Mangiferin improves early porcine embryonic development by reducing oxidative stress.

Mangiferin (MGN) is primarily found in the fruits, leaves, and bark of plants of the Anacardiaceae family, including mangoes. MGN exhibits various pharmacological effects, such as protection of the liver and gallbladder, anti-lipid peroxidation, and cancer prevention. This study aimed to investigate the effects of MGN supplementation during in vitro culture (IVC) on the antioxidant capacity of early porcine embryos and the underlying mechanisms involved. Porcine parthenotes in the IVC medium were exposed to different concentrations of MGN (0, 0.01, 0.1, and 1 µM). The addition of 0.1 µM MGN significantly increased the blastocyst formation rate of porcine embryos while reducing the apoptotic index and autophagy. Furthermore, the expression of antioxidation-related (SOD2, GPX1, NRF2, UCHL1), cell pluripotency (SOX2, NANOG), and mitochondria-related (TFAM, PGC1α) genes was upregulated. In contrast, the expression of apoptosis-related (CAS3, BAX) and autophagy-related (LC3B, ATG5) genes decreased after MGN supplementation. These findings suggest that MGN improves early porcine embryonic development by reducing oxidative stress-related genes.

Apigenin delays postovulatory oocyte aging by reducing oxidative stress through SIRT1 upregulation.

After ovulation, senescent oocytes inevitably experience reduced quality and defects in embryonic development. Apigenin (API) is a flavonoid with a wide range of pharmacological effects. Therefore, this study examined the protective effects of API on the quality of porcine oocytes during in-vitro ageing and the underlying mechanisms. The results showed that API treatment could reduce the activation rate after aging for 48 h. In addition, API significantly reduced reactive oxygen species, abnormal distribution of mitochondria, early apoptosis in ageing oocytes, increased glutathione, and mitochondrial adenosine triphosphate levels in ageing oocytes. Importantly, API increased the embryonic development rate in aged oocytes. We also examined molecular changes, finding decreased sirtuin 1 expression in in-vitro postovulatory oocytes, but API reversed this effect. Our results suggest that API attenuates the deterioration of oocyte quality during in-vitro ageing, possibly by reducing oxidative stress through the upregulation of sirtuin 1.

Improving the developmental competences of porcine parthenogenetic embryos by Notoginsenoside R1-induced enhancement of mitochondrial activity and alleviation of proapoptotic events.

Notoginsenoside R1 (NGR1), derived from the Panax notoginseng root and rhizome, exhibits diverse pharmacological influences on the brain, neurons, and osteoblasts, such as antioxidant effects, mitochondrial function protection, energy metabolism regulation, and inhibition of oxygen radicals, apoptosis, and cellular autophagy. However, its effect on early porcine embryonic development remains unclear. Therefore, we investigated NGR1's effects on blastocyst quality, reactive oxygen species (ROS) levels, glutathione (GSH) levels, mitochondrial function, and embryonic development-related gene expression in porcine embryos by introducing NGR1 during the in vitro culture (IVC) of early porcine embryos. Our results indicate that an addition of 1 µM NGR1 significantly increased glutathione (GSH) levels, blastocyst formation rate, and total cell number and proliferation capacity; decreased ROS levels and apoptosis rates in orphan-activated porcine embryos; and improved intracellular mitochondrial distribution, enhanced membrane potential, and reduced autophagy. In addition, pluripotency-related factor levels were elevated (NANOG and octamer-binding transcription factor 4 [OCT4]), antioxidant-related genes were upregulated (nuclear factor-erythroid 2-related factor 2 [NRF2]), and apoptosis- (caspase 3 [CAS3]) and autophagy-related genes (light chain 3 [LC3B]) were downregulated. These results indicate that NGR1 can enhance early porcine embryonic development by protecting mitochondrial function.

ID3 regulates progesterone synthesis in bovine cumulus cells through modulation of mitochondrial function.

DNA binding inhibitory factor 3 (ID3) has been shown to have a key role in maintaining proliferation and differentiation. It has been suggested that ID3 may also affect mammalian ovarian function. However, the specific roles and mechanisms are unclear. In this study, the expression level of ID3 in cumulus cells (CCs) was inhibited by siRNA, and the downstream regulatory network of ID3 was uncovered by high-throughput sequencing. The effects of ID3 inhibition on mitochondrial function, progesterone synthesis, and oocyte maturation were further explored. The GO and KEGG analysis results showed that after ID3 inhibition, differentially expressed genes, including StAR, CYP11A1, and HSD3B1, were involved in cholesterol-related processes and progesterone-mediated oocyte maturation. Apoptosis in CC was increased, while the phosphorylation level of ERK1/2 was inhibited. During this process, mitochondrial dynamics and function were disrupted. In addition, the first polar body extrusion rate, ATP production and antioxidation capacity were reduced, which suggested that ID3 inhibition led to poor oocyte maturation and quality. The results will provide a new basis for understanding the biological roles of ID3 as well as cumulus cells.

Customized liver organoids as an advanced in vitro modeling and drug discovery platform for non-alcoholic fatty liver diseases.

Non-alcoholic fatty liver disease (NAFLD) and its progressive form non-alcoholic steatohepatitis (NASH) have presented a major and common health concern worldwide due to their increasing prevalence and progressive development of severe pathological conditions such as cirrhosis and liver cancer. Although a large number of drug candidates for the treatment of NASH have entered clinical trial testing, all have not been released to market due to their limited efficacy, and there remains no approved treatment for NASH available to this day. Recently, organoid technology that produces 3D multicellular aggregates with a liver tissue-like cytoarchitecture and improved functionality has been suggested as a novel platform for modeling the human-specific complex pathophysiology of NAFLD and NASH. In this review, we describe the cellular crosstalk between each cellular compartment in the liver during the pathogenesis of NAFLD and NASH. We also summarize the current state of liver organoid technology, describing the cellular diversity that could be recapitulated in liver organoids and proposing a future direction for liver organoid technology as an in vitro platform for disease modeling and drug discovery for NAFLD and NASH.

Protective effect of onion peel extract on ageing mouse oocytes.

Mammalian oocytes not fertilized immediately after ovulation can undergo ageing and a rapid decline in quality. The addition of antioxidants can be an efficient approach to delaying the oocyte ageing process. Onion peel extract (OPE) contains quercetin and other flavonoids with natural antioxidant activities. In this study, we investigated the effect of OPE on mouse oocyte ageing and its mechanism of action. The oocytes were aged in vitro in M16 medium for 16 h after adding OPE at different concentrations (0, 50, 100, 200, and 500 µg/ml). The addition of 100 µg/ml OPE reduced the oocyte fragmentation rate, decreased the reactive oxygen species (ROS) level, increased the glutathione (GSH) level, and improved the mitochondrial membrane potential compared with the control group. The addition of OPE also increased the expression of SOD1, CAT, and GPX3 genes, and the caspase-3 activity in OPE-treated aged oocytes was significantly lower than that in untreated aged oocytes and similar to that in fresh oocytes. These results indicated that OPE delayed mouse oocyte ageing by reducing oxidative stress and apoptosis and enhancing mitochondrial function.

6-Gingerol Improves In Vitro Porcine Embryo Development by Reducing Oxidative Stress.

6-Gingerol, the main active ingredient in ginger, exhibits a variety of biological activities, such as antioxidant, anti-inflammatory, and anticancer activities, and can affect cell development. However, the effects of 6-gingerol on mammalian reproductive processes, especially early embryonic development, are unclear. This study explored whether 6-gingerol can be used to improve the quality of in vitro-cultured porcine embryos. The results showed that 5 µM 6-gingerol significantly increased the blastocyst formation rates of porcine early embryos. 6-Gingerol attenuated intracellular reactive oxygen species accumulation and autophagy, increased intracellular glutathione levels, and increased mitochondrial activity. In addition, 6-gingerol upregulated NANOG, SRY-box transcription factor 2, cytochrome c oxidase subunit II, mechanistic target of rapamycin kinase, and RPTOR independent companion of MTOR complex 2 while downregulating Caspase 3, baculoviral IAP repeat containing 5, autophagy related 12, and Beclin 1. Most importantly, 6-gingerol significantly increased the levels of p-extracellular regulated protein kinase 1/2 while reducing the levels of p-c-Jun N-terminal kinase 1/2/3 and p-p38. These results indicate that 6-gingerol can promote the development of porcine early embryos in vitro.

Rapid Screening and Comparison of Chimeric Lysins for Antibacterial Activity against Staphylococcus aureus Strains.

Chimeric lysins composed of various combinations of cell wall-lysing (enzymatic) and cell-wall-binding (CWB) domains of endolysins, autolysins, and bacteriocins have been developed as alternatives to or adjuvants of conventional antibiotics. The screening of multiple chimeric lysin candidates for activity via E. coli expression is not cost effective, and we previously reported on a simple cell-free expression system as an alternative. In this study, we sufficiently improved upon this cell-free expression system for use in screening activity via a turbidity reduction test, which is more appropriate than a colony reduction test when applied in multiple screening. Using the improved protocol, we screened and compared the antibacterial activity of chimeric lysin candidates and verified the relatively strong activity associated with the CHAP (cysteine, histidine-dependent amidohydrolase/peptidase) domain of secretory antigen SsaA-like protein (ALS2). ALS2 expressed in E. coli showed two major bands, and the smaller one (subprotein) was shown to be expressed by an innate downstream promoter and start codon (ATG). The introduction of synonymous mutations in the promoter resulted in clearly reduced expression of the subprotein, whereas missense mutations in the start codon abolished antibacterial activity as well as subprotein production. Interestingly, most of the S. aureus strains responsible for bovine mastitis were susceptible to ALS2, but those from human and chicken were less susceptible. Thus, the simple and rapid screening method can be applied to select functional chimeric lysins and define mutations affecting antibacterial activity, and ALS2 may be useful in itself and as a lead molecule to control bovine mastitis.

Chrysoeriol Improves In Vitro Porcine Embryo Development by Reducing Oxidative Stress and Autophagy.

Chrysoeriol (CHE) is a flavonoid substance that exists in many plants. It has various physiological and pharmacological effects, including anti-inflammatory, antioxidant, anti-tumor, and protective activity, especially for the cardiovascular system and liver. Among common livestock embryos, porcine embryos are often considered high-quality objects for studying the antioxidant mechanisms of oocytes. Because porcine embryos contain high levels of lipids, they are more vulnerable to external stimuli, which affect development. Our study explored the influence of CHE supplementation on oxidative stress in porcine oocytes and its possible mechanisms. Different concentrations of CHE (0, 0.1, 1, and 3 µM) were supplemented in the in vitro culture medium of the porcine oocytes. The results showed that supplementation with 1 µM CHE significantly increased the blastocyst rate and total cell number of embryos in vitro. After finding the beneficial effects of CHE, we measured reactive oxygen species (ROS), glutathione (GSH), and mitochondrial membrane potential (MMP) when the oocytes reached the 4-cell stage of development and determined the levels of apoptosis, cell proliferation, and autophagy at the blastocyst stage of development. The expression levels of some related genes were preliminarily detected by qRT-PCR. The results showed that the apoptosis of blastocysts in the CHE-treated culture also decreased compared with the untreated culture. Furthermore, CHE downregulated intracellular ROS and increased GSH in the embryos. CHE was also shown to improve the activity of mitochondria and inhibit the occurrence of autophagy. In addition, antioxidant-related genes (SOD1, SOD2, and CAT) and cell pluripotency-related genes (SOX2, OCT4, and NANOG) were upregulated. At the same time, apoptosis-related (Caspase 3) and autophagy-related (LC3B) genes showed a downward trend after supplementation with CHE. These results indicate that CHE improved the development of porcine embryos in vitro by reducing oxidative stress and autophagy levels.

TBX2 affects proliferation, apoptosis and cholesterol generation by regulating mitochondrial function and autophagy in bovine cumulus cell.

BACKGROUND: T-box transcription factor 2 (TBX2) is a member of T-box gene family whose members are highly conserved in evolution and encoding genes and are involved in the regulation of developmental processes. The encoding genes play an important role in growth and development. Although TBX2 has been widely studied in cancer cell growth and development, its biological functions in bovine cumulus cells remain unclear. OBJECTIVES: This study aimed to investigate the regulatory effects of TBX2 in bovine cumulus cells. METHODS: TBX2 gene was knockdown with siRNA to clarify the function in cellular physiological processes. Cell proliferation and cycle changes were determined by xCELLigence cell function analyzer and flow cytometry. Mitochondrial membrane potential and autophagy were detected by fluorescent dye staining and immunofluorescence techniques. Western blot and quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) were used to detect the expression changes of proliferation and autophagy-related proteins. Aadenosine triphosphate (ATP) production, glucose metabolism, and cholesterol synthesis of cumulus cells were measured by optical density and chemiluminescence analysis. RESULTS: After inhibition of TBX2, the cell cycle was disrupted. The levels of apoptosis, ratio of light chain 3 beta II/I, and reactive oxygen species were increased. The proliferation, expansion ability, ATP production, and the amount of cholesterol secreted by cumulus cells were significantly decreased. CONCLUSIONS: TBX2 plays important roles in regulating the cells' proliferation, expansion, apoptosis, and autophagy; maintaining the mitochondrial function and cholesterol generation of bovine cumulus cells.

Dihydromyricetin supplementation during in vitro culture improves porcine oocyte developmental competence by regulating oxidative stress.

Dihydromyricetin (DHM), a dihydroflavonoid compound, exhibits a variety of biological activities, including antitumor activity. However, the effects of DHM on mammalian reproductive processes, especially during early embryonic development, remain unclear. In this study, we added DHM to porcine zygotic medium to explore the influence and underlying mechanisms of DHM on the developmental competence of parthenogenetically activated porcine embryos. Supplementation with 5 µM DHM during in vitro culture (IVC) significantly improved blastocyst formation rate and increased the total number of cells in porcine embryos. Further, DHM supplementation also improved glutathione levels and mitochondrial membrane potential; reduced natural reactive oxygen species levels in blastomeres and apoptosis rate; upregulated Nanog, Oct4, SOD1, SOD2, Sirt1, and Bcl2 expression; and downregulated Beclin1, ATG12, and Bax expression. Collectively, DHM supplementation regulated oxidative stress during IVC and could act as a potential antioxidant during in vitro porcine oocytes maturation.

KRAS Affects the Lipid Composition by Regulating Mitochondrial Functions and MAPK Activation in Bovine Mammary Epithelial Cells.

Kirsten rat sarcoma viral oncogene homolog (KRAS), or guanosine triphosphatase KRAS, is a proto-oncogene that encodes the small guanosine triphosphatase transductor protein. Previous studies have found that KRAS can promote cytokine secretion, cell chemotaxis, and survival. However, its effects on milk fat synthesis in bovine mammary epithelial cells are unclear. In this study, the effects of KRAS inhibition on cell metabolism, autophagy, oxidative stress, endoplasmic reticulum stress, mitochondrial function, and lipid composition as well as the potential mechanisms were detected in an immortalized dairy cow mammary epithelial cell line (MAC-T). The results showed that inhibition of KRAS changed the lipid composition (especially the triglyceride level), mitochondrial functions, autophagy, and endoplasmic reticulum stress in cells. Moreover, KRAS inhibition regulated the levels of the mammalian target of rapamycin and mitogen-activated protein kinase (extracellular regulated protein kinases, c-Jun N-terminal kinases, p38) activation. These results indicated that regulation of KRAS would affect the synthesis and composition of milk fat. These results are also helpful for exploring the synthesis and secretion of milk fat at the molecular level and provide a theoretical basis for improving the percentage of fat in milk and the yield of milk from cows.

Wedelolactone facilitates the early development of parthenogenetically activated porcine embryos by reducing oxidative stress and inhibiting autophagy.

Wedelolactone (WDL) is a coumaryl ether compound extracted from the traditional Chinese medicinal plant, Eclipta prostrata L. It is a natural polyphenol that exhibits a variety of pharmacological activities, such as anti-inflammatory, anti-free radical, and antioxidant activities in the bone, brain, and ovary. However, its effect on embryonic development remains unknown. The present study explored the influence of WDL supplementation of porcine oocytes culture in vitro on embryonic development and the underlying mechanisms and its effect on the levels of Kelch-like ECH-associated protein 1/nuclear factor-erythroid 2-related factor 2/antioxidant response element (Keap1/Nrf2/ARE). The results showed that WDL (2.5 nM) significantly increased the blastocyst formation rate, mitochondrial activity, and proliferation ability while reducing the reactive oxygen species accumulation, apoptosis, and autophagy. These findings suggested that WDL can enhance the growth and development of early porcine embryos to alleviate oxidative stress and autophagy through regulating NRF2 and microtubule-associated protein 1 light chain 3 (MAP1LC3) gene expression levels.

Oroxin A reduces oxidative stress, apoptosis, and autophagy and improves the developmental competence of porcine embryos in vitro.

Oroxin A (OA) is a flavonoid isolated from Oroxylum indicum (L.) Kurz that has various biological activities, including antioxidant activities. This study aimed to examine the viability of using OA in an in vitro culture (IVC) medium for its antioxidant effects and related molecular mechanisms on porcine blastocyst development. In this study, we investigated the effects of OA on early porcine embryo development via terminal deoxynucleotidyl transferase dUTP nick-end labeling, 5-ethynyl-2'-deoxyuridine labeling, quantitative reverse transcription PCR, and immunocytochemistry. Embryos cultured in the IVC medium supplemented with 2.5 µM of OA had an increased blastocyst formation rate, total cell number, and proliferation capacity, along with a low apoptosis rate. OA supplementation decreased reactive oxygen species levels while increasing glutathione levels. OA-treated embryos exhibited an improved intracellular mitochondrial membrane potential and reduced autophagy. Moreover, levels of pluripotency- and antioxidant-related genes were upregulated, whereas those of apoptosis- and autophagy-related genes were downregulated by OA addition. In conclusion, OA improves preimplantation embryonic development by reducing oxidative stress and enhancing mitochondrial function.

KRAS Affects Adipogenic Differentiation by Regulating Autophagy and MAPK Activation in 3T3-L1 and C2C12 Cells.

Kirsten rat sarcoma 2 viral oncogene homolog (Kras) is a proto-oncogene that encodes the small GTPase transductor protein KRAS, which has previously been found to promote cytokine secretion, cell survival, and chemotaxis. However, its effects on preadipocyte differentiation and lipid accumulation are unclear. In this study, the effects of KRAS inhibition on proliferation, autophagy, and adipogenic differentiation as well as its potential mechanisms were analyzed in the 3T3-L1 and C2C12 cell lines. The results showed that KRAS was localized mainly in the nuclei of 3T3-L1 and C2C12 cells. Inhibition of KRAS altered mammalian target of rapamycin (Mtor), proliferating cell nuclear antigen (Pcna), Myc, peroxisome proliferator-activated receptor γ (PPARγ), CCAAT/enhancer binding protein beta (C/ebp-ß), diacylglycerol O-acyltransferase 1 (Dgat1), and stearoyl-coenzyme A desaturase 1 (Scd1) expression, thereby reducing cell proliferation capacity while inducing autophagy, enhancing differentiation of 3T3-L1 and C2C12 cells into mature adipocytes, and increasing adipogenesis and the capacity to store lipids. Moreover, during differentiation, KRAS inhibition reduced the levels of extracellular regulated protein kinases (ERK), c-Jun N-terminal kinase (JNK), p38, and phosphatidylinositol 3 kinase (PI3K) activation. These results show that KRAS has unique regulatory effects on cell proliferation, autophagy, adipogenic differentiation, and lipid accumulation.

Porcine circovirus 2 capsid protein produced in N. benthamiana forms virus-like particles that elicit production of virus-neutralizing antibodies in guinea pigs.

Porcine circovirus type 2 (PCV2) is a non-enveloped, icosahedral virus of the Circoviridae family, with a small, circular, single-stranded DNA genome. PCV2 infections cause substantial economic losses in the pig industry worldwide. Currently, commercially produced PCV2 vaccines are expensive, whereas plant-based expression systems can produce recombinant proteins at low cost for use as vaccines. In this study, recombinant PCV2 capsid protein (rCap) was transiently expressed in Nicotiana benthamiana and purified by metal affinity chromatography, with a yield of 102 mg from 1 kg plant leaves. Electron microscopy confirmed that purified rCap self-assembled into virus-like particles (VLPs) at neutral pH. It was shown to provoke a strong immune response in guinea pigs. The results indicate that plant systems can enable production of large amounts of proteins to serve as candidates for subunit vaccines.

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

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.

A classical swine fever virus E2 fusion protein produced in plants elicits a neutralizing humoral immune response in mice and pigs.

Classical swine fever (CSF) is one of the most important viral diseases of swine worldwide. Although live or attenuated virus vaccines have been used to control CSFV, it is difficult to distinguish vaccinated pigs from infected pigs; this leads to restrictions on import and export. Subunit vaccines based on the CSFV E2 glycoprotein have been developed using baculovirus or insect cell systems, but some weaknesses remain. Here, we describe production of an E2 recombinant protein using a Nicotiana benthamiana plant expression system. To do this, we took advantage of the ability of the swine Fc domain to increase solubility and stability of the fusion protein and to strengthen immune responses in target animals. N. benthamiana expressed high amounts of pFc2-fused E2 proteins, which were isolated and purified by affinity chromatography to yield a high pure recombinant protein in a cost-effective manner. Native-polyacrylamide gel electrophoresis and size exclusion chromatography confirmed that the pmE2:pFc2 fusion exists as a multimer rather than as a dimer. Injection of recombinant pmE2 protein into mice or piglets generated anti-pmE2 antibodies with efficient neutralizing activity against CSFV. These results suggest that a purified recombinant E2 protein produced in N. benthamiana generates high titers of neutralizing antibodies in vivo; as such, the protein could be developed as a subunit vaccine against CSFV.

Imperatorin Ameliorates the Aging-Associated Porcine Oocyte Meiotic Spindle Defects by Reducing Oxidative Stress and Protecting Mitochondrial Function.

Imperatorin (IMP) exhibits a variety of pharmacological properties, including antioxidant, anti-inflammatory, antibacterial, anti-cancer, and anti-hypertension activities. However, its effects on animal reproduction systems, especially oocyte development, maturation, and aging are not yet clear. In this study, the effects of IMP on oocyte development and aging as well as the underlying molecular mechanisms were explored. Oocytes were cultured for an additional 24 h for aging. Results revealed that the blastocyst formation and hatching rates of embryos, which were parthenogenetically activated aged oocytes, were significantly increased with IMP treatment (40 µM). Simultaneously, well-distributed cortical granules but no significant difference in zona pellucida hardness were observed after IMP treatment. During this stage, intracellular reactive oxygen species, apoptosis, and autophagy levels were decreased, while mitochondrial membrane potential, glutathione level, and activity of superoxide dismutase and catalase were increased. IMP-treated aged oocytes also showed significantly higher expression of MOS, CCNB1, BMP15, and GDF9 than non-IMP-treated aged oocytes although their levels were still lower than those in the fresh oocytes. These results suggest that IMP can effectively ameliorate the quality of aged porcine oocytes by reducing oxidative stress and protecting mitochondrial function.

Kaempferol alleviates the reduction of developmental competence during aging of porcine oocytes.

Kaempferol (KAE) is a natural flavonoid present in different plant species and exhibits anti-inflammatory, antioxidant, and anticancer therapeutic properties. In the present study, we investigated the influence and underlying mechanisms of KAE supplementation on porcine oocytes during in vitro aging. The results show that KAE treatment can alleviate the aging-related reduction of developmental competence. We observed that the blastocyst production rate in aged oocytes treated with 0.1 µM KAE was significantly higher than in untreated aging oocytes (36.78 ± 0.86% vs. 27.55 ± 2.60%, respectively, p < .05). The KAE-treated aging oocytes had significantly reduced levels of reactive oxygen species (p < .05). Furthermore, the mRNA levels of the embryonic pluripotency-related genes Oct4, NANOG, and ITGA5 were significantly increased in blastocysts derived from KAE-treated oocytes (p < .05). During excessive oocyte culture, KAE treatment maintained the mitochondrial membrane potential and reduced apoptosis; however, this was not observed in untreated aging oocytes. In conclusion, our results suggest that KAE treatment can alleviate the aging of porcine oocytes by reducing oxidative stress and improving mitochondrial function.