Reversal of the Effects of TNF-α on In Vitro Derived Bovine Embryos by Addition of a Prostaglandin Synthesis Inhibitor , Indomethacin by Lauren

Gast, Lauren Renae. Reversal of the Effects of TNF-α on In Vitro Derived Bovine Embryos by Addition of a Prostaglandin Synthesis Inhibitor, Indomethacin. (Dr. Scott Whisnant) Tumor Necrosis Factor alpha (TNFα) is a cytokine released by macrophage cells in response to local infection or disease. Mastitis, a common infectious disease in cattle, has been shown to increase the serum concentration of TNFα. This immune response pathway leads to an increase in other cytokines and prostaglandins which can compromise oocyte maturation and embryo development. The objective of this study was to investigate the possibility that the addition of TNF-α after fertilization of bovine oocytes in vitro will decrease embryo development to the blastocyst stage and that this effect can be reversed by the addition of a prostaglandin synthesis inhibitor, indomethacin. Ovaries were obtained from a local abattoir and immature cumulus oocyte complexes (COC) were aspirated from 210mm diameter follicles. For maturation, COCs were placed in M199 including fetal bovine serum (FBS), LH, FSH, estradiol, pyruvate, and gentamycin and incubated at 39 C, 100% humidity, and 5% CO2 for 18-20 hours. After maturation and fertilization, groups of 20-25 presumptive zygotes were randomly placed in either control development medium (M199 plus FBS), development medium containing 25 ng/ml of TNFα or development medium containing 25 ng/ml of TNFα plus 1 μg/ml indomethacin. All embryos were cultured at 39 C in an atmosphere of 5% CO2 in air and 100% humidity. Developmental data was collected by observation of blastocyst stages (early blastocyst, mid-blastocyst, late blastocyst, or hatching blastocyst) at day seven of culture (168 hours post fertilization [hpi]) and day nine of culture (216 hpi). At 168 hpi, bovine embryos exposed to 25 ng/ml TNF-α after fertilization showed a decrease in the percent of embryos that developed to the blastocyst stage (17.15 ± 2.36 % p<0.005) compared to the control (30.45 ± 2.36% p<0.005). The results collected on day seven of development (168 hpi) were significant (p<0.005). At day nine of development (216 hpi) the proportion of embryos developing to the blastocyst stage was not affected by treatment and was not significant, however a definite trend was detected (p< 0.08). There was no significant difference between the TNF-α treatment plus indomethacin group (25.75 ± 2.89% p<0.005) and the controls (30.45 ± 2.36% p<0.005), however these two groups were significantly different from the TNF-α treatment group alone (17.15 ± 2.36% p<0.005) on day seven of development. These results indicate that the inhibitory effect of 25 ng/ml TNF-α on the development of bovine embryos to the blastocyst stage operates through the increase of PGF-2α and this effect can be reversed by the addition of a prostaglandin synthesis inhibitor, indomethacin which blocks the biosynthetic pathway of PGF-2α from arachidonic acid. Reversal of the Effects of TNF-α on In Vitro Derived Bovine Embryos by Addition of a Prostaglandin Synthesis Inhibitor, Indomethacin by Lauren Renae Gast 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