Efeitos do α-pineno livre ou encapsulado em lipossomas durante o cultivo de folículos pré-antrais e maturação oocitária em bovinos

Abstract

Growing evidence has demonstrated that oxidative stress compromises in vitro culture (IVC) of ovarian follicles and in vitro maturation (IVM) of oocytes. Thus, strategies to reduce oxidative stress are essential to improve the quality of cultured follicles and oocytes. The objectives of this study were: 1) to investigate the effects of α-pinene during IVC of pre-antral follicles in bovine ovarian tissue; 2) to develop and characterize liposomes loaded with α-pinene and analyze their cytotoxicity in bovine cumulus cells (CCs) and evaluate their effects during IVM of bovine oocytes and subsequent parthenogenetic embryonic development. In Phase 1, ovarian tissue fragments were cultured for 6 days in α-MEM+ (control) or supplemented with 1.25, 2.5, 5, 10, or 20 µg/mL of α-pinene. In Phase 2, liposomes loaded with α-pinene (Lip-α-pinene) were produced using the lipid film hydration technique, characterized, and evaluated Regarding cytotoxicity in bovine cumulus oocyte complexes (COCs), cumulus oocyte complexes (COCs) were subsequently matured for 22-24 hours in maturation medium (control) or supplemented with Lip-white (empty liposome) or Lip-α-pinene at concentrations of 0.01, 1, or 100 µg/mL. After IVM, only mature oocytes proceeded to parthenogenetic activation and embryo culture. Data were analyzed using chi-square, Student's t-test, Kruskal-Wallis test, and ANOVA followed by Tukey's test (GraphPad Prism 9.0), with a significance level set at P < 0.05. In Phase 1, α-pinene at concentrations of 2.5, 5, or 10 µg/mL increased the percentage of normal follicles (P > 0.05) compared to the control, without influencing follicular growth (P < 0.05). α-pinene (10 µg/mL) maintained stromal cell density and collagen levels in cultured ovarian tissue similar to non-cultured tissues (P > 0.05) and increased mRNA levels for NRF2 and PRDX6 (P < 0.05) compared to the control. In Phase 2, Lip-α-pinene presented a size of 75.86 ± 0.95 nm, low polydispersity (0.26 ± 0.00), zeta potential of -31.55 ± 2.23 mV, encapsulation efficiency of 85 ± 2.51%, and Lip-α-pinene was not cytotoxic to CCs (P > 0.05). Lip-α-pinene did not affect the nuclear maturation rate (P > 0.05); however, 1 µg/mL of Lip-α-pinene preserved the ultrastructure of CCs and the oocyte, reduced ROS and lipid accumulation compared to the control, Lip-blank, and 100 µg/mL of Lip-α-pinene (P < 0.05). This result was accompanied by an increase in NRF2, SOD, and PRDX6 levels (P < 0.05). Furthermore, 1 and 100 µg/mL Lip-α-pinene increased cleavage rates and the number of cells per blastocyst (P < 0.05), but did not affect blastocyst formation and lipid content (P > 0.05). In conclusion, α-pinene preserves follicular structure, tissue integrity, and modulates antioxidant genes during ovarian tissue culture. When encapsulated, α-pinene increases the antioxidant capacity of oocytes by reducing ROS and increasing NRF2, SOD, and PRDX6 levels, improving the quality of oocytes and parthenogenetic embryos.