Production of piglets using intracytoplasmic sperm injection (ICSI) with flowcytometrically sorted boar semen and artificially activated oocytes.

The purpose of the present study was to develop a protocol for the successful production of piglets employing intracytoplasmic sperm injection (ICSI) with flowcytometrically sexed spermatozoa and artificially activated porcine oocytes. In vitro matured oocytes were fertilized by ICSI using non-sorted frozen/thawed epididymal semen. Oocytes were either activated by CaCl(2), Ca(2+)-ionophore or electrical pulse. Activation and fertilization rates of sperm injected oocytes stimulated by CaCl(2)-injection were significantly higher than those without activation (70.4% versus 45.9%; 49.9% versus 33.2%, respectively; P<0.001). Activation rate of sham injected oocytes increased in parallel (11.2% versus 26.3%, P<0.05), parthenogenetic development remained low (2.8% versus 8%). Co-incubation in Ca(2+)-ionophore did not improve activation rates as compared to non-activated oocytes (44.8% versus 42.5%). Fertilization rate decreased as compared to non-treated sperm injected oocytes (36.8% versus 24.5%, P<0.05). Activation of oocytes with a single electrical pulse resulted in significantly higher activation rates in all groups of oocytes as compared to non-stimulated ones (sperm injected oocytes: 65.6% versus 43.1%, P<0.001; sham injected oocytes: 48.5% versus 5.6%, P<0.001; control oocytes: 50.7% versus 0.0%, P<0.001). Fertilization rates (32.3% versus 48.2%) and parthenogenetic development (0.7% versus 38.9%, 0.0% versus 30.9%, P<0.001) increased significantly in parallel. In addition, in four replicates of flowcytometrically sorted Y-chromosome bearing spermatozoa were injected into in vivo matured oocytes, activated with 1.2 pl of a 30 mM CaCl(2) solution. On average 85.3 fertilized oocytes were transferred surgically into four recipients. Pregnancies delivered a total of 13 male piglets. These are the first piglets born from ICSI with sorted spermatozoa.