277 FERTILIZATION CAPABILITY OF BOAR SPERMATOZOA AFTER ALTERNATIVE SEMEN PRESERVATION METHODS AND INTRACYTOPLASMIC INJECTION INTO PORCINE OOCYTES

Long-term preservation of mammalian spermatozoa is regarded as being essential for the conservation of endangered species and for assisted reproduction. Although the method generally applied for semen preservation is storage at –196°C, liquid nitrogen and special equipment might not be available in every situation. Additionally, logistical difficulties make cryopreservation difficult under extreme conditions, such as in the wilderness. Fortunately, with the development of intracytoplasmic sperm injection (ICSI), preserved spermatozoa do not need to maintain their motility. The aim of the study was to investigate alternative preservation methods for spermatozoa without freezing in liquid nitrogen and to examine the effects of the preserved sperm on fertilization after injection into in vitro-matured pig oocytes. The pig was chosen as the model for investigations, and the alternative preservation methods included heat-drying, flame-drying, or freezing (–20°C, household refrigerator) of spermatozoa. After semen collection and swim up, sperm concentrations were adjusted to 0.5 to 1 × 105 cells mL–1. For heat-drying, aliquots of sperm suspension (50 µL) were dehumidified at 50, 56, or 90°C each for 45 min, or at 120°C for 20 min. Flame-drying was performed by flaming 5 µL of semen suspension or sperm-rich fraction for up to 2 s on glass slides. Dried semen samples were stored at 4°C. Further, sperm samples were frozen (100 µL/tube) without cryoprotectant and kept at –20°C. The specimens were stored up to 5 days. Before ICSI, heat-dried and flame-dried samples were rehydrated with 50 and 10 µL of ultrapure water (Millipore®, Millipore Corp., Billerica, MA, USA), respectively. Frozen sperm were thawed for 10 min at room temperature. Afterward, spermatozoa were injected into in vitro-matured pig oocytes at metaphase II, and sperm-injected oocytes were activated artificially by 10% ethanol. Subsequently, in vitro culture in mTCM-199® [20 µg mL–1 of insulin, 0.08 mg mL–1 of l-glutamine, 50 µg mL–1 of gentamicin, and 20% FCS (vol/vol)] with 10 µg mL–1 of cycloheximide was performed for 24 h. Afterward, oocytes were fixed, stained (aceto-orcein staining), and investigated for signs of fertilization. Positive fertilization criteria included extrusion of the second polar body, formation of two pronuclei, and the presence of a visible sperm tail. The fertilization ability of heat-dried samples achieved 9.0% (6/67), 8.8% (6/68), 2.7% (2/75), and 0% (0/60) at 50, 56, and 90°C for 45 min and 120°C for 20 min, respectively. Flame-drying reached a fertilization rate of 14.0% (6/43) v. 4.4% (3/69) for the sperm-rich fraction v. swim-up spermatozoa, respectively. With frozen semen (–20°C), 9.4% (13/138) fertilized oocytes were obtained. These results imply that ICSI with spermatozoa, which were alternatively preserved at various temperatures and circumstances in combination with artificial oocyte activation, may fertilize the oocytes. The best results were accomplished with the flame-dried sperm-rich fraction.