218 AN EVALUATION OF IN VITRO MATURATION OOCYTE ACTIVATION METHODS FOR IMPROVING OVINE INTRACYTOPLASMIC SPERM INJECTION EFFICIENCY
Authors: Hernández J. E. , Ducolomb Y. , Romo S. , Fierro R. , Kjelland M. E. , Betancourt M. 
Published: December 2015

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.