Reconstructed ovaries are emerging as a promising approach for assisted reproduction and species preservation due to their potential for in vitro follicle formation and oocyte production. However, efficient production of reconstructed ovaries remains technically limited by the requirement for securing a large number of gonadal somatic cells. Therefore, this study evaluated the feasibility of follicle formation under limited somatic cell conditions by forming ovarioids with a reduced number of somatic cells. The formation of follicle-like structures was confirmed using immunofluorescence analysis, and the expression of molecular markers related to apoptosis, autophagy, mitochondrial stability, and stress responses was analyzed. As a result, it was observed that the groups with a reduced number of gonad somatic cells (2000:20000 and 3000:30000) formed follicle-like structures similar to the control group. Furthermore, oocytes observed under all conditions ranged in diameter from 50 to 70 µm, corresponding to the primary oocyte stage. Additionally, it was observed that the microenvironment was partially reproduced through staining of LAMININ, N-CADHERIN, and CD44. However, as the number of somatic cells decreased, differences were observed in the expression patterns of follicle-related and stress-related molecules. In particular, compared to the control group, the 2000:20000 group showed a significant decrease in follicle-related factors (Nobox, Lhx8, Gdf9, Bmp15). These results suggest that the number of somatic cells and the composition of the microenvironment in a reconstructed ovarian model can affect the qualitative environment for follicle formation, providing a basic basis for optimizing the conditions for ovarian reconstitution.

Salidroside, a bioactive compound from Rhodiola rosea, shows potential in managing polycystic ovary syndrome (PCOS), a common endocrine-metabolic disorder. This study explored its therapeutic effects and mechanisms using a dehydroepiandrosterone (DHEA)-induced PCOS mouse model and granulosa cells (GCs). Salidroside was found to restore estrous cyclicity, improve ovarian morphology, and rebalance serum hormone levels. Mechanistically, it suppressed ROS-mediated AKT/NF-kappa B signaling, inhibited NLRP3 inflammasome activation, and reduced proinflammatory cytokines. Network pharmacology and molecular docking identified AKT as a core target, validated by CETSA and DARTS assays. Furthermore, salidroside disrupted NLRP3-driven latent TGF-beta 1 (LAP-TGF beta 1) activation, downregulated TGF-beta-SMAD2/3 signaling, and attenuated ovarian fibrosis along with abnormal hyaluronan synthase 2 (HAS2) expression. These results highlight salidroside as a promising natural candidate for alleviating PCOS through coordinated anti-inflammatory and antifibrotic mechanisms.

Background Polycystic ovary syndrome (PCOS) is a prevalent endocrine disorder that severely impacts female fertility. The natural dihydrochalcone trilobatin (TLB) has shown potential in managing metabolic diseases; however, its therapeutic effect on PCOS and the underlying mechanisms remain unexplored. Purpose To investigate the therapeutic potential of TLB for PCOS and to elucidate the mechanistic basis, focusing on glycolytic metabolism and mitochondrial function in granulosa cells (GCs). Methods A dehydroepiandrosterone (DHEA) -induced PCOS rat model was established and treated with two different doses of TLB or metformin.Therapeutic effects were assessed based on estrous cyclicity, ovarian morphology, serum hormone levels, and the HOMA-IR index. Key targets were identified through integrated metabolomics and bioinformatics analyses. The mechanism was further validated in vitro using dihydrotestosterone (DHT)-induced primary rat GCs, combined with molecular docking, cellular thermal shift assay (CETSA), drug affinity responsive target stability (DARTS) assay, co-immunoprecipitation (CO-IP), and Seahorse metabolic analysis. Results TLB administration alleviated estrous cycle disruption, improved ovarian morphology, and normalized serum testosterone, luteinizing hormone, and HOMA-IR index in PCOS rats. Metabolomics and bioinformatics analyses identified SIRT2 as a primary target of TLB. Mechanistically, TLB targets SIRT2, promoting the deacetylation of pyruvate kinase M2 (PKM2) at the K305 site. This deacetylation enhanced PKM2 tetramerization and enzymatic activity, thereby ameliorating the glycolytic imbalance in GCs, as evidenced by reduced pyruvate accumulation and restored lactate production. Furthermore, TLB, via the SIRT2/PKM2 axis, improved mitochondrial function, supported by the recovery of ATP production, mitochondrial membrane potential, and mitochondrial permeability transition pore integrity, ultimately suppressing GC apoptosis. Conclusion Our findings demonstrate that TLB alleviates ovarian PCOS phenotypes by targeting SIRT2 to promote PKM2 deacetylation at K305, which effectively corrects glycolytic flux and restores mitochondrial function in GCs.

Oviducts contain various nutrients that provide energy during oocyte development. This study aimed to improve the efficiency of in vitro reproduction using extracellular vesicles (EVs) produced by the oviduct epithelial cells of sika deer (Cervus nippon). Surprisingly, the uptake of deer oviduct epithelial cell extracellular vesicles (DOEC-EVs) by cumulus-oocyte complexes, which were encapsulated by dense cumulus cells (CCs), occurred only in CCs during maturation. Therefore, we hypothesized that DOEC-EVs are transported to oocytes through CCs to exert their effects. We first investigated the effects of DOEC-EVs on the expansion capacity of the cumulus-oocyte complexes, as well as cell cycle progression, proliferation, apoptosis, and lactate and pyruvate levels in CCs, and examined reactive oxygen species levels, mitochondrial function, and key gene expression. The results showed that DOEC-EVs regulated cell cycle progression, promoted proliferation, reduced apoptosis, and improved antioxidant capacity and glycolysis, and through the oocyte first polar body excretion rate, reactive oxygen levels, and mitochondrial membrane potential, it was shown that CC promoted in vitro oocyte maturation, improved the antioxidant capacity and mitochondrial function of oocytes, and promoted parthenogenetic embryo development. These results suggest that DOEC-EVs improve the efficiency of oocyte development in deer in vitro by acting on CCs, laying the foundation for further research on in vitro deer reproduction.

In vitro production techniques for bovine embryos can improve reproductive efficiency and expand quality breeding stock, but lipid metabolism disturbances during in vitro embryo culture can decrease embryo quality. Salidroside (SAL) is a glycoside extracted from the rhizome of the medicinal plant Rhodiola rosea that has antioxidant, antiaging, anti-inflammatory, and lipid metabolism-regulating effects. This study demonstrated that the addition of SAL to the culture medium of bovine embryos during in vitro culture increased the blastocyst rate and number of blastocyst cells and improved bovine blastocyst totipotency and proliferation. SAL reduced the lipid droplet content in bovine blastocysts and increased the levels of lipolysis-related genes (PNPLA2, LIPE, and MGLL). Fatty acids serve as ligands to activate PPARα and promote the transcription and expression of downstream fatty acid β-oxidation-related genes (CPT1A, CPT2, ACOX1, and ACOX2). SAL reduced the ROS level, increased the GSH level, increased the expression of antioxidant-related proteins (Nrf2 and downstream HO-1), and increased the levels of antioxidant enzyme-related genes (GPx1, SOD1, SOD2, and CAT) in bovine embryos. SAL increased the mitochondrial membrane potential and mitochondrial function, number, and distribution; facilitated mitochondria‒lipid droplet interactions; increased fatty acid availability to mitochondria; and further enhanced fatty acid β-oxidation. In conclusion, SAL not only acts as an antioxidant to reduce oxidative stress generated during in vitro bovine embryo culture but also promotes lipolysis to produce free fatty acids (FFA) to activate PPARα, enhances fatty acid β-oxidation, regulates lipid metabolism, and reduces the lipid content in blastocysts, thereby improving embryo developmental competence.

The endoplasmic reticulum and mitochondria are interconnected through the MAM structure, and mitochondrial fusion protein 2 (MFN2) is a key regulatory factor. In this study, tunicamycin (TM) was used to induce endoplasmic reticulum stress in bovine embryos to explore its effects on MFN2 expression, mitochondrial function and mitochondrial autophagy. The results showed that TM treatment significantly reduced the blastocyst rate and proliferation capacity of embryos, inhibited the expression of pluripotency genes (SOX2, CDX2, OCT4), and upregulated key proteins of the UPR pathway. The expression of MFN2 and MAM region E3 ubiquitin ligase (HRD1) was significantly increased, PINK1 expression was downregulated, and Parkin localization on the mitochondrial membrane was reduced. Colocalization analysis and the reduction of LC3-II ratio indicated that mitochondrial autophagy was blocked. At the same time, mitochondrial membrane potential, ATP content and functional genes (PGC-1, TFAM) expression were downregulated, OXPHOS key enzymes were inhibited, and glycolysis was compensated. The mitochondrial apoptosis marker cytochrome C was released, Caspase3 was upregulated, and the PI positive rate increased. In summary, ER stress inhibits mitophagy through HRD1 -mediated PINK1 degradation, leading to the accumulation of mitochondrial damage, aggravating energy metabolism disorders and apoptosis, and ultimately inhibiting the in vitro development of bovine embryos.

Fel d1, the major cat allergen responsible for over 90% of human IgE-mediated allergies, has an incompletely defined physiological role. To explore its function and assess the feasibility of producing hypoallergenic cats, we knocked out the CH2 domain of Fel d1 using CRISPR/Cas9 in feline skin cells. An optimized sgRNA introduced a frameshift mutation, with knockout efficiency validated by sequencing, qRT-PCR, and Western blot. Transcriptomic alterations were profiled by RNA-seq, and functional consequences were investigated via GO, KEGG, and GSEA analyses. Key findings were confirmed by qPCR, and phenotypes were assessed using CCK-8, EdU, and flow cytometry. The approach successfully generated a three-base insertion, resulting in near-complete loss of CH2 mRNA and Fel d1 protein. RNA-seq identified 3,469 differentially expressed genes (DEGs), with significant enrichment in pathways for hypertrophic cardiomyopathy (HCM) and rheumatoid arthritis (RA). Key genes in these pathways (e.g., TGFB2, MYBPC3, MMP3, and TLR4) were upregulated, and CH2 deletion impaired proliferation while increasing apoptosis. We conclude that CH2 deletion, while effectively abolishing the major allergen, triggers unintended transcriptomic reprogramming linked to pathological states. This underscores the necessity of comprehensive safety profiling, including transcriptomics, prior to applying gene-edited cells in SCNT-based development of hypoallergenic cats.

This study investigated the role of mitochondrial fusion protein-2 (MFN2) in bovine embryonic development and its relationship with endoplasmic reticulum (ER) stress, aiming to increase the efficiency of in vitro embryo culture. Western blot analysis revealed that MFN2 expression peaked at the 2-cell stage, decreased at the 4-cell stage, and gradually increased from the 6-8-cell stage to the blastocyst stage. Inhibiting MFN2 at the zygote stage reduced blastocyst formation and proliferation, and this damage was partially reversed by the ER stress protective agent TUDCA. MFN2 inhibition also led to the decreased formation of the inner cell mass (ICM) and reduced expression of the totipotent genes CDX2 and SOX2. Additionally, reactive oxygen species (ROS) levels increased following MFN2 inhibition but decreased after TUDCA treatment. The expression of antioxidative stress-related genes (SOD and CAT) was downregulated after MFN2 inhibition but upregulated following TUDCA treatment. Furthermore, MFN2 inhibition reduced ER fluorescence intensity and increased the expression of UPR signaling markers (GRP78, XBP1, CHOP, IRE1, and ATF6), indicating increased ER stress. TUDCA administration reversed these effects, restoring MFN2 levels and reducing apoptosis. In conclusion, MFN2 is essential for bovine embryonic development because it regulates ER stress and maintains cell function, with MFN2 deficiency leading to developmental disorders and cell damage. ER stress protectors such as TUDCA can effectively mitigate these negative effects, highlighting a potential strategy for improving in vitro embryo culture efficiency.

Oxidative stress poses a challenge to in vitro embryo culture. As a flavonoid, galangin (GAL) has been shown to have antioxidant effects, but the effect and antioxidant capacity of GAL in the in vitro development of porcine parthenogenetic embryos are still unknown. In this study, we demonstrated that 1 µM GAL significantly increased the blastocyst rate, decreased the accumulation of intracellular reactive oxygen species (ROS), increased the glutathione (GSH) level, and enhanced mitochondrial function in early porcine embryos. Nuclear factor erythroid-2-related factor 2 (Nrf2) was identified as the target gene of GAL via network pharmacology, and the transcript levels of related antioxidant enzymes (HO-1, NQO1, SOD2, and CAT) were found to be increased. Since Nrf2 has seven domains, we constructed Nrf2 mutants lacking different domains in vitro. We found that GAL specifically binds to the Neh1 domain of Nrf2. Subsequent embryonic experiments demonstrated that the antioxidant effect of GAL was abolished after Nrf2 deletion. These results suggest that GAL can directly bind to Nrf2 to regulate the level of oxidative stress and improve mitochondrial function in embryos.

Methoxychlor (MXC) is a widely used organochlorine pesticide primarily targeting pests. However, MXC has been found to negatively impact the reproductive system of both humans and animals, triggering oxidative stress and apoptosis. Melatonin (MLT), an endogenous hormone, possesses various benefits, including circadian rhythm regulation and anti-inflammatory and antioxidative stress effects. Moreover, MLT plays a crucial role in the development of animal germ cells and embryos. This study aimed to investigate the impact of MLT on porcine oocytes exposed to MXC. The experimental findings revealed that 200 μM MXC had detrimental effects on the maturation of porcine oocytes. However, the addition of 10-8 M MLT mitigated the toxic effects of MXC. MXC induced oxidative stress in porcine oocytes, leading to an increase in reactive oxygen species and impairing mitochondrial function. Consequently, oocyte quality was affected, resulting in elevated levels of early apoptosis and DNA damage, ultimately negatively impacting subsequent embryonic development. However, the addition of MLT showed the potential to ameliorate the damage caused by MXC. In conclusion, our results suggest that MLT exhibits a protective effect against MXC-induced damage to porcine oocyte maturation.

It is important to study the bacteria that cause endometritis to identify effective therapeutic drugs for dairy cows. In this study, 20% oxytetracycline was used to treat Holstein cows (n = 6) with severe endometritis. Additional 10 Holstein cows (5 for healthy cows, 5 for cows with mild endometritis) were also selected. At the same time, changes in bacterial communities were monitored by high-throughput sequencing. The results show that Escherichia coli, Staphylococcus aureus and other common pathogenic bacteria could be detected by traditional methods in cows both with and without endometritis. However, 16S sequencing results show that changes in the abundance of these bacteria were not significant. Endometritis is often caused by mixed infections in the uterus. Oxytetracycline did not completely remove existing bacteria. However, oxytetracycline could effectively inhibit endometritis and had a significant inhibitory effect on the genera Bacteroides, Trueperella, Peptoniphilus, Parvimonas, Porphyromonas, and Fusobacterium but had no significant inhibitory effect on the bacterial genera Marinospirillum, Erysipelothrix, and Enteractinococcus. During oxytetracycline treatment, the cell motility, endocrine system, exogenous system, glycan biosynthesis and metabolism, lipid metabolism, metabolism of terpenoids, polyketides, cofactors and vitamins, signal transduction, and transport and catabolism pathways were affected.

Transforming growth factor-beta (TGF-β) plays a critical role in regulating trophoblast invasion and proliferation. Growth differentiation factor-8 (GDF-8) is a member of the TGF-β superfamily and is categorized as a myostatin subtype. It is primarily a secreted protein synthesized in skeletal muscle cells. It is expressed in the placenta, reproductive tissues, and cells. In this study, we investigated the role of GDF-8 in the development and hatching rate of bovine embryos. We noted a notable elevation (p < 0.05) in the development and hatching rates compared to the control embryos. Furthermore, the GDF-8 group showed a significantly improved total cell number (p < 0.05) and an increase in trophectoderm ratio inner cell mass (trophectoderm: inner cell mass) cells (p < 0.001) compared to the control group. Additionally, blastocysts treated with GDF-8 exhibited significantly higher mRNA levels of caudal-type homeobox 2 (CDX2) (p < 0.05). The trophoblast invasion area was significantly larger in the GDF-8 group than in the control group (p < 0.01). Furthermore, qRT-PCR analysis revealed significantly higher mRNA levels (p < 0.05) of matrix metalloproteinases 9 (MMP9) and follistatin-like 3(FSTL3), both of which are associated with the ALK5-SMAD2/3 signaling pathway, in the GDF-8 group than those in the control group. The mRNA expression levels of genes related to tight junctions (TJ) and adherent junctions were higher in the GDF-8 group than those in the control group (p < 0.05). After 24 h of thawing, blastocysts were analyzed using 4-kDa FITC-dextran, which revealed a higher TJ integrity in the GDF-8 group (p < 0.01). Thus, GDF-8 plays a crucial role in bovine embryonic development, in vitro implantation, and cryotolerance.

The prevalence of porcine enteric coronaviruses (PECs), including transmissible gastroenteritis virus (TGEV), swine acute diarrhea syndrome coronavirus (SADS-CoV), porcine delta coronavirus (PDCoV), and porcine epidemic diarrhea virus (PEDV), poses a serious threat to animal and public health. Here, we aimed to further optimize the porcine aminopeptidase N (pAPN) gene editing strategy to explore the balance between individual antiviral properties and the biological functions of pAPN in pigs. Finally, APN-chimeric gene-edited pigs were produced through a CRISPR/Cas9-mediated knock-in strategy. Further reproductive tests indicated that these gene-edited pigs exhibited normal pregnancy rates and viability. Notably, in vitro viral challenge assays further demonstrated that porcine kidney epithelial cells isolated from F1-generation gene-edited pigs could effectively inhibit TGEV infection. This study is the first to report the generation of APN-chimeric pigs, which may provide a natural host animal for characterizing PEC infection with APN and help in the development of better antiviral solutions.

Salidroside (Sal) possesses several pharmacological activities, such as antiaging, and anti-inflammatory, antioxidant, anticancer activities, and proliferation-promoting activities, but the effects of Sal on oocytes have rarely been reported. In the present study, we evaluated the beneficial effects of Sal, which is mainly found in the roots of Rhodiola. Porcine cumulus oocyte complexes were cultured in IVM medium supplemented (with 250 μmol/L) with Sal or not supplemented with Sal. The maturation rate in the Sal group increased from 88.34 ± 4.32% to 94.12 ± 2.29%, and the blastocyst rate in the Sal group increased from 30.35 ± 3.20% to 52.14 ± 7.32% compared with that in the control group. The experimental groups showed significant improvements in the cumulus expansion area. Sal reduced oocyte levels of reactive oxygen species (ROS) and enhanced intracellular GSH levels. Sal supplementation enhanced the mitochondrial membrane potential (MMP), ATP level, and mtDNA copy number, which shows that Sal enhances the cytoplasmic maturation of oocytes. Oocytes in the Sal group exhibited slowed apoptosis and reduced DNA breakage. Cell cycle signals and oocyte meiosis play important roles in oocyte maturation. The mRNA expressions of the MAPK pathway and MAPK phosphorylation increased significantly in the Sal group. The mRNA expression of the oocyte meiosis gene also increased significantly. These results show that Sal enhances the nuclear maturation of oocytes. Moreover, Sal increased the number of blastocyst cells, the proliferation of blastocysts, and the expressions of pluripotency genes. Sal down-regulated apoptosis-related genes and the apoptotic cell rate of blastocysts. In summary, our results demonstrate that Sal is helpful to improving the quality of porcine oocytes in vitro, and their subsequent embryonic development.

Introduction: Methoxychlor (MXC) is an organochlorine pesticide (OCP) that was formerly used worldwide as an insecticide against pests and mosquitoes. However, MXC is not biodegradable and has lipophilic characteristics; thus, it accumulates in organisms and affects reproductive function. MXC, as an estrogenic compound, promotes oxidative stress, induces oxidative stress damage to ovarian follicles, and causes miscarriages and stillbirths in females. In this research endeavor, our primary objective was to explore the ramifications of MXC regarding the developmental processes occurring during the initial stages of embryogenesis in pigs.Methods: In this study, we counted the blastocyst rate of early embryos cultured in vitro. We also examined the reactive oxygen species level, glutathione level, mitochondrial membrane potential, mitochondrial copy number and ATP level in four-cell stage embryos. Finally, apoptosis and DNA damage in blastocyst cells, as well as pluripotency-related and apoptosis-related genes in blastocyst cells were detected. The above experiments were used to evaluate the changes of MXC damage on early parthenogenetic embryo development.Results and Discussion: The results showed that early embryos exposed to MXC had a significantly lower cleavage rate, blastocyst rate, hatching rate, and total cell count compared with the control group. It was also of note that MXC not only increased the levels of reactive oxygen species (ROS), but also decreased the mitochondrial membrane potential (ΔΨm) and mitochondrial copy number during the development of early embryos. In addition, after MXC treatment, blastocyst apoptosis and DNA damage were increased, decreased cell proliferation, and the expression of pluripotency-related genes SOX2, NANOG, and OCT4 was down-regulated, while the expression of apoptosis-related genes BAX/BCL-2 and Caspase9 was up-regulated. Our results clearly show that MXC can have deleterious effects on the developmental processes of early porcine embryos, establishing the toxicity of MXC to the reproductive system. In addition, the study of this toxic effect may lead to greater concern about pesticide residues in humans and the use of safer pesticides, thus potentially preventing physiological diseases caused by chemical exposure.

As an antioxidant, procyanidin B1(PB1) can improve the development of somatic cell nuclear transfer (SCNT) embryos; PB1 reduces the level of oxidative stress (OS) during the in vitro development of SCNT embryos by decreasing the level of reactive oxygen species (ROS) and increasing the level of glutathione (GSH) and mitochondrial membrane potential (MMP). Metabolite hydrogen peroxide (H2O2) produces OS. Catalase (CAT) can degrade hydrogen peroxide so that it produces less toxic water (H2O) and oxygen (O-2) in order to reduce the harm caused by H2O2. Therefore, we tested the CAT level in the in vitro development of SCNT embryos; it was found that PB1 can increase the expression of CAT, indicating that PB1 can offset the harm caused by oxidative stress by increasing the level of CAT. Moreover, if H2O2 accumulates excessively, it produces radical-(HO-) through Fe2+/3+ and damage to DNA. The damage caused to the DNA is mainly repaired by the protein encoded by the DNA damage repair gene. Therefore, we tested the expression of the DNA damage repair gene, OGG1. It was found that PB1 can increase the expression of OGG1 and increase the expression of protein. Through the above test, we proved that PB1 can improve the repairability of DNA damage. DNA damage can lead to cell apoptosis; therefore, we also tested the level of apoptosis of blastocysts, and we found that PB1 reduced the level of apoptosis. In summary, our results show that PB1 reduces the accumulation of H2O2 by decreasing the level of OS during the in vitro development of SCNT embryos and improves the repairability of DNA damage to reduce cell apoptosis. Our results have important significance for the improvement of the development of SCNT embryos in vitro and provide important reference significance for diseases that can be treated using SCNT technology.

m6A is one of the most common and abundant modifications of RNA molecules present in eukaryotes. The methyltransferase complex, consisting of methyltransferase-like 3 (METTL3), METTL14, and WTAP, is responsible for the m6A modification of RNA. WTAP was identified as an mRNA splicing regulator. Its role as a regulatory subunit of the m6A methyltransferase complex in embryonic development remains largely unknown. To investigate the role of WTAP in porcine early embryonic development, si-WTAP was microinjected into porcine parthenogenetic zygotes. WTAP knockdown significantly reduced the blastocyst rate and global m6A levels, but did not affect the cleavage rate. Betaine was supplemented into the in vitro culture (IVC) to increase the m6A levels. Betaine significantly increased the global m6A levels but did not affect the blastocyst rate. Furthermore, the pluripotency genes, including OCT4, SOX2, and NANOG, were downregulated following WTAP knockdown. The apoptotic genes BAX and CASPASE 3 were upregulated, while the anti-apoptotic gene BCL2 was downregulated in WTAP knockdown blastocysts. TUNEL staining revealed that the number of apoptotic cells was significantly increased following WTAP knockdown. Our study indicated that WTAP has an indispensable role in porcine early embryonic development.

Abstract Hydroxyurea (HU) is an FDA-approved drug used to treat a variety of diseases, especially malignancies, but is harmful to fertility. We used porcine oocytes as an experimental model to study the effect of HU during oocyte maturation. Exposure of cumulus–oocyte complexes (COCs) to 20 µM (P<0.01) and 50 µM (P<0.001) HU reduced oocyte maturation. Exposure to 20 µM HU induced approximately 1.5- and 2-fold increases in Caspase-3 (P<0.001) and P53 (P<0.01) gene expression levels in cumulus cells, respectively, increased Caspase-3 (P<0.01) and P53 (P<0.001) protein expression levels in metaphase II (MII) oocytes and increased the percentage of apoptotic cumulus cells (P<0.001). In addition, HU decreased the mitochondrial membrane potential (Δφm) (P<0.01 and P<0.001) and glutathione (GSH) levels (P<0.01 and P<0.001) of both cumulus cells and MII oocytes, while increasing their reactive oxygen species (ROS) levels (P<0.001). Following parthenogenetic activation of embryos derived from MII oocytes, exposure to 20 µM HU significantly reduced total blastocyst cell numbers (P<0.001) and increased apoptosis of blastocyst cells (P<0.001). Moreover, HU exposure reduced the rate of development of two-celled, four- to eight-celled, blastocyst, and hatching stages after parthenogenetic activation (P<0.05). Our findings indicate that exposure to 20 µM HU caused significant oxidative stress and apoptosis of MII oocytes during maturation, which affected their developmental ability. These results provide valuable information for safety assessments of HU.

Leonurine (LEO) is pseudoalkaloid that has been isolated from motherwort. It has been found to have various biological activities, including an antioxidant capacity. This study aimed to confirm whether LEO could be used in porcine in vitro culture (IVC) medium for its antioxidant effect and related molecular mechanisms. The results showed that embryos in IVC medium supplemented with 40 μM LEO had an increased blastocyst formation rate, total cell number, and proliferation capacity and a low apoptosis rate. LEO supplementation decreased reactive oxygen species levels and increased glutathione levels. Moreover, LEO-treated embryos exhibited improved intracellular mitochondrial membrane potential and reduced autophagy. In addition, pluripotency related gene was up-regulated while apoptosis and autophagy related genes were down-regulated with LEO supplementation. These results suggest that LEO has a beneficial effect on pre-implantation embryo development by reducing oxidative stress and enhancing mitochondrial function.