Search Results (105)

To achieve the goals of productivity and sustainability across diverse livestock systems, reproductive factors play a pivotal role. Historically, reproductive research has primarily focused on females, as they are responsible for maintaining pregnancy and delivering offspring following oocyte fertilization. However, since the early 2000s, the biological significance of sperm RNAs has been increasingly recognized in various livestock species. These RNAs contribute both genetically and epigenetically at the time of fertilization and during early embryonic development. Multiple types of sperm RNA have been identified in bovine, porcine, ovine, buffalo, and caprine spermatozoa. Notably, transcriptomic profiling has shown potential to differentiate between high- and low-fertility males, even when conventional semen quality values appear normal in both groups. This opens the possibility for more accurate identification of highly fertile sires. Nevertheless, a definitive marker or set of markers has yet to be established, likely due to the transcriptome’s sensitivity to environmental conditions and to the variability in evaluation methodologies. Therefore, global scientific efforts should aim to establish standardized, robust protocols, as sperm RNA represents a promising avenue for enhancing the sustainability of animal production systems. Full article

Oocytes and cumulus cells undergo meiotic resumption and proliferation via gonadotropins and growth factors during maturation, and various small G proteins are activated when COCs undergo physiological changes. This study investigated the influence of gonadotropins and growth factors on Ras and its GTPases during porcine COC maturation. Unmatured COCs were treated with FSH, LH, or EGF for 44 h. The mRNA expression levels of the Ras subfamily (H-Ras, K-Ras, N-Ras, and R-Ras), its GTPases (RASA1 and SOS1), and proliferation factors (ERK, CCNB1, and Cdc2) were analyzed using RT-PCR. In contrast to other Ras subfamilies, R-Ras expression is upregulated during COC maturation. We evaluated the effects of FSH, LH, and EGF at various concentrations that most effectively regulated the expression of R-Ras and GTPases. The results demonstrated that 0.5 µg/mL FSH, 10 IU/mL human chorionic gonadotropin (hCG), and 10 ng/mL EGF effectively enhanced R-Ras expression and cell proliferation. FSH supplementation during porcine COC maturation significantly upregulated R-Ras and ERK expression, independent of LH and EGF, and downregulated Cdc2 expression. These results indicated that FSH regulates R-Ras expression, thereby promoting cell proliferation during COC maturation. These results provide fundamental knowledge for understanding the role of Ras and its family members in the development of follicular environments in pigs. Full article

Both the livestock and biomedical fields require a large supply of high-quality mature oocytes. However, the in vitro maturation (IVM) process often leads to an accumulation of reactive oxygen species (ROS), which can cause defects in oocyte meiosis and embryo development, ultimately compromising oocyte quality. Urolithin A (UA), known for its antioxidant properties, has not been thoroughly investigated for its potential to mitigate the negative effects of oxidative stress during the in vitro culturing of oocytes, and its underlying mechanism is not well understood. In this study, an in vitro oxidative stress model was established using porcine oocytes treated with H₂O₂, followed by exposure to varying concentrations of UA. The results revealed that 30 μM UA significantly improved both the quality of oocyte culture and the developmental potential of the resulting embryos. UA was found to enhance oocyte autophagy, reduce oxidative stress-induced mitochondrial damage, and restore mitochondrial function. Additionally, it lowered ROS and DNA damage levels in the oocytes, maintained proper spindle/chromosome alignment and actin cytoskeleton structure, promoted nuclear maturation, prevented abnormal cortical granule distribution, and supported oocyte cytoplasmic maturation. As a result, UA alleviated oxidative stress-induced defects in oocyte maturation and cumulus cell expansion, thereby improving the developmental potential and quality of parthenogenetic embryos. After supplementation with UA, pig parthenogenetic embryo pluripotency-related genes (Nanog and Sox2) and antiapoptotic genes (Bcl2) were upregulated, while proapoptotic genes (Bax) were downregulated. In conclusion, this study suggests that adding UA during IVM can effectively mitigate the adverse effects of oxidative stress on porcine oocytes, presenting a promising strategy for enhancing their developmental potential in vitro. Full article

One of the major factors causing reduced developmental capacity of aged porcine oocytes is the induction of oxidative stress during oocyte aging. Tauroursodeoxycholic acid (TUDCA) supports cellular function by acting as an antioxidant and free radical scavenger. The aim of this study is to evaluate whether exogenous supplementation of TUDCA to the porcine in vitro maturation system can ameliorate the compromised quality of aged oocytes by mitigating free radical production. We found that TUDCA was able to effectively maintain normal oocyte morphology, cortical granule distribution, and spindle structure during postovulatory aging. Additionally, the blastocyst rate and total cell number in blastocysts were significantly increased in aged porcine oocytes treated with TUDCA. Importantly, aged porcine oocytes treated with TUDCA reduced ROS levels, increased the expression levels of GSH and SOD1 genes, and improved the mitochondrial membrane potential ratio. Further study demonstrated that TUDCA significantly alleviated apoptosis in aged porcine oocytes, confirmed by the decreased Caspase 3 levels and ratio of BAX to BCL2. Interestingly, TUDCA could effectively alleviate the phenomenon of endoplasmic reticulum stress triggered during the oocyte aging process. Taking these findings together, our study demonstrates that TUDCA supplementation beneficially affects the quality of aged porcine oocytes by suppressing oxidative stress, apoptosis, and endoplasmic reticulum stress. Full article

Triclosan (TCS) is a highly effective broad-spectrum antibacterial agent; however, the specific roles of TCS in oocyte maturation remain poorly understood. This research investigated the influence of TCS on biologically active processes during the in vitro maturation of porcine oocytes. Our results demonstrated that TCS significantly decreased the maturation rate of porcine oocytes in a concentration-dependent manner and impaired cumulus expansion. These detrimental effects were mediated by the disruption of mitochondrial function and distribution, leading to oxidative stress characterized by an accumulation of reactive oxygen species (ROS), a decrease in the expression of the antioxidant enzymes SOD2 and GSH, reduced ATP production, and a loss of mitochondrial membrane potential (ΔΨm). We also observed interference with endoplasmic reticulum (ER) distribution, disturbances in Ca²⁺ homeostasis, and fluctuations in ER stress, as evidenced by reduced expression of ER stress-related proteins. Furthermore, TCS exposure induced autophagy, as indicated by the levels of SQSTM1 (P62) and LC3-II. Additionally, TCS increased apoptosis rates, corresponding with a downregulation of Bcl-2 expression. Collectively, our findings suggest that exposure to TCS can impair cytoplasmic function, thereby affecting oocyte quality. Full article

Oocyte quality is crucial for successful fertilization and subsequent embryonic development. Post-ovulatory aging leads to reduced oocyte quality and impaired embryogenesis, representing an unavoidable challenge in terms of certain assisted reproductive techniques. Diosmetin (DIOS), a natural flavonoid found in lemons, spearmint, and spider moss, exhibits antioxidant, anti-inflammatory, and anti-apoptotic properties. However, its effects on the aging of mature porcine oocytes in vitro remain unexplored. This study investigated the impact of DIOS on porcine oocyte aging. In the IVM medium, fresh oocytes were cultured for 44 h, while aging oocytes were cultured for 68 h. Following the addition of varying DIOS concentrations (0.01, 0.1, and 1 μM) to the IVM medium, the DIOS-treated aging oocyte group was cultured for 68 h. The results demonstrated that 0.1 μM DIOS significantly improved the blastocyst rates and cell counts, reduced the reactive oxygen species (ROS), elevated the glutathione (GSH) levels, enhanced the mitochondrial function, and decreased the markers of autophagy (LC3B), apoptosis (annexin V), endoplasmic reticulum stress (CHOP), and senescence (SA-β-Gal). Furthermore, DIOS treatment upregulated the expression of relevant genes compared to the aged group. These findings suggest that DIOS effectively delays porcine oocyte aging. Full article

(1) Background: Nucleosomes represent the essential structural units of chromatin and serve as key regulators of cell function and gene expression. Oocytes in the germinal vesicle (GV) stage will later undergo meiosis and become haploid cells ready for fertilization, while somatic cells undergo mitosis after DNA replication. (2) Purpose: To furnish theoretical insights and data that support the process of cell reprogramming after nuclear transplantation. (3) Methods: We compared the nucleosome occupancy, distribution, and transcription of genes between two types of cells: fully grown GV oocytes from big follicles (BF) and somatic cells (porcine embryonic fibroblast, PEF). (4) Results: The nucleosome occupancy in the promoter of BF was 4.85%, which was significantly higher than that of 3.3% in PEF (p < 0.05), and the nucleosome distribution showed a noticeable increase surrounding transcriptional start sites (TSSs) in BF. Next, we reanalyzed the currently published transcriptome of fully grown GV oocytes and PEF, and a total of 51 genes in BF and 80 genes in PEF were identified as being uniquely expressed. The nucleosome distribution around gene TSSs correlated with expression levels in somatic cells, yet the results in BF differed from those in PEF. (5) Conclusion: This study uncovers the dynamic nature and significance of nucleosome positioning and chromatin organization across various cell types, providing a basis for nuclear transplantation. Full article

Parabens are widely used in various industries, which are including chemical, pharmaceutical, food, cosmetic, and plastic processing industries. Among these, methyl paraben (MP) serves as an antimicrobial preservative in processed foods, pharmaceuticals, and cosmetics, and it is particularly detected in baby care products. Studies indicate that MP functions as an endocrine-disrupting compound with estrogenic properties, negatively affecting mitochondrial bioenergetics and antioxidant activity in testicular germ cells. However, limited information exists regarding studies on the effects of MP in oocytes. The aim of this study was to investigate the specific mechanism and the toxic effects of MP during oocyte maturation cultured in vitro using a porcine oocyte model. The results indicated that MP (50 μM) inhibited oocyte expansion, significantly reducing the expression of expansion-related genes MAPK1 and ERK1, and decreased the first polar body extrusion significantly as well. ATP levels decreased, reactive oxygen species (ROS) levels remained unchanged, and glutathione (GSH) levels decreased significantly, resulting in an elevated ROS/GSH ratio. The expression of antioxidant genes SOD1 and GPX was significantly decreased. Additionally, a significant decrease in levels of mitochondrial production and biosynthesis protein PGC1α+β, whereas levels of antioxidant-related protein Nrf2 and related gene expression were significantly increased. Autophagy protein LC3B and gene expression significantly decreased, and apoptosis assay indicated a significant increase in levels of caspase3 protein and apoptosis-related genes. These results demonstrated the negative effect of MP on oocyte maturation. In conclusion, our findings indicate that MP disrupts redox balance and induces mitochondrial dysfunction during meiosis in porcine oocytes, resulting in the inhibition of meiotic progression. The present study reveals the mechanism underlying the effects of methyl para-hydroxybenzoate on oocyte maturation. Full article

The physiological state of Granulosa cells (GCs) is intricately linked to the growth and development of oocytes. Oxidative stress has been found to cause damage to GCs in vitro. Astaxanthin (AST), a well-known natural ketone-type carotenoid, has demonstrated strong antioxidant properties. This study investigates the impact of astaxanthin supplementation on the physiological state of porcine ovarian granulosa cells cultured in vitro. Variations in morphology, apoptosis, reactive oxygen species (ROS) levels, and the expression of apoptosis and anti-oxidation-related genes in porcine GCs from different passages were observed. Significant morphological changes, increases in apoptosis, and decreases in antioxidant capacity resulting from passage were observed. Subsequently, treatment with 5 μmol/L astaxanthin significantly enhanced cell viability, proliferation, antioxidant capacity and mitochondrial function while also regulating the estradiol (E2) and progesterone (P4) levels. Additionally, the gene expression of antioxidation, E2, and P4 synthesis markers was assessed, revealing reduced apoptosis and ROS levels in porcine GCs. In conclusion, supplementation with 5 μmol/L astaxanthin in vitro effectively enhances the physiological condition of porcine GCs and optimizes the culture system for these cells in vitro. Optimizing the culture system of porcine GCs in vitro can simulate the function of granulosa cells in vivo and provide a theoretical reference for further promoting follicular development, which is beneficial to improving sow fertility in actual production. Full article

Isoorientin (ISO) is a natural lignan glycoside flavonoid found in various plants, including Charcot and Stonecrop. ISO exhibits diverse physiological and pharmacological effects, such as antioxidative, anti-inflammatory, hepatoprotective, antiviral, antianxiety, and anti-myocardial ischaemic properties, as well as lipid metabolism regulation. This study investigated the impact of ISO supplementation on oxidative stress and lipid accumulation in porcine early embryos, along with its underlying mechanisms. Porcine embryos were cultured in vitro under different concentrations of ISO (0, 1, 10, and 100 nM). The results revealed that 10 nM ISO significantly enhanced the blastocyst rate and total embryonic cell count in vitro. ISO-treated embryos exhibited reduced reactive oxygen species levels and elevated glutathione levels compared to the untreated group. In addition, ISO treatment significantly increased the expression of the key antioxidant regulator Nrf2, improved mitochondrial function, and reduced lipid droplet accumulation. Concurrently, early embryo autophagy and apoptosis levels decreased. Furthermore, ISO treatment upregulated antioxidant-related genes (SOD1, SOD2, and CAT) and mitochondrial biogenesis related genes (NRF1, NRF2, and SIRT1), while downregulating lipid synthesis-related genes (SREBP1 and FASN). Additionally, lipid hydrolysis-related genes (ACADS) were elevated. These findings collectively suggest that ISO may facilitate early embryonic development in pigs by ameliorating oxidative stress and lipid metabolism. Full article

Assisted reproduction technology (ART) procedures are often impacted by post-ovulatory aging (POA), which can lead to reduced fertilization rates and impaired embryo development. This study used RNA sequencing analysis and experimental validation to study the similarities and differences between in vivo- and vitro-matured porcine oocytes before and after POA. Differentially expressed genes (DEGs) between fresh in vivo-matured oocyte (F_vivo) and aged in vivo-matured oocyte (A_vivo) and DEGs between fresh in vitro-matured oocyte (F_vitro) and aged in vitro-matured oocyte (A_vitro) were intersected to explore the co-effects of POA. It was found that “organelles”, especially “mitochondria”, were significantly enriched Gene Ontology (GO) terms. The expression of genes related to the “electron transport chain” and “cell redox homeostasis” pathways related to mitochondrial function significantly showed low expression patterns in both A_vivo and A_vitro groups. Weighted correlation network analysis was carried out to explore gene expression modules specific to A_vivo. Trait–module association analysis showed that the red modules were most associated with in vivo aging. There are 959 genes in the red module, mainly enriched in “RNA binding”, “mRNA metabolic process”, etc., as well as in GO terms, and “spliceosome” and “nucleotide excision repair” pathways. DNAJC7, IK, and DDX18 were at the hub of the gene regulatory network. Subsequently, the functions of DDX18 and DNAJC7 were verified by knocking down their expression at the germinal vesicle (GV) and Metaphase II (MII) stages, respectively. Knockdown at the GV stage caused cell cycle disorders and increase the rate of abnormal spindle. Knockdown at the MII stage resulted in the inefficiency of the antioxidant melatonin, increasing the level of intracellular oxidative stress, and in mitochondrial dysfunction. In summary, POA affects the organelle function of oocytes. A_vivo oocytes have some unique gene expression patterns. These genes may be potential anti-aging targets. This study provides a better understanding of the detailed mechanism of POA and potential strategies for improving the success rates of assisted reproductive technologies in pigs and other mammalian species. Full article

The in vitro maturation efficiency of porcine oocytes is relatively low, and this limits the production of in vitro porcine embryos. Since melatonin is involved in mammalian reproductive physiology, in this study, we have explored whether endogenously produced melatonin can help in porcine oocyte in vitro maturation. We have found, for the first time in the literature, that mitochondria are the major sites for melatonin biosynthesis in porcine oocytes. This mitochondrially originated melatonin reduces ROS production and increases the activity of the mitochondrial respiratory electron transport chain, mitochondrial biogenesis, mitochondrial membrane potential, and ATP production. Therefore, melatonin improves the quality of oocytes and their in vitro maturation. In contrast, the reduced melatonin level caused by siRNA to knockdown AANAT (siAANAT) is associated with the abnormal distribution of mitochondria, decreasing the ATP level of porcine oocytes and inhibiting their in vitro maturation. These abnormalities can be rescued by melatonin supplementation. In addition, we found that siAANAT switches the mitochondrial oxidative phosphorylation to glycolysis, a Warburg effect. This metabolic alteration can also be corrected by melatonin supplementation. All these activities of melatonin appear to be mediated by its membrane receptors since the non-selective melatonin receptor antagonist Luzindole can blunt the effects of melatonin. Taken together, the mitochondria of porcine oocytes can synthesize melatonin and improve the quality of oocyte maturation. These results provide an insight from a novel aspect to study oocyte maturation under in vitro conditions. Full article

Antioxidants protect cellular function and structure by neutralizing the oxidative stress caused by increased reactive oxygen species (ROS) during sperm freezing. Studies on cryopreservation using various antioxidants have demonstrated encouraging results. Many studies have used antioxidants to increase the efficiency of sperm freezing and to improve the success rate of artificial insemination and pregnancy. Manganese (III) tetrakis (4-benzoic acid) porphyrin chloride (MnTBAP) is a newly synthesized antioxidant with positive effects on sperm morphology and capacitation in humans, rams, and stallions. In this study, porcine semen was treated with 0, 50, 100, and 150 μM of MnTBAP based on a Tris–egg-yolk extender and frozen to determine whether MnTBAP can assist the status of sperm during cryopreservation. First, motility was assessed using the computer-assisted sperm analysis (CASA) system, with the 100 μM treatment group showing the highest motile rate (66.8%) compared with that of the other groups (control, 51.1%; 50 μM and 150 μM, 59.6%); therefore, the remaining analyses were conducted comparing the two groups (control vs. 100 μM group; p < 0.01). Second, fluorescence staining was applied to examine the control and 100 μM groups using fluorescence microscopy. The viability (41.7% vs. 62.4%) and the acrosome integrity (77.9% vs. 86.4%) differed significantly (p < 0.05). In addition, the mitochondrial membrane potential (MMP) was 46.5% vs. 51.9%; the fragmentation rate, estimated using the Sperm-sus-Halomax kit, was 63.4% vs. 57.4%; and the detected caspase activity was 30.1% vs. 22.9%. These tended to be higher in the treated group but did not differ significantly. Third, measurements using FACSLyric revealed that the 100 μM treatment group exhibited a state of elevated normal lipid arrangement within the plasma membrane and diminished levels of apoptosis and ROS (p < 0.01). We assessed the expression of genes relevant to antioxidant effectiveness using real-time RT-qPCR. Our findings indicated significant alterations in the expression levels of various mRNA species, with the exception of NOX5 (p < 0.05). Finally, the straws were dissolved and used to treat matured denuded oocytes to investigate the effect on fertilization and embryo development in vitro. The cleavage rate was (77.6% vs. 84.1%), and the blastocyst rate was 9.7% vs. 11.4% (p < 0.05). In conclusion, these results suggest that MnTBAP positively affected sperm freeze–thawing, improving the fertilization capacity, and leading to increased embryo development. Full article

Recent studies have established that exosomes (EXs) derived from follicular fluid (FF) can promote oocyte development. However, the specific sources of these EXs and their regulatory mechanisms remain elusive. It is universally acknowledged that oocyte development requires signal communication between granulosa cells (GCs) and oocytes. However, the role of GC-secreted EXs and their functions are poorly understood. This study aimed to investigate the role of porcine granulosa-cell-derived exosomes (GC-EXs) in oocyte development. In this study, we constructed an in vitro model of porcine GCs and collected and identified GC-EXs. We confirmed that porcine GCs can secrete EXs and investigated the role of GC-EXs in regulating oocyte development by supplementing them to cumulus–oocyte complexes (COCs) cultured in vitro. Specifically, GC-EXs increase the cumulus expansion index (CEI), promote the expansion of the cumulus, alleviate reactive oxygen species (ROS), and increase mitochondrial membrane potential (MMP), resulting in improved oocyte development. Additionally, we conducted small RNA sequencing of GC-EXs and hypothesized that miR-148a-3p, the highest-expressed microRNA (miRNA), may be the key miRNA. Our study determined that transfection of miR-148a-3p mimics exerts effects comparable to the addition of EXs. Meanwhile, bioinformatics prediction, dual luciferase reporter gene assay, and RT-qPCR identified DOCK6 as the target gene of miR-148a-3p. In summary, our results demonstrated that GC-EXs may improve oocyte antioxidant capacity and promote oocyte development through miR-148a-3p by targeting DOCK6. Full article

Understanding the complex interplay between genetics and environmental factors is vital for enhancing livestock production efficiency while safeguarding animal health. Despite extensive studies on production-specific genes in livestock, exploring how epigenetic mechanisms and heritable modifications govern animal growth and development remains an under-explored frontier with potential implications across all life stages. This study focuses on the GBAF chromatin remodeling complex and evaluates its presence during embryonic and fetal development in swine. Immunocytochemistry and co-immunoprecipitation techniques were employed to investigate the presence and interactions of GBAF subunits BRD9 and GLTSCR1 in porcine oocytes, preimplantation embryos, and cell lines, and transcriptional dynamics of GBAF subunits across these key developmental stages were analyzed using existing RNA-seq datasets. BRD9 and GLTSCR1 were identified across all represented stages, and an interaction between GLTSCR1 and BAF170 was shown in PTr2 and PFF cells. Our findings highlight the ubiquitous presence of GBAF in porcine early development and the potentially novel association between GLTSCR1 and BAF170 in swine. The transcriptional dynamics findings may suggest GBAF-specific contributions during key developmental events. This study contributes to the growing understanding of epigenetic regulators in both swine and mammalian development, emphasizing the implications of GBAF as a modulator of key developmental events. Full article