Tumor necrosis factor alpha inhibits in vitro bovine embryo development through a prostaglandin mediated mechanism

Mastitis and other inflammatory diseases occur in cattle leading to not only decreased milk production and increased costs but to a reduction in reproductive performance [1,2]. Cytokines, such as tumor necrosis factor α (TNFα), play a role in the immune response and are increased in the serum of cows diagnosed with clinical mastitis [2]. This release of cytokines can have detrimental effects on endometrial and oviductal tissue leading to decreased embryo development. Addition of TNFα to oocyte maturation media in vitro reduced the development of bovine cumulus oocyte complexes (COC) to the blastocyst stage [3]. This effect of TNFα could be mediated by prostaglandins. Cyclo-oxygenase-2 (COX2), an inducible enzyme responsible for the first step in prostaglandin production from arachidonic acid is upregulated by cytokines [4]. Exposure of bovine embryos to prostaglandin F2 α (PGF2α) inhibited development to the blastocyst stage in vitro [5] and progesterone supplemented cows exposed to PGF2α had reduced embryo quality [6]. Bovine oocytes expressed TNFα mRNA and lower TNFα mRNA levels were associated with more oocytes developing to the blastocyst stage on day 8 [7]. The objective of the current experiment was to determine if addition of indomethacin, an inhibitor of the COX enzyme, could block the detrimental effects of TNFα on development of bovine embryos.Tissue culture medium (TCM-199) was purchased from Gibco-BRL (Grand Island, NY). Fatty-acid free BSA was purchased from Boehringer-Mannheim (Indianapolis, IN). Recombinant bovine TNFα was purchased from Pierce Endogen (Rockford, IL) and reconstituted in sterile saline to a final concentration of 4 ng/mL. All other reagents and media supplements were of tissue culture grade and were obtained from Sigma Chemical Co. (St. Louis, MO). Indomethacin was reconstituted to a 1000X stock solution of 1 μg/mL in sterile absolute ethanol. With the exception of the fertilization medium, all culture media contained 50 μg/mL of gent