Available at: https://digitalcommons.calpoly.edu/theses/1924
Date of Award
MS in Agriculture - Animal Science
Fernando Campos Chillon
Bovine in vitro produced embryos suffer from poor developmental competence and altered metabolism which hinders their cryotolerance. Overall, the goals of this thesis were to improve oocyte and embryo culture with the antioxidant polydatin and to optimize slow freezing procedures. This thesis was designed as three experiments, and in each experiment, oocytes were aspirated from abattoir ovaries, matured for 23h, fertilized with semen from 1 of 3 bulls, and cultured in synthetic oviductal (SOF) based medium (SCF1) in 38.5 °C in 5% O2, 5% CO2 and 90% N2. Stage 7 blastocysts were stained with Nile Red for lipid content or Mitotracker Red CMX-Rosamine for mitochondrial activity or Cell Rox Green for reactive oxygen species. Ten images per embryo were acquired using confocal microscopy at 5-µM step size and detected fluorescence by IMAGE PRO software. Embryos were also slow frozen in ethylene glycol and analyzed for post-thaw re-expansion after 24 and 48 hours. Experiment one was a 2x2x2 factorial design to test two culture media (SCF1 and a conventional SOF media), the use of a sucrose dehydration prior to slow freezing (2 min in 0 or 0.6 sucrose), and the length of equilibration in ethylene glycol prior to slow freezing (10 or 20 min). We determined that embryos cultured in SCF1 had increased blastocyst rate, mitochondrial activity, and cryotolerance and decreased lipid accumulation (pin vitro.