We previously developed an in vitro production system for porcine embryos and reported that the addition of glutamine and hypotaurine during in vitro culture improved blastocyst yield and the total number of cells in the blastocysts. Glutamine and hypotaurine might reduce oxidative stress, allowing the development of embryos cultured in vitro, because glutamine reportedly protects embryos against oxidative stress by helping to maintain intracellular levels of cysteine, a precursor of glutathione (GSH), and hypotaurine is a potent antioxidant. In the present study we evaluated the effects of the presence of glutamine and hypotaurine from Day 2 (Day 0 = the day of in vitro fertilization) to Day 3 on oxidative stress during in vitro development of porcine embryos. Porcine cumulus-oocytes complexes from prepubertal gilts were matured and fertilized in vitro using frozen-thawed ejaculated boar semen (Yoshioka et al. 2003 Biol. Reprod. 69, 2092–2099). Presumptive zygotes were cultured in porcine zygote medium (PZM)-5 (Suzuki et al., 2002) containing 2 mM of glutamine and 5 mM of hypotaurine as a basal culture medium until Day 2. The cleaved embryos were then transferred into one of four media prepared as follows: (1) containing no glutamine or hypotaurine (G-H-), (2) containing glutamine (G+H-), (3) containing hypotaurine (G-H+), (4) containing glutamine and hypotaurine (G+H+) (= PZM-5), and cultured for 24 h. After culture, the total number of cells, intracellular GSH content, and level of hydrogen peroxide (H₂O₂), which is a reactive oxygen species, in the cleaved embryos were examined. Some cleaved embryos were cultured in PZM-5 from Day 3 until Day 5 and the percentage of embryos that developed into blastocysts and the total number of cells in the blastocysts were investigated. Intracellular GSH content and H₂O₂ level on Day 3 were determined by a dithionitrobenzoic acid-glutathione disulfide (DTNB-GSSG) reductase recycling assay and dichlorohydrofluorescein diacetate (DCHFDA)-based assay, respectively. Data were statistically analyzed by ANOVA and Fisher's PLSD test. The total number of cells (4.3 to 4.4 cells) and intracellular GSH content (2.3 to 2.9 pmol/embryo) in the cleaved embryos on Day 3 did not differ among treatments. On Day 3, the intracellular H₂O₂ level of the cleaved embryos cultured in G+H+ decreased by 49% compared with those cultured in G-H- (100%) (P < 0.05). On Day 5, the percentage of embryos that developed into blastocysts in G+H+ (52%, 47/90) and G+H- (41%, 36/88) was significantly higher than in G-H- (11%, 11/90) and G-H+ (21%, 19/89) (P < 0.05). The total number of cells in the Day 5 blastocysts from G+H+ (34.5 cells) was significantly (P < 0.05) greater than in those from G-H- (25.8 cells). These results suggest that the presence of glutamine and hypotaurine in PZM-5 from Day 2 to Day 3 improves the subsequent development of porcine embryos into blastocysts by reducing intracellular H₂O₂ levels.

This work was supported by MAFF, Japan.