Lethal effect of high concentrations of parecoxib and flunixin meglumine on the in vitro culture of bovine embryos.

Abstract

The aim of this experiment was to evaluate the effects of cycloxigenase inhibitor drugs, i.e. flunixin meglumine (FM) and parecoxib (P), on the development of bovine embryos cultured in vitro until the blastocyst and hatched blastocyst stages. Immature oocytes were aspirated from slaughterhouse ovaries and morphologically selected for IVM (Monteiro FM et al. 2007 Anim. Reprod. 4, 51&#x2013;58). Twenty hours after maturation (39&#xb0;C and 5% CO2 in air), matured oocytes were transferred to fertilization media, inseminated with frozen&#x2013;thawed semen, and incubated for 10 to 12 h. Presumptive zygotes (PZ) were then transferred to TCM 199 HEPES medium, vortexed to remove cumulus cells and finally to drops of IVC media (SOFaaci plus 5% BFS [Gibco] with 13 mm sodium pyruvate). Each drop of IVC medium had appropriate concentrations of FM (0.14/n = 123; 1.4/n = 122; 14/n = 117; 140/n = 44 or 1400 &#x3bc;g mL-1/n = 44 PZ) or P (0.09/n = 134; 0.9/n = 109; 9/n = 118; 90/n = 113 or 900 &#x3bc;g mL-1/n = 45 PZ), besides extra drops as control groups (CFM; n = 124 and CP; n = 149 PZ). Based on published data from bovine (FM) and human (P) administered concentrations, it was calculated the blood concentration to a bovine weighing 450 kg (FM = 14 and P = 9 &#x3bc;g mL-1). Both drugs were used from available commercial preparations, and in a pilot test, there were no deleterious effects of the solvent itself on the blastocyst and hatched blastocyst rates. During culture, petri dishes containing PZ/embryos were kept into plastic bags, under controlled atmosphere of 5% O2, 5% CO2 and 90% N2 at 39&#xb0;C. There were 11 replicates for each treatment. In all drops (both drug concentrations and control group) the blastocyst and hatched blastocyst rates (BR and HBR, respectively) were evaluated at 144 and 192 h after fertilization, respectively. Statistical analysis was performed with ANOVA on ranks (Dunn's test a posteriori and significance being considered when P &lt; 0.05; BioEstat version 5.0). According to the results, FM (1400 and 140 &#x3bc;g mL-1) and P (900 &#x3bc;g mL-1) concentrations were toxic enough for a complete inhibition of in vitro bovine embryo development. There were no significant differences among the other drug concentrations and their respective control group, on the BR (27.7 &#xb1; 3.9; 29.6 &#xb1; 3.4 and 29.8% &#xb1; 4.8) and HBR (13.5 &#xb1; 4.4; 15.6 &#xb1; 3.8 and 22.1% &#xb1; 5.1), respectively to 0.14; 1.4 and 14 &#x3bc;g mL-1 for FM; on BR (26.0 &#xb1; 2.6; 18.2 &#xb1; 4.6; 25.8 &#xb1; 5.9 and 23.2% &#xb1; 4.8) and HBR (14.1 &#xb1; 3.3; 10.2 &#xb1; 3.3; 16.8 &#xb1; 3.8 and 12.0% &#xb1; 3.4), respectively to 0.09; 0.9; 9 and 90 &#x3bc;g mL-1 for P; and on BR (35.3 &#xb1; 5.2 and 36.5% &#xb1; 3.4) and HBR (26.6 &#xb1; 4.5 and 19.8% &#xb1; 3.6), respectively for CFM and CP. The results suggest that, during in vitro bovine embryo culture, there was no significant toxicity of either drug, with exception of the complete lethal concentrations of 140 and 1400 &#x3bc;g mL-1 (flunixin meglumine) and 900 &#x3bc;g mL-1 (parecoxib) on blastocyst production.<fn-group><fn>Supported by FAPESP &#x2013; Brazil (MFGN 06/06491-2 and 07/07705-9; EMR 07/04284-2; RAS; CFS and RALS) and CAPES &#x2013; Brazil.</fn></fn-group>