130 THE EFFECT OF ALTERED ENERGY SUBSTRATE CONCENTRATIONS ON THE DEVELOPMENT OF DIPLOID PARTHENOGENETIC PORCINE EMBRYOS CREATED FROM OOCYTES FROM GILTS AND SOWS

Abstract

There is building evidence that altering the concentration of energy substrates in the culture medium to more closely approximate the concentrations thought to be present in the reproductive tract improves porcine embryo development. The aim of this experiment was to examine the development of porcine parthenogenetic embryos in such a modified version of NCSU23. The embryos were created from both sow- and gilt-derived oocytes to see whether the source of oocytes influences how the embryos respond. Ovaries from slaughtered sows or prepubertal gilts were collected, follicles (3–6 mm) were aspirated and oocytes surrounded by at least three layers of compact cumulus cells were collected and matured in TCM199 containing cysteamine, insulin, FSH, EGF, and 10% sow follicular fluid, for approximately 40 h. Cumulus cells were removed and good quality mature oocytes with a visible polar body were electrically activated (approximately 44 h after the start of maturation) by 2 DC pulses (1.5 kVcm⁻¹, 60 μs) applied 1 s apart, cultured in NCSU23 + 7.5 μg/mL cytochalasin B for 3 h and then placed into the treatment droplets. All long-term culture was in 50-μL droplets under mineral oil in an atmosphere of 5%CO₂, 5%O₂, 90%N₂ at 38.5°C for a total of 6 days (144 h). The treatments were: NCSU23 (N23); a modified NCSU23 containing 0.6 mM glucose, 0.2 mM pyruvate, and 5.7 mM lactate (PLG); or the modified NCSU23 for the first 48 h and then NCSU23 for the remaining 4 days (dPLG). All treatments were transferred into fresh droplets of the appropriate medium at 48 h. Heat inactivated fetal bovine serum (10%) was added early on Day 5. On Day 6, the embryos were assessed for morphological development and those embryos judged to be good blastocysts were stained for cell count. Experiments using sow or gilt oocytes were conducted and analyzed separately. Morphological data were analyzsed by χ² and cell number data by ANOVA with the LSD test used to determine differences between treatments. There were no statistical differences in the percentage of activated sow or gilt oocytes developing to blastocysts (sow: N23 51.3%, PLG 48.9%, dPLG 55.1%; and gilt: N23 25.6%, PLG 38.2%, dPLG 38.6%). The blastocysts produced in the dPLG treatment contained significantly more cells than those produced in the N23 treatment for both sow- (mean ± SEM; N23 47.1 ± 2.1; dPLG 53.8 ± 1.7; P < 0.001) and gilt (N23 39.3 ± 3.4; dPLG 50.5 ± 3.0; P < 0.05)-derived embryos. The number of cells per blastocyst produced in the PLG treatment did not differ significantly from the other treatments (sow: 49.9 ± 2.0; gilt: 43.4 ± 2.8). These data demonstrate that culturing for the first 48 h with the modified energy substrate concentrations improved the cell number of the resulting blastocysts but did not affect the proportion developing into blastocysts by day 6 of culture. This effect appeared to be consistent whether sow- or gilt-derived parthenogenetic embryos were used.