Production of piglets derived from in vitro-produced Blastocysts fertilized and cultured in chemically defined media: Effects of theophylline, adenosine, and cysteine during in vitro fertilization

To further develop defined conditions for in vitro fertilization (IVF) and in vitro culture (IVC) of in vitro-matured porcine oocytes, we evaluated the effects of theophylline, adenosine, and cysteine in a chemically defined medium during IVF. Viability to full term of in vitro-produced blastocysts after lVF and IVC in chemically defined medium was also investigated by embryo transfer to recipients. A chemically defined medium, porcine gamate medium (PGM), was modified from porcine zygote medium (PZM-4), which was previously established. PGM was used as a basal medium for lVF and PZM-4 was for the culture of presumptive zygotes. Addition of 2.5 mM theophylline to PGM significantly increased the percentage of male pronuclear formation compared with controls (no addition). Addition of 1 muM adenosine to PGM supplemented either with or without 2.5 mM theophylline significantly reduced the number of penetrated spermatozoa compared with controls (no addition of adenosine). Supplementation with 0.2 muM cysteine in PGM containing both 2.5 mM theophylline and 1 muM adenosine further increased the percentage of development to the blastocyst stage, compared with no supplementation of cysteine, but there was no difference in fertilization parameters, such as monospermy and pronuclear formation, regardless of presence or absence of theophylline and adenosine. When Day 5 blastocysts were transferred into four recipients (20-25 blastocysts per recipient), all recipients became pregnant and farrowed a total of 21 live piglets. The present results clearly demonstrate that porcine blastocysts can be produced by lVF and IVC in chemically defined media and that they can develop to full term after embryo transfer.