Effect of Serum and Hormone Supplementation on the Competence of Oocytes Selected by Brilliant Cresyl Blue Staining

KUCHENBROD, LAUREN MARIE. Effect of Serum and Hormone Supplementation on the Competence of Oocytes Selected by Brilliant Cresyl Blue Staining. (Under the direction of Charlotte E. Farin and Peter W. Farin). Successful production of viable blastocysts in vitro depends on the utilization of a culture system that ensures oocyte maturation and the acquisition of developmental competence. A new method for evaluation of oocyte quality is assessment of glucose-6-phosphate dehydrogenase (G6PDH) activity prior to culture using brilliant cresyl blue (BCB) stain. The objective of this study was to determine if the status of maturity (BCB+, BCB–) prior to culture alters the response of bovine cumulus-oocyte complexes (COC) to serum (estrus cow serum (ECS)), hormone (follicle stimulating hormone (FSH) and epidermal growth factor (EGF)) and heat inactivation status of serum. Bovine COC were incubated in 26μM BCB in TL-Hepes for 90 minutes. They were then designated as BCB+ or BCB– and matured in one of the following treatments in modified synthetic oviduct fluid supplemented with polyvinyl alcohol and myoinositol (mSOF+PVA) for 20 h: control, FSH (0.2 U/ml) + EGF (50 ng/ml), 10% ECS, and FSH+EGF+ECS. COC were then fertilized for 20 h. Presumptive zygotes were denuded and cultured in mSOF+PVA for 72 h. Presumptive zygotes were then moved to mSOF + 10% ECS for culture to 7 d post-insemination. Serum and hormone had an additive effect on cumulus expansion. Heat inactivation of that serum also improved cumulus expansion. The presence of serum during maturation improved the ability of BCB– oocytes to resume meiosis whereas BCB+ oocytes exhibited similar rates of germinal vesicle breakdown (GVBD) irrespective of the presence of serum. Serum also improved the proportion of oocytes to reach metaphase II (MII). Heat inactivation of serum reduced the proportion of oocytes cleaving by 48 h post-insemination (hpi) compared to non-treated serum. This difference was even greater for cleavage to at least the 4-cell stage by 48 hpi. The presence of serum during maturation resulted in a higher proportion of BCB+ oocytes cleaving to at least the 4-cell stage by 48 hpi. BCB– oocytes treated with heat inactivated serum in the absence of hormone yielded greater blastocyst development compared to treatment with non-treated serum in the absence of hormone. Conversely, treatment of BCB+ oocytes with heat inactivated serum in the absence of hormone yielded lower blastocyst development compare treatment with non-treated serum in the absence of hormone. BCB– oocytes treated with heat inactivated serum in the absence of hormone yielded greater blastocyst development compared to BCB+ oocytes under the same conditions. However, the addition of hormone during maturation eliminated the differential effect of heat inactivated serum on blastocyst production with respect to BCB status. Heat inactivation of serum also resulted in a higher proportion of blastocysts developing from cleaved BCB– oocytes compared to those developing from cleaved BCB+ oocytes. There was a greater proportion of blastocysts of combined quality grades 1 and 2 produced in the presence of heat inactivated serum compared to those produced in non-treated serum. There are differential effects of supplementation of maturation medium and of heat inactivation of serum on oocytes selected by brilliant cresyl blue staining. These findings suggest that although BCB– oocytes are less mature at time of recovery compared to BCB+ oocytes, their developmental competence can be improved by culture conditions. Effect of Serum and Hormone Supplementation on the Competence of Oocytes Selected by Brilliant Cresyl Blue Staining by Lauren Marie Kuchenbrod A thesis submitted to the Graduate Faculty of North Carolina State University in partial fulfillment of the requirements for the Degree of Master of Science