The effect of activation protocols on the development of cloned goat embryos

This study was conducted to compare the developmental competence of somatic nuclear transfer (NT) embryos, after either ionomycin or ethanol activation, in locally bred goats. Donor cells were prepared from the ear skin fibroblasts of a female goat. Cells, at passage 3-8, starved by culturing in 0.5% FCS for 4-8 d, were used for NT. Immature oocytes were obtained from FSH-stimulated goats and matured for 22 hr before enucleation and NT. After fusion, the reconstructed embryos were activated with either ionomycin or ethanol followed by culturing in 6-dimethylaminopurine (6-DMAP) and cytochalasin B (CB), for 3 hr. In experiment I, the fused NT embryos (n=63, ionomycin and n=68, ethanol treatments, respectively) were cultured in B2 with a Vero co-culture system and their developmental competence was evaluated through to Day 9. In experiment II, the NT embryos at the 2-4 cell stage on Day 2 derived from each treatment (ionomycin n=46, and ethanol n=37), were transferred into 10 synchronous recipients. There were no significant differences between the NT embryos derived from the ionomycin and ethanol groups, in fusion (86.3% versus 82.9%), cleavage (90.5% versus 82.4%) and for morula/blastocyst development rates (9.5% versus 5.9%). Sixty percent (3/5) of the recipients from ionomycin became pregnant by midterm (2.5 mts) while only 20% (1/5) from ethanol treatment was pregnant by Day 45. The results demonstrate that activation with either ionomycin or ethanol in combination with 6-DMAP-CB treatment does not affect the development of cloned goat embryos.     

Isotopologue fractionation during microbial reduction of N2O within soil mesocosms as a function of water-filled pore space

Isotopologue analyses of N₂O within soil mesocosm experiments were used to evaluate the influence of N₂O reduction on isotope fractionation. We investigated fractionation during N₂O reduction at 60%, 80% and 100% water-filled pore space (WFPS) and found net isotope effects (NIE) for δ¹⁵N of 4.2–7.8‰, δ¹⁸O of 12.5–19.1‰, δ¹⁵N^α of 6.4–9.7‰ and δ¹⁵N^β of 2.0–5.9‰. Consequently, N₂O reduction has a marked affect on isotopologue values and the importance of this process in flux chamber studies should not be ignored. With the exception of SP (the difference between the δ¹⁵N of the central, α, and terminal, β, atoms) inverse relationships between the NIE, reaction rate and reaction rate constant and WFPS were observed. Isotopic discrimination in SP during N₂O reduction was small and the average NIE for the treatments varied between 2.9‰ and 4.5‰. A strong correlation was evident between δ¹⁸O vs. δ¹⁵N and δ¹⁸O vs. δ¹⁵N^α during reduction with slopes of 2.6 and 1.9, respectively, which contrasts from a slope of <1 commonly observed for mixing between soil-derived and atmospheric N₂O in flux chambers.