277ENERGY REQUIREMENT DURING DEVELOPMENT TO THE BLASTOCYST STAGE OF PORCINE EMBRYOS PRODUCED IN VITRO

Abstract

A successful in vitro production (IVP) of porcine blastocysts, which enables piglet production after transfer to recipients, was reported (Kikuchi et al., 2002 Biol. Reprod. 66, 1033–1041). Generally, in the IVP system, both glucose and glutamine as energy sources were included in vitro culture (IVC) medium from Day 2 (Day 0=the day of in vitro fertilization) until Day 6. However, the exact requirement of these substances for the development to the blastocyst stage of IVP embryos has not yet been clarified. The objective of the present study was to evaluate whether these two substances are necessary for embryonic development to the blastocyst stage in culture during the period. Porcine cumulus-oocyte complexes were matured for 46h and fertilized in vitro as reported by Kikuchi et al. (see above). After removal of cumulus cells and spermatozoa, the oocytes were cultured subsequently in NCSU-37 supplemented with pyruvate and lactate (IVC-PyrLac) for 2 days. Then they were cultured until Day 6 in other IVC medium prepared as follows (1–6); Basic IVC medium (BM) was a modified NCSU-37 consisting of 108.7mM NaCl, 4.8mM KCl, 1.7mM CaCl2, 1.2mMKH2PO4, 1.2mM MgSO4, 25.1mM NaHCO3 and 4mgmL−1 fatty acid-free BSA. Then one or more of the following energy sources were supplemented to BM;; (1) 12mM sorbitol (SigmaUltra), 5.55mM glucose (Wako special grade) and 1.0mM glutamine (Sigma) (NCSU-37/Gln+), (2) 19.2mM sorbitol and 1.0mM glutamine (IVC-Sorbitol/Gln+); (3) 19.2mM mannitol (SigmaUltra) and 1.0mM glutamine (IVC-Mannitol/Gln+), (4) 12mM sorbitol and 5.55mM glucose (NCSU-37/Gln−); 5) 19.2mM sorbitol (IVC-Sorbitol/Gln−); and 6) 19.2mM mannitol (IVC-Mannitol/Gln−). The osmolarity of these media was adjusted to 283–285 osmolg−1. All embryos were fixed as whole mounts, stained and evaluated. The rate of blastocysts in NCSU-37/Gln+ (26.8%) was significantly higher (P<0.05; by analysis of variance and Duncan’s multiple range test) than those in IVC-Sorbitol/Gln+, IVC-Mannitol/Gln+ and NCSU-37/Gln− (19.0%, 17.0% and 15.5%, respectively). A remarkable decrease in the rates in IVC-Sorbitol/Gln− and IVC-Mannitol/Gln− (P<0.05; 1.4% and 2.0%, respectively) was observed. The cell numbers of NCSU-37/Gln+, IVC-Sorbitol/Gln+, IVC-Mannitol/Gln+ and NCSU-37/Gln− (55.5, 52.0, 49.6 and 58.7, respectively) had a tendency to be higher than those of IVC-Sorbitol/Gln− and IVC-Mannitol/Gln− (38.0 and 35.2, respectively). These results confirm that the supplementation of maturation medium with at least one energy source (glucose or glutamine) promotes embryonic development in vitro to the blastocyst stage, that the combination of both sources improves the chance of the embryonic survival, and that porcine embryos do not utilize sorbitol or mannitol as an energy source. The importance of glucose and glutamine is suggested for the development to the blastocyst stage of porcine IVP embryos.