205 Effect of different temperatures and storage intervals on pig oocyte developmental rate following in vitro maturation

Oocytes are more sensitive to temperature changes than other germ cells and somatic cells. This study was designed to examine whether storing pig oocytes before in vitro maturation (IVM) affects cytoplasmic and nuclear maturation. Ovaries were collected from a local abattoir and transported to the laboratory (37°C) within an hour. Oocytes were retrieved from the ovary using slicing method. A group of 200 oocytes was used per treatment. Good quality oocytes were stored in an appendoffs tube containing holding solution (ViGRO™ Holding Plus) at 5°C, 18°C, 24°C, and 38.5°C for 5, 30, 60, or 120 min, respectively. After oocytes’ storage at different temperatures and period, they were then subjected into IVM (500 µL of North Carolina State University 23) media covered with mineral oil. Oocytes were then incubated at 38.5°C for 44 h with 5% CO₂ and 100% humidity. Analysis of variance was used to analyse data using the GLM procedure, treatment means were compared with the least significance difference test. After 44 h of IVM, the cytoplasm damage was evaluated by visualising parameters such as cracked cytoplasm, darkish in colour resulting to lyse, and cytoplasm shrinkage as well as polar body extrusion in all groups, with the aid of Oosight Imaging System (Hamilton Thorne) connected to an inverted microscope. There was no significant difference of cumulus-oocyte complexes (COCs) expanded stored at 5°C for 30 min (57.6 ± 14.3) and 24°C for 30 min (55.9 ± 21.3). There was no significant difference of COCs expanded stored at 5°C for 60 min (53.8 ± 15.0), 18°C for 120 min (52.1 ± 8.9), 24°C for 5 min (53.2 ± 18.1), or 24°C for 60 min (54.4 ± 15.9). There was significant difference on oocytes’ damaged cytoplasm at 24°C for 30 min (4.4 ± 3.9), 24°C for 60 min (8.1 ± 5.4), and 38.5°C for control (0). There was significant difference on oocytes’ polar body extrusion stored at 5°C for 120 min (6.2 ± 5.2), 18°C for 60 min (7.6 ± 6.6), 24°C for 120 min (12.4 ± 7.9), and 38.5°C for control (25.5 ± 7.6). No significant difference was observed on oocytes’ polar body stored at 5°C for 5 min (11.5 ± 4.2), 5°C for 30 min (10.5 ± 4.7), 5°C for 60 min (9.2 ± 5.2), 18°C for 30 min (10.8 ± 5.2), 18°C for 120 min (12.1 ± 7.1), or 24°C for 30 min (12.2 ± 6.3). In conclusion, storing pig oocytes at 5°C, 18°C, and 24°C was low in survivability after maturation. Pig oocytes did not survive storage temperatures after IVM as compared to 38.5°C. Further research is necessary concerning pig oocytes storage to determine its practicality when used in conjunction with in vitro maturation.

The authors acknowledge the Agricultural Research Council for financial support.