Time-dependent intrafollicular cyclooxygenase (COX) inhibition with prostaglandin: Effects on bovine ovulation and follicular fluid extracellular vesicles miRNAs.

Prostaglandins (PGs), specifically prostaglandin E2 (PGE2) and prostaglandin F2α (PGF2α), are critical regulators of the periovulatory cascade in cattle. While their roles are established, their precise, time-sensitive application for ovulation induction and rescue remains under investigation. We evaluated whether exogenous PGE2 and PGF2α, alone or combined, modulate ovulation and whether flunixin meglumine (FM), a nonsteroidal anti-inflammatory drug and COX inhibitor, reliably generates ovulation-block models. Experiments were conducted in cyclic Bos taurus cows. Estrous cycles were synchronized using intravaginal progesterone (P4) device (IVD), estradiol benzoate, cloprostenol sodium, and GnRH analogs. In Experiment 1, the effects of intrafollicular (IFI) of PGE2 alone or in combination with PGF2α on ovulation timing and luteal P4 production were evaluated. Experiments 2-4 examined FM-induced ovulation blockade via intramuscular (IM), intraovarian (IO), or IFI administration. Experiments 5-6 examined whether IFI prostaglandins reverse FM-induced blockade at defined periovulatory windows (16 h and 22 h post-GnRH). Experiment 7 evaluated extracellular vesicles (EV) from follicular fluid (FF) after systemic FM, including miRNA profiling. Ovulation timing was monitored by ultrasonography and luteal function by serum P4. In Experiment 1, IFI PGE2 or PGE2+PGF2α did not advance ovulation (median 66 h for PG groups vs. 42 h for control), but PGE2 increased serum P4 (P = 0.0014). Systemic IM FM (≈75-80 % ovulation) and IO FM (100 % ovulation) did not consistently block ovulation (Experiments 2-4), whereas IFI FM at 22 h post-GnRH inhibited ovulation in all treated cows (Experiment 6). Co-administration of prostaglandins at 16 h did not significantly reverse FM blockade (Experiment 5), but PGE2 partially restored ovulation at 22 h (44 % ovulated; P = 0.04), closer to the physiological PG peak (Experiment 6). Four miRNAs (bta-miR-584, bta-miR-369-5p, bta-miR-218, bta-miR-411c-5p) were downregulated in FF EV after FM, which were mapped to eight pathways pertinent to follicular rupture and PG-linked mechanisms. In conclusion, exogenous prostaglandins did not hasten ovulation, although PGE2 exerted a luteotropic effect. IFI FM administered near the late periovulatory window effectively blocked follicular rupture, and timely IFI PGE2 partially rescued FM-inhibited ovulation. Systemic FM also modulated FF EV miRNAs, suggesting pathway-level mechanisms underlying ovulatory failure.