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.

In vitro produced bovine embryos are critical to the cattle industry. However, these embryos have altered morphology, epigenetics, and metabolism when compared to their in vivo counterparts. The aim of this thesis was to alter maturation and culture media to improve the developmental competence of in vitro bovine embryos. This thesis is comprised of three experiments and one proof of concept study. Each experiment followed the same general layout: oocyte aspiration from Jersey or Holstein ovaries, oocyte maturation for 24 hours, fertilization with bull semen for 24 hours, then embryo culture for 7-8 days in 38.5 °C in 5% O2, 5% CO2 and 90% N2. A proportion of stage 7 grade 1 blastocysts were fixed and stained with Nile Red to evaluate lipid content, Mitotracker Red CMX-Rosamine to measure mitochondrial activity, or Cell Rox Green to assess reactive oxygen species (ROS). Using a confocal microscope, images were taken of each stained embryo to detect and measure fluorescence. Any stage 7 embryos that were not imaged were slow frozen and evaluated for re-expansion when thawed. Experiment 1 was a one-way treatment designed to compare the conventional maturation media (control) containing fetal bovine serum (FBS), to an alternative media replacing FBS with a human platelet lysate serum substitute (SS). Both abattoir and ovum pick up (OPU) oocytes were used. The results suggested that maturing in vitro and OPU oocytes with serum substitute maintained developmental competence, including a similar yield of embryos and re-expansion rate. Resulting in vitro SS embryos had lower lipid content (p<0.05) and ROS levels compared (p<0.05) to the FBS control. Experiment 2 was a 2x2 factorial design testing how the addition of FGF2, LIF, and IGF1 cytokines to maturation and culture media affected in vitro embryo development. The first factor was maturation media (Mcon: industry standard and Mcyt: added cytokines) and the second factor was culture media (Ccon: industry standard and Ccyt: added cytokines). The two maturation media crossed with the two culture media equated to four treatments, including a control. The results suggested that cytokine addition had no effect on blastocyst rate or re-expansion rate. The combination of MCyt x CCyt media produced the lowest lipid levels (p<0.05) while the MCon x CCon treatment led to the highest mitochondrial activity (p<0.05). Experiment 3 was a 2x2 factorial design testing how the addition of melatonin to cytokine supplemented maturation media affected embryo developmental competence. The maturation factor had two levels: no supplementation (NoM) and melatonin with cytokine supplementation (MM). The culture factor had two levels: no supplementation (NoC) and cytokine supplementation (CC). We found no difference in blastocyst or re-expansion rate between any treatments. NoM showed higher mitochondrial activity than MM (p<0.05). NoC showed higher mitochondrial activity than CC (p<0.05). The NoM x NoC treatment showed the highest lipid levels of any treatment (p<0.05). The NoM x NoC treatment showed the highest mitochondrial activity of any treatment (p<0.05). The final part to this thesis focused on the preliminary use of phasor-fluorescence lifetime imaging microscopy (FLIM) and deep imaging via emission recovery (DIVER) technologies to autofluoresce endogenous compounds and predict the viability of an embryo without the use of invasive labels. In conjunction with the University of California Irvine, we tested the technologies on morula and blastocyst stage embryos to see if developmental competence was altered. Results suggested FLIM successfully captured NADH levels and DIVER successfully captured ROS and lipid content. Future studies are planned to fully investigate the effects of the microscopes on development and to accurately predict bovine embryo viability for transfer. Overall, human platelet lysate was a successful replacement for FBS, likely due to its similar content of protein and growth factors. Neither cytokine nor melatonin supplementation had conclusive results, further trials are needed to fully determine effectiveness.

The production of embryos has been described as a revolutionary process with the ability to make cattle systems more successful. However, despite constant research done in the field of embryology, there remains a discrepancy between the quality of in vitro produced (IVP) and in vivo derived (IVD) embryos. This difference is potentially associated with the lack of synchronization between nuclear and cytoplasmic maturation events within the oocyte, which is carefully mediated in the ovarian environment and the cumulus oocyte complex (COC). The purpose of this thesis was to utilize a pre-maturation culture system to keep oocytes in an arrested germinal vesicle (GV) state before subjecting them to maturation. In the first half of the experiment, oocytes were pre-matured in C-type natriuretic peptide (CNP) supplemented medium for 12 hours. Following pre-maturation, oocytes were transferred to amphiregulin (AREG) for another 12 hours to develop. This procedure is known as CAPA-AREG. After fertilization and a 7- day culture, embryos were assessed for cleavage rate and blastocyst rate. Embryos were also subjected to staining in order to evaluate lipid content and mitochondrial activity via confocal microscopy and ImageJ software. Hurdles in acquiring data also encouraged an assessment on the IVF laboratory and how procedures can be optimized. Overall, embryology lab procedures must be strictly followed, and the laboratory environment must be maintained to the best of the staff’s ability to increase success rates. Due to mishaps in the laboratory, the effectiveness of CNP and AREG on improving bovine oocyte maturation and embryo development is still inconclusive. Further research is required to determine if CNP and AREG can be utilized in future bovine IVF procedures.