Carnosic acid improves porcine early embryonic development by inhibiting the accumulation of reactive oxygen species

Abstract

Carnosic acid (CA), a natural catechol rosin diterpene, is used as an additive in animal feeds and human foods. However, the effects of CA on mammalian reproductive processes, especially early embryonic development, are unclear. In this study, we added CA to parthenogenetically activated porcine embryos in an in vitro culture medium to explore the influence of CA on apoptosis, proliferation, blastocyst formation, reactive oxygen species (ROS) levels, glutathione (GSH) levels, mitochondrial membrane potential, and embryonic development-related gene expression. The results showed that supplementation with 10 μM CA during in vitro culture significantly improved the cleavage rates, blastocyst formation rates, hatching rates, and total numbers of cells of parthenogenetically activated porcine embryos compared with no supplementation. More importantly, supplementation with CA also improved GSH levels and mitochondrial membrane potential, reduced natural ROS levels in blastomeres, upregulated Nanog, Sox2, Gata4, Cox2, Itga5, and Rictor expression, and downregulated Birc5 and Caspase3 expression. These results suggest that CA can improve early porcine embryonic development by regulating oxidative stress. This study elucidates the effects of CA on early embryonic development and their potential mechanisms, and provides new applications for improving the quality of in vitro-developed embryos.

Keywords: Antioxidant; Carnosic acid; Embryo development; Oxidative stress; Porcine.

Fig. 1.

Blastocyst rates on Day 5 and Day 6 after treatment with different carnosic acid (CA) concentrations. (A) Embryonic development on Day 5 and Day 6 after treatment with different CA concentrations. Scale bar = 400 μm. (B) Blastocyst rates in the groups treated with 0 μM (n = 160), 5 μM (n = 173), 10 μM (n = 180), 25 μM (n = 182), and 50 μM (n = 168) CA. R = 3. Significant differences are represented with ** (P < 0.01).

Fig. 2.

Effects of carnosic acid (CA) on the development of parthenogenetic embryos. (A) Embryonic development in the negative control (NC) and CA-treated groups on different days. Scale bar = 400 μm. (B) Cleavage rates of embryos in the NC group (n = 214) and the CA-treated group (n = 217). R = 4. (C) Blastocyst formation rates in the NC group (n = 254) and the CA-treated group (n = 298) on different days. R = 5. (D) Average diameters of blastocysts on Day 6 in the NC group (n = 107) and the CA-treated group (n = 133). R = 4. Plot depicting the distributions of embryo diameter for black dots in the NC and CA-treated groups. (E) Hatching rates in the NC group (n = 254) and the CA-treated group (n = 298). R = 5. Significant differences are represented with * (P < 0.05) and ** (P < 0.01).

Fig. 3.

Total cell numbers and apoptotic nuclei in blastocysts with or without carnosic acid (CA) treatment. (A) Apoptotic nuclei in the negative control (NC) and CA-treated groups. The white arrows indicate nuclei positively stained for apoptosis. Scale bar = 100 μm. (B) and (C) Total cell numbers and apoptosis rates in blastocysts with (n = 57) or without (n = 73) CA treatment, respectively. R = 3. Black dots represent the tested values. Significant differences are represented with * (P < 0.05) and ** (P < 0.01).

Fig. 4.

Effects of carnosic acid (CA) on oxidation resistance in 4-cell-stage embryos. (A) Fluorescence intensity of H₂DCFDA staining in different groups. Scale bar = 200 μm. (B) Fluorescence intensity of CMF₂HC staining in the negative control (NC) and CA-treated groups. Scale bar = 200 μm. (C) Relative reactive oxygen species (ROS) levels in 4-cell-stage embryos. Plot depicting the distributions of relative embryo ROS levels for blue (n = 86), green (n = 104), red (n = 90), and orange (n = 74)-colored dots in different groups. Significant differences are represented with different capital letters (P < 0.01). R = 5. (D) Relative GSH levels of 4-cell-stage embryos in the NC (n = 91) and CA-treated (n = 93) groups. R = 3. Significant differences are represented with different capital letters (P < 0.01) and ** (P < 0.01).

Fig. 5.

Carnosic acid (CA) enhances mitochondrial activity. (A) JC-1 staining in 4-cell-stage embryos with or without CA treatment. (B) Relative fluorescence levels of JC-1 in the negative control (NC) (n = 126) and CA-treated (n = 140) groups. R = 5. Black dots represent the tested values. Scale bar = 100 μm. Significant differences are represented with ** (P < 0.01).

Fig. 6.

Differential gene expression in blastocysts. Gene expression levels were analyzed in porcine blastocysts with or without carnosic acid (CA) treatment on Day 7. Significant differences are represented with * (P < 0.05) and ** (P < 0.01).