Abstract This study investigated the effect of Folliculinum 6 cH on the oocyte meiosis resumption and viability rates, progesterone production and mitochondrial activity after in vitro maturation of cumulus-oocyte complexes (COCs) in sheep. Sheep ovaries were collected at a local slaughterhouse and COCs were recovered by slicing technique. The selected COCs were maturated in TCM199 (Control treatment), or control medium supplemented with 0.05% ethanol (v/v) (the vehicle of the homeopathic preparation – Ethanol treatment) or with Folliculinum 6 cH. After 24 h of in vitro maturation (IVM), oocytes were mechanically denuded and incubated with Hoechst 33342 and MitoTracker (0.5 μM) Orange CMTMRos for analysis of viability and chromatin configuration, and mitochondrial activity, respectively. The results showed that Folliculinum 6 cH addition increased oocyte degeneration and reduced meiotic resumption compared to the control (P < 0.05). Interestingly, the percentages meiotic resumption and oocyte maturation were lower in the Folliculinum 6 cH treatment compared to its vehicle (Ethanol treatment) (P < 0.05). On the other hand, when the treatments were compared, higher mitochondrial activity was observed in the Ethanol treatment (P < 0.05). In conclusion, contrary to its vehicle, the addition of Folliculinum 6 cH to the IVM medium promoted oocyte degeneration and affected negatively the mitochondrial distribution, impairing meiosis resumption.

ABSTRACT Most anticancer drugs like doxorubicin (DXR) have low specificity that results in undesirable effects especially when it comes to collateral effects on reproduction. Plants are excellent sources when searching for new drugs. Auxemma oncocalyx (A. oncocalyx) and its main component Oncocalyxone A (onco A) have anti-tumoral activity and are less toxic than DXR in reproductive parameters. However, there are no studies on the action of these drugs regarding the porcine in vitro oocyte competence and embryo development. The aim of this study was to evaluate the effect of A. oncocalyx and onco A exposure during in vitro maturation (IVM) of oocytes (Experiment 1) or in vitro embryo culture (IVC) (Experiment 2) on the oocyte developmental competence. For experiment 1, COCs were distributed in IVM medium alone (control) or supplemented with DXR (0.3 (g/mL), A. oncocalyx (1.2 (g/mL) and onco A (1 (g/mL). Then, oocytes were submitted to in vitro fertilization (IVF) and in vitro embryo culture. For experiment 2, zygotes were cultured with DXR, A. oncocalyx and onco A for 7 days. Viability, maturation, fertilization and embryo developmental parameters were evaluated in both experiments. In experiment 1; DXR, A. oncocalyx and onco A reduced (P<0.05) oocyte viability and IVM efficiency. Onco A increased (P<0.05) the meiotic resumption. After IVF, all drugs reduced (P<0.05) viability, IVF efficiency and percentage of cleaved embryos, nevertheless, only DXR decreased the percentage of blastocyst. In experiment 2; all drugs reduced (P<0.05) the percentage of penetration, but only DXR and onco A decreased (P<0.05) IVF efficiency. DXR and A. oncocalyx decreased (P<0.05) the percentage of cleaved embryo, but had no effect on blastocyst formation. In conclusion, the addition of DXR during IVM or IVC negatively affected the IVF efficiency and cleavage rate. In addition, the exposure of COCs to DXR only during IVM was more detrimental to oocyte viability and blastocyst formation than A. oncocalyx and onco A.

RESUMEN La mayoría de los medicamentos contra el cáncer como la doxorrubicina (DXR) tienen una baja especificidad que produce efectos indeseables, especialmente cuando se trata de efectos colaterales en la reproducción. Las plantas son excelentes fuentes cuando se buscan nuevos medicamentos. Auxemma oncocalyx (A. oncocalyx) y su componente principal Oncocalyxone A (onco A) tienen actividad antitumoral y son menos tóxicos que DXR en los parámetros reproductivos. Sin embargo, no hay estudios sobre la acción de estos medicamentos con respecto a la competencia de los ovocitos in vitro porcina y el desarrollo embrionario. El objetivo de este estudio fue evaluar el efecto de la exposición a A. oncocalyx y onco A durante la maduración in vitro (IVM) de los ovocitos (Experimento 1) o el cultivo de embriones in vitro (IVC) (Experimento 2) en la competencia del desarrollo de los ovocitos. Para el experimento 1, los COC se distribuyeron en medio IVM solo (control) o suplementado con DXR (0.3 µg / mL), A. oncocalyx (1.2 µg / mL) y onco A (1 µg / mL). Luego, los ovocitos se sometieron a fertilización in vitro (FIV) y cultivo embrionario in vitro. Para el experimento 2, los cigotos se cultivaron con DXR, A. oncocalyx y onco A durante 7 días. Se evaluaron los parámetros de viabilidad, maduración, fertilización y desarrollo embrionario en ambos experimentos. En el experimento 1; DXR, A. oncocalyx y onco A redujeron la viabilidad de los ovocitos (P <0.05) y la eficiencia de IVM. Onco A aumentó (P <0.05) la reanudación meiótica. Después de la FIV, todos los medicamentos redujeron la viabilidad (P <0.05), la eficacia de la FIV y el porcentaje de embriones clivados, sin embargo, solo DXR disminuyó el porcentaje de blastocistos. En el experimento 2; todos los medicamentos redujeron (P <0.05) el porcentaje de penetración, pero solo DXR y onco A disminuyeron (P <0.05) la eficacia de la FIV. DXR y A. oncocalyx disminuyeron (P <0.05) el porcentaje de embriones clivados, pero no tuvieron efecto sobre la formación de blastocistos. En conclusión, la adición de DXR durante IVM o IVC afectó negativamente la eficiencia de IVF y la tasa de clivaje. Además, la exposición de los COCs a DXR solo durante la IVM fue más perjudicial para la viabilidad de los ovocitos y la formación de blastocistos que A. oncocalyx y onco A.