Oestrus resynchronisation (RES, Resynch) programmes for non-pregnant cows allow shortening the period between an unsuccessful insemination and the next attempt on the same cow. The protocol of oestrus RES may be started after ruling out pregnancy by means of ultrasonography carried out 28 days after insemination or after performing a test for pregnancy-specific glycoproteins (PAG) in blood or milk. The Resynch protocol can be based on a double application of prostaglandins, the OvSynch protocol, or hormonal therapy with exogenous sources of progesterone (CIDR intravaginal devices). The efficiency of the method depends on the functional state of the ovaries, the diameter of the corpus luteum, external factors, and the health and maturity of the cows. The present paper constitutes a comparison of research findings concerning the effectiveness of RES programmes.

OBJECTIVE To evaluate safety and toxicokinetic profiles associated with daily oral administration of grapiprant, a new analgesic that selectively blocks the prostaglandin E2 EP4 receptor, to cats. ANIMALS 24 healthy domestic shorthair cats (12 males and 12 females). PROCEDURES Cats were randomly assigned (3 of each sex/group) to receive a placebo capsule or grapiprant at 3, 9, or 15 mg/kg, administered PO once daily for 28 days, beginning on day 0. Food consumption and behavior were observed daily, body weight was measured weekly, and clinicopathologic tests were performed on blood and urine samples collected on days −7, 14, and 25. Blood samples for toxicokinetic analyses were collected after treatment on days 0 and 27. Cats were euthanized on day 28, and full necropsies and histologic evaluations were performed. RESULTS Grapiprant rapidly reached peak serum concentrations and maintained substantial concentrations throughout the 28-day period. By day 27, maximum serum concentrations ranged from 683 ng/mL to 4,950 ng/mL, which were attained by 1 to 4 hours after administration. Serum half-lives on day 27 ranged from approximately 2 to 14 hours (median, approx 5 to 6 hours). Grapiprant was well tolerated, and no adverse effects were detected at doses ≤ 15 mg/kg. No significant effects of grapiprant were identified on body weight, food consumption, clinicopathologic variables, or gross or histologic necropsy findings. CONCLUSIONS AND CLINICAL RELEVANCE Results suggested the safety of daily oral administration of grapiprant to cats. Additional studies are needed to evaluate the efficacy of grapiprant for treatment of cats with osteoarthritis.

OBJECTIVE To evaluate ex vivo cyclooxygenase (COX) inhibition and compare in vitro and ex vivo COX-1 inhibition by flunixin meglumine and firocoxib in horses. ANIMALS 4 healthy horses for in vitro experiments and 12 healthy horses (6 males and 6 females; 5 Thoroughbreds, 5 Warmbloods, and 2 ponies) undergoing elective surgery for ex vivo experiments. PROCEDURES 12 horses received flunixin meglumine (1.1 mg/kg, IV, q 12 h) or firocoxib (0.09 mg/kg, IV, q 24 h). Blood samples were collected before (baseline) and 2 and 24 hours after NSAID administration. Prostanoids (thromboxane B2, prostaglandin E2, and prostaglandin E metabolites) served as indicators of COX activity, and serum drug concentrations were measured by use of high-performance liquid chromatography. An in vitro coagulation-induced thromboxane B2 assay was used to calculate drug concentration-COX-1 inhibition curves. Effect of time and treatment on COX activity was determined. Agreement between in vitro and ex vivo measurement of COX activity was assessed with Bland-Altman analysis. RESULTS At 2 and 24 hours after NSAID administration, COX-1 activity was reduced, compared with baseline activity, for the flunixin meglumine group only and relative COX-1 activity was significantly greater for the firocoxib group, compared with that for the flunixin meglumine group. There was no significant change in COX-2 activity after surgery for either group. Bland-Altman analysis revealed poor agreement between in vitro and ex vivo measurement of COX-1 activity. CONCLUSIONS AND CLINICAL RELEVANCE Compared with flunixin meglumine, firocoxib had COX-1-sparing effects ex vivo in equine patients that underwent elective surgery.

OBJECTIVE To validate primer sets for use in reverse transcription quantitative PCR assays to measure gene expression of cytosolic phospholipase A2 (cPLA2) and microsomal prostaglandin E2 synthase 1 (mPGES1) in equine mononuclear cells and determine the effects of firocoxib, a selective cyclooxygenase 2 (COX-2) inhibitor, on COX-2, cPLA2, and mPGES1 gene expression following incubation of mononuclear cells with lipopolysaccharide (LPS). ANIMALS 8 healthy adult horses. PROCEDURES Peripheral blood mononuclear cells were isolated by density gradient centrifugation and incubated at 37°C with medium alone, firocoxib (100 ng/mL), LPS (1 ng/mL or 1 μg/mL), or combinations of firocoxib and both LPS concentrations. After 4 hours, supernatants were collected and tested for prostaglandin E2 (PGE2) concentration with an enzyme inhibition assay, and gene expression in cell lysates was measured with PCR assays. RESULTS Primer pairs for cPLA2 and mPGES1 yielded single products on dissociation curve analyses, with mean assay efficiencies of 102% and 100%, respectively. Incubation with firocoxib and LPS significantly decreased PGE2 supernatant concentrations and significantly reduced COX-2 and mPGES1 gene expression, compared with values following incubation with LPS alone. CONCLUSIONS AND CLINICAL RELEVANCE Primer sets for mPGES1 and cPLA2 gene expression in equine mononuclear cells were successfully validated. Firocoxib significantly decreased LPS-induced COX-2 and mPGES1 expression, suggesting that it may be useful in the control of diseases in which expression of these genes is upregulated.

Objective-To assess effects of in vitro meloxicam exposure on metabolism in articular chondrocytes from dogs with naturally occurring osteoarthritis Sample-Femoral head cartilage from 16 dogs undergoing total hip replacement Procedures-Articular cartilage samples were obtained. Tissue sulfated glycosaminoglycan (SGAG), collagen, and DNA concentrations were measured. Collagen, SGAG, chondroitin sulfate 846, NO, prostaglandin E2 (PGE2), and matrix metalloproteinase (MMP)-2, MMP-3, MMP-9, and MMP-13 concentrations in culture medium were analyzed. Aggrecan, collagen II, MMP-2, MMP-3, MMP-9, MMP-13, ADAM metallopeptidase with thrombospondin type 1 motif (ADAMTS)-4, ADAMTS-5, tissue inhibitor of metalloproteinase (TIMP)-1, TIMP-2, TIMP-3, interleukin-1β, tumor necrosis factor-α, cyclooxygenase-1, cyclooxygenase-2, and nducible nitric oxide synthase gene expression were evaluated. Comparisons between tissues cultured without (control) and with meloxicam at concentrations of 0.3, 3.0, and 30.0 μg/mL for up to 30 days were performed by means of repeated-measures analysis. Results-Meloxicam had no effect on chondrocyte SGAG, collagen, or DNA concentrations. Expression of ADAMTS-5 was significantly decreased in all groups on all days, compared with the day 0 value. On day 3, culture medium PGE2 concentrations were significantly lower in all meloxicam-treated groups, compared with values for controls, and values remained low. Culture medium MMP-3 concentrations were significantly lower on day 30 than on day 3 in all meloxicam-treated groups. Conclusions and Clinical Relevance-Results suggested that in vitro meloxicam treatment of osteoarthritic canine cartilage for up to 30 days did not induce matrix degradation or stimulate MMP production. Meloxicam lowered PGE2 release from this tissue, and effects on tissue chondrocyte content and matrix composition were neutral.

Objective: To determine the effects of 2 conjugated linoleic acid (CLA) isomers (cis-9, trans-11 and trans-10, cis-12) on synthesis of prostaglandin (PG) E2 and F2α and expression of prostaglandin H synthase-2 (PGHS-2) of adult and fetal bovine endometrial epithelial cells in vitro. Sample: Primary cultures of endometrial epithelial cells obtained from 4 adult cows and 4 fetal bovine carcasses. Procedures: Cells were exposed to 0, 50, 100, or 200μM cis-9, trans-11 or trans-10, cis-12 CLA isomers for 24 hours. Culture media collected before and after 6 hours of stimulation of cells with phorbol 12-myristate 13-acetate were assayed to detect PGE2 and PGF2α via ELISA. After stimulation, cells were collected for western blot analysis to quantify PGHS-2. Results: Concentrations of PGF2α and PGE2 were significantly lower in culture media of adult and fetal endometrial epithelial cells exposed to any concentration of either CLA than they were in media of cells not exposed to CLAs. The trans-10, cis-12 CLA isomer seemed to decrease PG production more markedly than did the cis-9, trans-11 CLA isomer. Most concentrations of both CLAs significantly reduced culture media PGE2:PGF2α concentration ratios of cells. Exposure of cells to CLAs did not affect expression of PGHS-2 protein. Conclusions and Clinical Relevance: Results of this study indicated CLAs significantly decreased PGF2α and PGE2 concentrations and PGE2:PGF2α concentration ratios for cultures of adult and fetal endometrial epithelial cells with no apparent effect on PGHS-2 expression. Similar effects in cows could have effects on maternal recognition of pregnancy and immune function.

Objective: To determine concentration-dependent effects of tiludronate on cartilage explants incubated with or without recombinant equine interleukin-1β (rEq IL-1). Sample: Articular cartilage explants from the femorotibial joints of 3 young adult horses. Procedures: Cartilage explants were incubated with 1 of 6 concentrations (0, 0.19, 1.9, 19, 190, or 1,900 mg/L) of tiludronate and with or without rEq IL-1 (0.01 ng/mL) for 96 hours. Prostaglandin E2 (PGE2) concentrations in culture medium and explant digests were analyzed via PGE2 enzyme immunoassay. Sulfated glycosaminoglycan (sGAG) concentrations in culture medium were quantified via 1,9-dimethylmethylene blue assay. Chondrocyte apoptosis in paraffin embedded explant sections was measured via terminal deoxynucleotidyl transferase-mediated dUTP nick end–labeling assay. Relative gene expression of matrix metalloproteinases (MMPs), interleukin (IL)-6, and IL-8 was determined via the comparative cycle threshold method. Results: rEq IL-1 increased PGE2 concentration, sGAG release from explants, chondrocyte apoptosis, and MMP gene expression. Lower tiludronate concentrations reduced rEq IL-1–induced sGAG release and chondrocyte apoptosis, whereas the higher tiludronate concentrations increased sGAG release and chondrocyte apoptosis. At the highest tiludronate concentration evaluated, IL-8 gene expression was increased independent of whether rEq IL-1 was present. Conclusions and Clinical Relevance: Tiludronate had biphasic concentration-dependent effects on cartilage explants that were independent of PGE2 secretion or MMP gene expression. Low tiludronate concentrations had some chondroprotective effects, whereas high tiludronate concentrations were detrimental to equine articular cartilage. Administration of tiludronate intra-articularly to horses may be detrimental, dependent on the dose used. In vivo studies are needed before intra-articular tiludronate administration to horses can be recommended.

Objective: To determine whether incubation of cruciate ligament cells with acetylsalicylic acid, carprofen, meloxicam, or robenacoxib provides protection against apoptosis induced by sodium nitroprusside (SNP). Sample: Explants of cranial (CCL) and caudal (CaCL) cruciate ligaments from eight 1-day-old Beagles. Procedures: Primary cultures of CCL and CaCL cells were created via enzymatic dissociation of cruciate explants. Purified cell cultures were incubated for 2 hours without (controls) or with 1 of 3 concentrations of 1 of 4 NSAIDs (10, 100, or 200 μg of acetylsalicylic acid/mL; 0.1, 1, or 10 μg of carprofen/mL; 0.1, 1, or 10 μg of meloxicam/mL; or 0.1, 1, or 10 μg of robenacoxib/mL) and subsequently incubated for 18 hours with 1 of 3 concentrations of SNP in an attempt to induce mild, moderate, or severe cytotoxic effects. Cell viability and apoptosis were analyzed via a cell proliferation assay and flow cytometry, respectively. Prostaglandin E2 concentrations were measured via an ELISA. Results: Cytoprotective effects of NSAIDs were dependent on the extent of SNP-induced apoptosis and were greatest in CCL and CaCL cell cultures with moderate SNP-induced cytotoxic effects. Preincubation with an NSAID improved cell viability by 15% to 45% when CCL and CaCL cells were subsequently incubated with SNP. Carprofen (10 μg/mL) had the greatest cytoprotective effects for CCL and CaCL cells. Incubation with NSAIDs resulted in a nonsignificant decrease in PGE2 production from SNP-damaged cells. Conclusions and Clinical Relevance: Results indicated that carprofen, meloxicam, and robenacoxib may reduce apoptosis in cells originating from canine cruciate ligaments.

Objective—To compare secretory responses to prostaglandin (PG) E2 in mucosa obtained from the proximal and distal portions of the colon of dogs. Sample—Colonic mucosa from cadavers of 18 clinically normal adult dogs. Procedures—Short-circuit current (ISC) and maximum change in ISC (ΔIsc) in response to administration of 1μM PGE2 were measured across mucosa obtained from the proximal and distal portions of the colon. Responses were evaluated in mucosa (n = 6 dogs) incubated in Ussing chambers with or without 1 mM amiloride or without chloride in the Ringer's bathing solution. Responses were also evaluated in mucosa (n = 9 dogs) incubated with or without pretreatment with 1 μM indomethacin, with or without amiloride in the subsequent bathing solution. Histologic changes in mucosa from 3 dogs were assessed over time. Results—ISC and ΔISC were significantly reduced when chloride was removed from, but not when amiloride was added to, the bathing solution and were significantly reduced after pretreatment with indomethacin. The ΔISC was significantly greater in mucosa from the distal portion of the colon than in the proximal portion of the colon. Histologic changes after incubation for 3 hours were minimal. Conclusions and Clinical Relevance—ISC and ΔISC resulted from electrogenic chloride secretion. Chloride secretion was reduced when release of PGs was prevented by indomethacin and was induced by administration of PGE2. Chloride secretion in response to PGE2 was greater in mucosa from the distal portion of the colon than in mucosa from the proximal portion of the colon.

Objective-To determine the effects of recombinant equine interleukin -1beta (reIL-1beta) and 4 anti-inflammatory compounds on the expression and activity of cyclooxygenase (COX)-2 in cultured equine chondrocytes. Sample Population-Articular cartilage from 9 young adult horses. Procedure-Reverse transcriptase-polymerase chain reaction methods were used to amplify a portion of equine COX-2 to prepare a cDNA probe. Northern blot analysis was used to quantify the expression of COX-2 in first-passage cultures of equine articular chondrocytes propagated in media containing dexamethasone (DEX), phenylbutazone (PBZ), polysulfated glycosaminoglycan, and hyaluronan, each at concentrations of 10 and 100 micrograms/ml and each with or without reIL-1beta. A commercial immunoassay was used to determine prostaglandin E2 (PGE2) concentrations in conditioned medium of similarly treated cells to quantify COX-2 activity. Results-Addition of reIL-1beta increased the expression of COX-2 in a dose-dependent manner, which was paralleled by an increased concentration of PGE2 in culture medium. Concentration of PGE2 in spent medium from reIL-1beta-treated chondrocytes was significantly reduced by DEX and PBZ; however, only DEX significantly reduced gene expression of COX-2. Conclusions and Clinical Relevance-Prostaglandin E2 is considered to be an important mediator in the pathophysiologic processes of arthritis, and cultured chondrocytes respond to interleukin-1 with enhanced expression and activity of COX-2. Palliative relief in affected horses is probably attributable, in part, to inhibition of PGE2 synthesis; however, analysis of these data suggests that of the 4 compounds tested, only DEX affects pretranslational regulation of the COX-2 gene in cultured equine chondrocytes.