Effect of culture timing on in vitro growth of porcine oocytes derived from pre-antral follicles

Abstract

Recently, there are several studies have been conducted to modify the best conditions in the in vitro culture of porcine oocyte–granulosa cell complexes (OGCs) derived from 0.4 – 0.7 mm pre-antral follicles. On the other hand, the research on OGCs’ in vitro growth (IVG) primarily focused on the culturing method but not the culture duration. Hence, this research was carried out in order to identify the ideal timing for the in vitro growth process of OGCs collected from pre-antral follicles. The OGCs were cultured in vitro in three different time marks, including 7, 10, and 14 days. After each time mark, the survival rate of oocytes, diameter, and antrum formation status were examined. As a result, oocytes’ survival rate was quite high on day 7 (84.26%) and day 10 of IVG, but decreased markedly on day 14 (67.86%). Antrum formation pattern followed the survival rate, with most antrum observed on day 7 and day 10, and nearly disappeared on day 14 of the culturing process. However, the oocyte size on day 10 and day 14 was slightly larger than on day 7. Subsequently, the chromatin configuration and histone modifications were obtained by applying the orcein staining and immunofluorescent staining of oocytes after each time mark. The oocytes expressed all chromatin stages, including the filamentous chromatin (FC) stage, stringy chromatin (SC) stage, and germinal vesicle (GV) stage, as well as spontaneous maturation stages. GV stages were primarily found in the later days of culturing (day 10 and day 14), while the FC and SC stages were mainly observed on day 7. Expression of acetylation of histone H3 at lysine 9 was also examined with immunofluorescent staining. As a result, the intensity of histone acetylation was highest on day 10 of IVG. All the data above were compared with fully-grown oocytes derived from large antral follicles ranging from 4-6 mm. In conclusion, day 10 was the best duration for culturing the OGCs on the attachment dish in vitro due to the highest rate of antrum formation and the chromosome configuration to attain the meiotic competence later.