, NCSU-37 and TCM-199 respectively. There was a significant (p<0.05) difference in the total fertilization rate of the control (75.8±17.2) and the combination of DMSO and EG (50.3±21.4). Pig oocytes are very sensitive to lower temperatures after IVM. The NCSU-23 and NCSU-37 media enhance pig oocytes polar body extrusion. The combination of DMSO and EG affected the pronucleus since the cryoprotectants have toxicity when used on pig oocytes after maturation.