218 AN EVALUATION OF IN VITRO MATURATION OOCYTE ACTIVATION METHODS FOR IMPROVING OVINE INTRACYTOPLASMIC SPERM INJECTION EFFICIENCY

Abstract

Given previous low sperm decondensation rates and poor oocyte activation in sheep ICSI (10–20%), we evaluated activation techniques for IVM/ICSI. Incubations were performed in a 5% CO₂ cell incubator at 38.5°C and saturated humidity. Sheep ovaries were collected at an abattoir and transported <3 h to the laboratory. Follicular fluid was aspirated from 2–8 mm follicles using an 18-gauge needle and syringe with 1 mL of modified Tyrode’s medium supplemented with 10 mM sodium lactate, 10 mM HEPES, and 0.1% polyvinyl alcohol (TL-HEPES-PVA, 7.3–7.4 pH), with 200 IU mL^–1 heparin. Cumulus-oocyte complexes (COC) with compact cumulus mass and uniform cytoplasm were selected from the follicular fluid and washed 3× in 500-µL drops of maturation medium (TCM 199) with Earle’s salts and 26.2 mM sodium bicarbonate and l-glutamine with 0.1% polyvinyl alcohol, 0.91 mM sodium pyruvate, 3.05 mM d-glucose, 0.57 mM cysteine, and 10 ng mL^–1 epidermal growth factor. Next, 500 µL of maturation medium with 0.5 μg mL^–1 LH, 0.5 μg mL^–1 FSH, and 10% (vol/vol) of FCS was placed in sterile 4-well plates with 20 to 30 COC/well and with mineral oil for 24 h incubation. The COC were placed in a 500-µL drop of TCM 199-HEPES (TCM 199-H) with 300 IU of hyaluronidase for 3 min and washed (3×) in TCM 199-H. Next, 20 to 30 oocytes were placed in 250-µL droplets of TCM 199-H under a microscope to identify the first polar body (PB). Oocytes with PB were placed in 100-µL droplets of modified Tris-buffered medium (mTBM) for 1 to 4 h of incubation. The groups formed were (1) control: oocytes manipulated as in ICSI but no injection, (2) false injection: oocyte pierced but no sperm insertion, (3) chemical activation (c-a): 7% ethanol (7%Et) × 5 min, (4) c-a: 50 µM calcium ionophore (CaI) × 10 min, (5) c-a: 5 µM ionomicine (Io) × 5 min, (6) ICSI, and (7) 7%Et × 5 min + ICSI. For ICSI, 2 straws of frozen semen from a proven ram were thawed and diluted 1 : 10 with TCM 199-H and 3 mg mL^–1 BSA, and then centrifuged 3 min at 200 × g. The sperm pellet was diluted with 100 µL of TCM 199-H, and 2 mL of TCM 199-H was added to a 45° bent tube for a 1-h swim-up. Next, 500 µL of supernatant was diluted to 1 × 10⁶ sperm mL^–1 and 10 µL added to 10 µL of 10% polyvinylpyrrolidone (PVP). Five oocytes at a time were placed in a Petri dish with a 10-µL drop of TCM-199-H, with 1% gentamycin, 2% serum, and one 2-µL drop of sperm suspension-PVP. Groups of 10 to 20 oocytes were activated in 100-mL drops of respective chemical in TCM 199-H at 20 to 22°C. Oocytes were washed (3×) in mTBM and set in 200 mL of mTBM for 18 to 20 h of incubation. Oocytes were stained with 10 μg mL^–1 Hoechst 33258 for 15 min to assess pronucleus formation. Pearson χ² tests showed statistical differences (α = 0.05) among the groups (χ² = 123.165, P < 0.001); for example, groups 1 and 7 (χ² = 68.179, P < 0.001) and 6 and 7 (χ² = 42.842, P < 0.001). Results (oocytes, percentage activated) for each group were (1) n = 151, 13.2%, (2) n = 78, 32%, (3) n = 393, 53.6%, (4) n = 350, 46.8%, (5) n = 78, 42.3%, (6) n = 200, 24.5%, and (7) n = 123, 60.9%. The highest percentage of oocyte activation was achieved using 7%Et × 5 min + ICSI.