Non-steroidal anti-inflammatory drugs (NSAIDs) are a group of medications widely used in human and veterinary medicine for the control and management of pain, fever, and immune-related conditions [1], [2]. In general, these medications target pathways associated with the release of proinflammatory mediators as a response to tissue damage, by blocking the activity of cyclooxygenases 1 and 2 (COX-1 and -2), and thus the formation and release of prostaglandins, including PGE and PGF2α [3], [4]. Many of the NSAIDs used clinically in veterinary medicine are nonspecific COX inhibitors, resulting not only in the decrease of pro-inflammatory responses in affected tissues, but also in a decrease of beneficial activities of arachidonic acid-derivatives, such as mucous production and blood flow of the gastrointestinal mucosa [5], [6], [7]. Such side effects have been described in various species, including horses, and are related with the occurrence of gastric ulcers and colitis, particularly when NSAIDs are used for prolonged periods of time [8], [9], [10], [11].

The NSAID phenylbutazone (Bute), is a medication commonly used for managing musculoskeletal-associated pain in horses [2]. Often, mares enrolled in assisted reproductive technologies (ARTs) programs are treated for prolonged periods of time with Bute due to chronic lameness. Various studies have indicated that treatment with NSAIDs, including Bute or flunixin meglumine (FM), during the periovulatory phase of mares is associated with an increased incidence of ovulation failure resulting in anovulatory hemorrhagic follicles (AHFs) [12], [13], [14]. A study by Cuervo-Arango [13] reported that administration of 1.7 mg/kg BW FM at every 12 h intervals for 1.5 days (i.e., 0, 12, 24, and 36 h) following treatment with an ovulation-synchronizing agent (i.e., human chorionic gonadotrophin – hCG [1500 IU]) resulted in >80% rates of AHF formation, whereas mares treated with FM after 28h post hCG treatment did not have formation of AHFs. As the FM dose utilized in that work was higher than the therapeutic recommended dose (i.e., 1.1 mg/kg BW), it is plausible that the occurrence of AHFs in these mares may result not only because of the timing between hCG administration and NSAID therapy, but also due to the dose and frequency utilized. A study by Lima et al., [14] reported that mares treated for up to 4 days following deslorelin administration with recommended doses of the NSAIDs phenylbutazone (a non-selective COX inhibitor; 4.4 mg/kg BW) or meloxicam (a COX-2-selective inhibitor; 0.6 mg/kg BW) resulted in 82 – 91% of AHFs formation. Hence, these results may indicate that the administration of NSAIDs, irrespectively of the dose, result in ovulation failure in mares during the follicular dominance phase. For women enrolled in superovulation and in vivo-matured oocyte retrieval programs for in vitro embryo production, NSAIDs are often administered to reduce the incidence of spontaneous ovulations before an oocyte retrieval is performed [15], [16]. Such results in mares and women have been attributed to inhibition of prostaglandin synthesis within the dominant follicle, as this hormone has been shown to increase its levels as a by-product of the activation of inducible cyclo-oxygenase 2 (COX-2) increased expression levels at the periovulatory phase in multiple species, including horses [17], [18], [19], Apparently, a negative effect of NSAIDs on the developmental competence (i.e., in vitro maturation and blastocyst formation) of human oocytes has not been described yet. To date, no studies have determined the effects of NSAIDs, and particularly Bute, on the developmental competence of in vitro-matured equine oocytes subjected to Intracytoplasmic Sperm Injection (ICSI).

In the current study, a Preliminary Study and a Main Experiment were conducted to: 1) determine the effect of Bute administration for 10 days, at a double frequency (twice per day), on the in vitro competence of equine oocytes retrieved from post-mortem ovaries; 2) determine the effect of Bute administration for 10 days, at a single frequency (once per day), on the in vitro competence of equine oocytes retrieved from live mares. We hypothesized that oocytes recovered either from post-mortem ovaries in mares treated for 10 days with phenylbutazone, or live mares treated for 10 days with phenylbutazone, would have similar in vitro competence (in vitro maturation, cleavage, and blastocyst rates) after ICSI than oocytes recovered from non-treated mares.