151 EFFECTS OF HEXOSES SUPPLEMENTED IN THE MATURATION AND PRE IMPLANTATION MEDIUM ON THE IN VITRO DEVELOPMENT OF PORCINE OOCYTES

Abstract

The present study was conducted to determine the effects of hexoses on in vitro maturation, fertilization, and development of porcine oocytes. In the first experiment, porcine oocytes were matured in modified North Carolina State University (NCSU)-37 medium supplemented with 0.6 mM cysteine, 1 mM dibutyryl cyclic AMP (dbc AMP), 10 IU/mL equine chorionic gonadotrophin, 10 IU/mL human chorionic gonadotrophin, 50 μ/mL gentamycin (Sigma-Aldrich, Tokyo, Japan), 10% (v/v) porcine follicular fluid, and various hexoses (glucose, fructose, and galactose) at various concentrations of 0 (control), 2.5, 5.5, and 10 mM. They were subsequently cultured in the maturation medium without hormones and dbcAMP for an additional 22 h. Fertilization was performed according to Kikuchi et al. (2002 Biol. Reprod. 66, 1033–1041); 15 oocytes were co-incubated with 1 million frozen thawed sperm/mL in fertilization medium for 5 h. Supplementation of either glucose (2.5 or 5.5 mM) or fructose (5.5 mM) in the maturation medium significantly increased the percentages of maturation to metaphase II (68.5%, 79.4%, and 70.2%, respectively) and monospermic fertilization of oocytes (55.0%, 64.5%, and 58.9%, respectively), as compared with control group (metaphase II: 52.8%; monospermic: 42.7%; P < 0.05). Supplementation of galactose had no effect on the meiotic maturation and monospermic fertilization of oocytes. In the second experiment, presumptive zygotes were cultured in modified NCSU-37 supplemented with 4 mg/mL BSA, 0.17 mM sodium pyruvate, 2.73 mM sodium lactate, and 50 μg/mL gentamycin. The cleaved embryos were collected at Day 3 after in vitro fertilization and then cultured for a further 4 days in modified NCSU-37 medium supplemented with 5.5 mM of glucose, fructose, or galactose. The percentages of blastocyst formation, calculated from cleaved embryos, were significantly higher in the glucose and fructose groups (20.4% and 18.4%, respectively) than in the galactose group (12.9%) and the non-supplemented control group (9.2%). Although fructose supplementation did not accelerate blastocyst formation, it did significantly increase the mean cell number of blastocysts (48.0 ± 2.9 vs. 38.6 ± 1.8) and reduced the index of DNA-fragmented nuclei in the blastocysts stained by the TUNEL method (7.6 ± 0.9 vs. 11.8 ± 0.9), as compared with glucose supplementation. In the present study, although all hexoses were used through the glycolysis pathway, supplementation of galactose in the maturation and embryo culture medium had no beneficial effect on development. In contrast, both glucose and fructose enhanced development with an optimal concentration of 5.5 mM. Replacement of glucose with fructose did not accelerate blastocyst formation but did enhance embryo quality in terms of increasing cell number and decreasing the number of fragmented nuclei.