This study investigated the effects of astaxanthin (ASTA) on testosterone synthesis and mitochondrial function in testicular Leydig cells of aging roosters. ASTA significantly enhanced Leydig cell viability (P < 0.05) and increased testosterone production at concentrations of 2.5-20 μg/mL (P < 0.05), with the optimal effect observed at 5 μg/mL (P < 0.01). At this concentration, ASTA significantly upregulated the mRNA and protein expression of key steroidogenic enzymes, steroidogenic acute regulatory (StAR), cholesterol side-chain cleavage cytochrome (P450scc), 3β-hydroxysteroid dehydrogenase (3β-HSD), 17β-hydroxysteroid dehydrogenase (17β-HSD) (P < 0.01), and steroidogenic factor-1 (SF-1) (P < 0.05). ASTA also significantly elevated the activities and mRNA expression of antioxidant enzymes superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-PX) (P < 0.01), reduced reactive oxygen species (ROS) levels (P < 0.05), and decreased malondialdehyde (MDA) content (P < 0.01). Furthermore, ASTA treatment significantly improved mitochondrial membrane potential (MMP), adenosine triphosphate (ATP) content and mitochondrial DNA (mtDNA) copy number (P < 0.01), increased the expression of mitochondrial biogenesis regulators peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α), nuclear respiratory factor-1 (NRF1), and mitochondrial transcription factor A (TFAM) (P < 0.01), and significantly suppressed apoptosis (P < 0.05), as evidenced by increased B-cell lymphoma-2 (Bcl-2) expression (P < 0.01) and reduced expression of Bcl-2-associated X protein (Bax), cysteinyl aspartate specific proteinase-3 (caspase-3), and apoptosis-inducing factor (AIF) (P < 0.01). These results indicate that ASTA enhances testosterone synthesis in aging rooster Leydig cells by reducing oxidative stress, improving mitochondrial function and biogenesis, upregulating steroidogenic genes, and inhibiting mitochondrial-related apoptosis.

Inosine monophosphate (IMP) is a major flavor compound in meat. Given the observed positive correlation between adenylate kinase 1 (AK1) expression and IMP content in Beijing-You chickens (BJYs), we investigated the role of AK1 in regulating myoblast proliferation and differentiation. Cell counting kit-8 (CCK-8) assays, 5-ethynyl-2 '-deoxyuridine (EdU) incorporation, flow cytometry, and Western blotting showed that AK1 promoted myogenic differentiation and suppressed the proliferative activity of myoblasts. During the proliferation of myoblasts, glutamine phosphoribosylpyrophosphate amidotransferase (GPAT) was suppressed by AK1 and adenylosuccinate synthase (ADSS) was enhanced, which led to reduced IMP accumulation and inhibited uric acid (UA) and adenosine triphosphate (ATP) production. During myoblast differentiation, the protein expression of de novo IMP synthesis genes, including GPAT, 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase/ IMP cyclohydrolase (PurH), and adenylosuccinate lyase (ADSL), was upregulated, while ADSS was down-regulated by AK1, resulting in enhanced IMP deposition and stimulated production of UA and ATP. In summary, this study elucidates the regulatory role of AK1 in coordinating the proliferation and differentiation of myoblasts, as well as its stage-specific modulation of IMP anabolism. These findings provide theoretical insights into the molecular mechanisms governing IMP deposition and offer potential guidance for improving meat flavor quality in chicken through molecular breeding strategies.

Bovine viral diarrhea virus (BVDV) infects bovine trophoblast cells (BTCs) to induce abortion, stillbirth and other reproductive disorders, and severely threatens the global cattle industry. Exosomes serve as vital viral transmission carriers mediating immune evasion. Tumor susceptibility gene 101 (TSG101), a core ESCRT component, regulates exosome biogenesis and cargo sorting. Nevertheless, the mechanism of TSG101 in exosome-mediated BVDV transmission across BTCs remains unknown. In this study, BTCs were used as cell models to explore the role and mechanism of TSG101 in BVDV transmission. BVDV infection caused obvious cytopathic effects (CPE) in BTCs, with viral particles packaged in exosomes to facilitate immune evasion and transmission. BVDV also upregulated TSG101 and exosomal CD63, and TSG101 colocalized with BVDV E2. TSG101 knockdown suppressed BVDV proliferation and exosome-mediated transmission, and impaired CD63 trafficking into exosomes. Co-IP and immunofluorescence assays further confirmed the interaction between BVDV E2 and TSG101. Molecular docking and 100-ns MD simulation showed that E2 and TSG101 formed a stable binding complex, with key interacting residues identified at the contact surface. TSG101 mutations at Y110K, K118A, and E284A exerted distinct effects. TSG101 Y110K and K118A restricted BVDV replication by impairing TSG101 function, while the E284A mutation preserved TSG101 responsiveness, promoted viral proliferation. These findings expand understanding of BVDV E2-mediated ESCRT homeostasis regulation and provide a molecular basis for targeting the TSG101 to limit BVDV vertical transmission in BTCs.

This study investigated the effects of dietary lycopene supplementation on semen quality, testicular histology, antioxidant capacity, and reproductive hormone levels in aging breeder roosters. A total of 96 roosters were randomly divided into four groups and supplemented with 0, 50, 100, and 200 mg/kg of lycopene for six weeks. Lycopene significantly improved semen volume, sperm concentration, motility, viability, and morphological parameters at all doses (P < 0.05). The 200 mg/kg group exhibited the highest semen volume by week 6 (0.44 mL, P < 0.05). Sperm concentration increased significantly in the 100 mg/kg group (P < 0.05), and motility was highest in the 200 mg/kg group by week 4 (92.08%, P < 0.05). Testicular histology also showed significant improvement, with a notable increase in seminiferous tubule area at 200 mg/kg (0.11 mm², P < 0.01), while Leydig cell density followed a quadratic response, peaking at 100 mg/kg (44.60 cells/mm², P < 0.01). Plasma LH and testosterone levels peaked at 100 mg/kg, with significant increases of 12.81% and 43.37%, respectively (P < 0.01). Lycopene enhanced antioxidant capacity across seminal plasma, plasma, and testicular tissues, with significant increases in T-SOD, GSH-Px, and CAT activities (P < 0.05). MDA levels were significantly reduced, especially at 100 mg/kg (P < 0.01). Lycopene supplementation also improved mitochondrial function in sperm, as indicated by enhanced mitochondrial membrane potential at 100 mg/kg (P < 0.01) and reduced reactive oxygen species levels and sperm apoptosis (P < 0.05). In conclusion, lycopene supplementation, particularly at 100 mg/kg, significantly improves semen quality, testicular health, antioxidant capacity, and reproductive hormone levels in aging breeder roosters, with potential applications in enhancing reproductive performance.

Insulin-like growth factor 2 (IGF2) is essential for cell growth and differentiation and functions through the IGF2 receptor (IGF2R) to regulate embryonic and placental development. Exosomes that are synthesized and released from cells and play important roles in embryogenesis and placental development rely on the IGF2R for sorting and transport. However, the role of the imprinted Igf2-Igr2r axis and exosomes in the co-regulation of early placental development remains unknown. Cotyledon villi were collected from bovine placentas at different gestational ages, and the localization and expression of IGF2, IGF2R, and exosomal marker proteins were detected. Furthermore, the expression of exosomal marker factors was detected after the expression of IGF2R or IGF2 was inhibited through RNA interference or the addition of inhibitors, respectively. Our results demonstrated that IGF2, IGF2R, and the exosomal markers CD63, CD9, TSG101, and Rab11 are mainly located on the cell membrane of mononuclear trophoblast cells and binuclear trophoblast cells, which make up the cotyledon villi of the bovine placenta. The expressions of IGF2, IGF2R, and the exosomal marker proteins CD63, CD9, TSG101, and Rab11 showed a significant upward trend with increased gestation duration. Additionally, both Igf2r-knockdown and suppressing the expression of IGF2 with chromeceptin (IGF2 inhibitor) led to the downregulation of exosomal marker proteins in both bovine placental trophoblast cells (BTCs) and BTC-derived exosomes. Our study confirmed that the imprinted Igf2-Igf2r axis participates in the early development of cotyledon villi in the bovine placenta by manipulating exosome biogenesis, providing evidence for improving disorders during placental development.

BPA is a globally ubiquitous industrial compound that harms male reproductive health by causing abnormal sperm development and subsequent spermatogonia loss, yet the underlying mechanisms of BPA-induced spermatogenesis disorder remain unclear. Here, we explored BPA's effects on adolescent male mice and GC-1 cells by gavaging mice with BPA at doses of 20, 200, or 2000 μg/kg/d for 4 weeks and treating GC-1 cells with 10 μM BPA for 12 h to establish a damage model. The results revealed that BPA induced spermatogenesis disorder via ferroptosis, which was associated with the activation of excessive mitophagy. RNA-seq analysis elucidated that upregulated BCAT1 plays a key role in this process. Specifically, downregulation of BCAT1 alleviated BPA-induced mitophagy, whereas overexpression of BCAT1 exacerbated these effects. Moreover, the occurrence of BPA-induced spermatogenesis disorder is regulated by the binding of PINK1 via targeting SER227 to BCAT1. Additionally, quercetin, a potential BCAT1 ligand, reduced BCAT1 expression and mitigated BPA-induced mitophagy and ferroptosis both in vitro and in vivo. In summary, our results reveal that quercetin effectively inhibits BPA-induced mitophagy activation, thereby reducing ferroptosis in spermatogonia cells. This study highlights BCAT1 as a potential therapeutic target and provides novel insights into BPA-induced testicular toxicity and therapeutic strategy development.

This study aimed to compare fat deposition, muscle tissue structure, and abdominal fat lipid metabolism among 150-day-old Beijing You chickens, 450-day-old Beijing You chickens, and 150-day-old Ross 308 broilers. Slaughter performance analysis revealed that Beijing You chickens exhibited significantly higher fat deposition than Ross 308 broilers, with continuous accumulation observed with increasing age. Histological observations showed that the breast muscle fibers of Beijing You chickens were significantly smaller than those of Ross 308 broilers of the same age, while the diameters of abdominal adipocytes were significantly larger and increased further with age. Lipidomic profiling of abdominal fat identified 613 significantly different lipid species between Beijing You chickens and Ross 308 broilers, and 250 lipid species differing between the two age groups of Beijing You chickens. Among them, 123 lipid species were differentially abundant in both comparisons, mainly enriched in glycerophospholipid and sphingolipid metabolism pathways, suggesting their key roles in lipid metabolic remodeling. These findings reveal distinct differences in lipid metabolism and muscle development among chicken breeds and provide a theoretical basis for understanding the molecular mechanisms underlying flavor, tenderness, and metabolic regulation, as well as a valuable reference for molecular breeding of high-quality poultry.

OBJECTIVE:The content of intramuscular fat (IMF) is closely linked to meat quality, and the mechanism of IMF deposition is complex. Despite numerous transcriptomic studies on IMF, variations in sample sizes and data analysis methods have produced inconsistent gene expression patterns and results. To identify the pivotal genes influencing pig IMF content, we performed a meta-analysis on 10 pig muscle transcriptome datasets with a total of eighty samples, forty with high and forty with low IMF samples. METHODS:DESeq2 has been used to analyze the high and low IMF groups for 10 datasets each, resulting in the differentially expressed genes (DEGs) for each dataset. To identify key genes affecting IMF content, we performed a meta-analysis of the differential expression results from the 10 datasets using MetaVolcanoR. Subsequently, we conducted protein-protein interaction network analysis, Gene Ontology and Kyoto encyclopedia of genes and genomes functional enrichment analysis, and quantitative trait locus (QTL) analysis on the DEGs. RESULTS:The meta-analysis identified 129 DEGs, comprising 71 upregulated and 58 downregulated DEGs in the high IMF group. The DEGs exhibited enrichment in processes associated with adipocyte differentiation and fat anabolism. QTL analysis demonstrated that five DEGs, including FASN and SCD, corresponded to six QTLs associated with IMF. CONCLUSION:The findings suggest that meta-analysis effectively integrates data from multiple datasets, resulting in more reliable outcomes. This approach enabled the identification of the core gene cluster comprising FASN, SCD, and PLIN1, LEP, and G0S2, which influence IMF content in pigs.

Astaxanthin (AST), a potent antioxidant, has shown promise in improving poultry production metrics. This study investigated the effects of dietary AST on carcass traits, breast muscle composition, intestinal barrier function, cecal microbiota, and metabolomics in overfed Pekin ducks. A total of 150 one-day-old male Pekin ducks were assigned to five groups: Control (CON, basal diet), low-dose AST (LDG, 40 mg/kg), medium-dose (MDG, 80 mg/kg), high-dose (HDG, 120 mg/kg), and an ad libitum-fed group (ALG), and the feeding trial lasted for 42 days. Our findings revealed that AST significantly enhanced live and carcass weights, increased breast muscle mass in the HD group, and promoted subcutaneous fat deposition MD group, (P < 0.05). Supplementation of AST improved breast muscle amino acid profiles, especially phenylalanine, alanine, arginine, lysine, and tryptophan, and shifted fatty acid composition by reducing saturated fatty acids and increasing mono- and polyunsaturated fatty acids, notably in the MDG (P < 0.05). Astaxanthin reduced serum total and low-density lipoprotein cholesterol while increasing high-density lipoprotein cholesterol (P < 0.05). Furthermore, astaxanthin enhanced gut barrier integrity by lowering serum nitric oxide, endotoxin, and D-lactate levels and upregulating jejunal Mucin-2 and occludin gene expression (P < 0.05). Microbiota analysis revealed increases in Bacteroidota, Firmicutes, and Actinobacteria, with beneficial associations between gut integrity and Weissella and Lactobacillus, and negative correlations with Faecalibacterium. Metabolomics identified enrichment of glycerophospholipid, tryptophan, glutathione, glycine‑serine-threonine, and aminoacyl-tRNA biosynthesis pathways. Key upregulated metabolites included 5-methoxyindoleacetate and glyceric acid, while Lysophosphatidylcholines (LysoPCs) were downregulated. 6-hydroxymelatonin and creatine were enriched but not significant. Microbiota-metabolome correlations indicated negative associations between Weissella and LysoPCs, Faecalibacterium and creatine, and a positive link between Collinsella and glyceric acid (P < 0.05), favoring gut health. Overall, AST improved carcass traits and intestinal function in overfed ducks via microbiota and metabolic modulation.

Intramuscular fat (IMF) is an important indicator for evaluating meat quality. Transcriptome sequencing (RNA-seq) is widely used for the study of IMF deposition. Machine learning (ML) is a new big data fitting method that can effectively fit complex data, accurately identify samples and genes, and it plays an important role in omics research. Therefore, this study aimed to analyze RNA-seq data by ML method to identify differentially expressed genes (DEGs) affecting IMF deposition in pigs. In this study, a total of 74 RNA-seq data from muscle tissue samples were used. A total of 155 DEGs were identified using a limma package between the two groups. 100 and 11 significant genes were identified by support vector machine recursive feature elimination (SVM-RFE) and random forest (RF) models, respectively. A total of six intersecting genes were in both models. KEGG pathway enrichment analysis of the intersecting genes revealed that these genes were enriched in pathways associated with lipid deposition. These pathways include α-linolenic acid metabolism, linoleic acid metabolism, ether lipid metabolism, arachidonic acid metabolism, and glycerophospholipid metabolism. Four key genes affecting intramuscular fat deposition, PLA2G6, MPV17, NUDT2, and ND4L, were identified based on significant pathways. The results of this study are important for the elucidation of the molecular regulatory mechanism of intramuscular fat deposition and the effective improvement of IMF content in pigs.

The distribution of adipose depots in different body parts affects pig production value and human health, governed by complex epigenomic mechanisms. Limited studies on pig adipose depots have hindered the genetic improvement of fat-related economic traits and their biomedical applications. To address this issue, we generated epigenomic maps for backfat, belly fat, groin fat, and intermuscular fat (IMF) in Meishan pigs, integrating ChIP-seq, ATAC-seq, RNA-seq, Hi-C, and public whole-genome sequencing data. Our results reveal that belly/backfat share similar chromatin states, while groin fat/IMF exhibit distinct H3K27ac modification, super-enhancer (SE) dynamics, and open chromatin landscapes compared to belly/backfat. The spatially specific expressions of adipogenic transcription factors (TFs), such as lipid synthesis-related TFs PPARA and SOX6, which are highly expressed in back/belly fat, and adipocyte differentiation TF KLF4 was driven by a groin fat specific SE, underlie these chromatin state disparities. These results also suggest enhanced lipid synthesis in belly/backfat and adipocyte differentiation in groin fat. Moreover, candidate functional variants identified in IMF-gained H3K27ac peaks are primarily associated with meat quality traits. Genes linked to pig backfat thickness may also serve as candidate genes for human obesity due to the conserved cis-regulatory elements and gene expression patterns between humans and pigs. Overall, our epigenomic landscape enhances understanding of adipose depot regulation in mammals, facilitating cross-species insights and precision breeding.

Astaxanthin (ASTA), a potent antioxidant with anti-inflammatory, anti-proliferative, and anti-apoptotic effects, has garnered attention for its potential to enhance the nutritional value of eggs. This study aimed to evaluate the effects of dietary ASTA on egg yolk nutritional composition, sensory attributes, and related molecular mechanisms. We assessed how varying levels of ASTA supplementation (0, 0.01 %, 0.02 %, and 0.04 %) influenced yolk color, texture, and nutrient profile. The results showed that dietary ASTA had no significant effect on laying productive performance but enhanced yolk color in a dose-dependent manner (P < 0.05). Texture analysis showed that ASTA reduced the cohesiveness and chewiness of yolks (P < 0.05), while electronic tongue analysis confirmed no significant impact on flavor. ASTA did not significantly alter amino acid composition but increased total fatty acid content in the 0.02 % and 0.04 % groups (P < 0.05). Compared with the control group, the content of vitamin E in egg yolk significantly (P < 0.05) decreased in the ASTA groups, and the contents of vitamin D in egg yolk significantly (P < 0.05) decreased in the 0.01 % and 0.04 % ASTA groups, which corresponded with inhibited expression of alpha-tocopherol transfer protein (TTPA) and vitamin D receptor (VDR) mRNA, as well as reduced VDR protein levels. ASTA further upregulated the expression of nutrient-transport proteins, including very low-density lipoprotein (VLDL) and retinol-binding protein 4 (RBP4) (P < 0.05), demonstrating its role in modulating nutrient transport and distribution. This research underscores the importance of balancing ASTA levels to optimize both nutrient enhancement and preservation of existing beneficial compounds in fortified eggs. Our research findings provide valuable insights for the production of ASTA-enriched eggs, enabling consumers to select nutritionally fortified eggs according to their own nutritional needs.

The present study aimed to investigate the direct effects of α-Linolenic acid (ALA) on the in vitro production of testosterone and the expression of key enzymes and proteins related to steroidogenesis in Leydig cells of roosters. METHODS:Purified primary Leydig cells isolated from 65-week-old roosters were purified and treated with different concentrations of ALA treatments: (0 μm/L [control], solvent control group (DMSO), 20 μM/L, 40 μM/L, and 80 μM/L) and cell counting-8 (CCK-8) for cell viability assay, Enzyme-linked immunosorbent assay (ELISA) kit for the determination of testosterone in cell supernatants, quantitative (real-time) PCR, and analysis of activities of antioxidants catalase (CAT), superoxide dismutase (SOD) and malondialdehyde (MDA), evaluation of mitochondrial membrane potential, pro- and anti-apoptotic proteins/genes Bcl-2, Bcl-2-associated X protein (Bax), apoptosis-inducing factor (AIF) were done respectively. RESULTS:Our results showed that ALA significantly increased testosterone secretion in primary rooster Leydig cells (P < 0.05), and 40 μM/L is the optimal dose. Leydig cells supplemented with ALA (20, 40, 80 μM) increased the expression of key enzymes and proteins 3β-hydroxysteroid dehydrogenase (3β-HSD), steroidogenic acute regulatory protein (StAR), cholesterol side-chain cleavage enzyme (P450scc) concerning steroidogenesis, enhanced antioxidant capability, improved mitochondrial biogenesis, and markedly improved the mitochondrial membrane potential (P < 0.05). Furthermore, the expression of the apoptosis-suppressive gene Bcl-2 was significantly increased, but Bax and AIF expression was decreased in the ALA group compared to that in the control group (P < 0.05). CONCLUSION:ALA promoted testosterone production, enhanced steroidogenic enzyme expression, improved mitochondrial function, and antioxidant capacity, and reduced apoptosis in primary rooster Leydig cells, with 40 μM/L identified as the optimal concentration.

SCOPE:Aging in males can lead to declines in testosterone production, essential for maintaining male reproductive health. METHODS AND RESULTS:To investigate the effects of dietary supplementation with alpha-linolenic acid (ALA) on testosterone production in aged breeder roosters and understand the underlying molecular mechanisms involved. An in vivo model is established to investigate the effects of dietary ALA supplementation on testosterone production in aged breeder roosters, and the Leydig cell culture is used to identify the potential molecular mechanism. Dietary supplementation with ALA increases in plasma testosterone. Congruently, ALA supplementation enhances the expression of testosterone biosynthesis-related enzymes. ALA supplementation exerts anti-apoptotic effects in testicular mitochondria, as evidenced by a lower expression of pro-apoptotic factors and a higher expression of the anti-apoptotic factor B-cell lymphoma 2 (Bcl-2). Moreover, In Leydig cells, ALA supplementation promotes mitochondrial biogenesis genes. The proposed mechanism is that ALA activates the sirtuin1 (SIRT1) pathway and is supported by higher SIRT1 transcript and protein in Leydig cells. Furthermore, blocking SIRT1 with siRNA reverses ALA's effects on testosterone biosynthesis and mitochondrial function-related genes. CONCLUSION:These findings indicate that dietary supplementation with ALA can improve testosterone production in aged breeder roosters, possibly by modulation of mitochondrial function via activating the SIRT1 pathway.

Chickens play a crucial role as the primary global source of eggs and poultry, and the quality of rooster semen significantly impacts poultry reproductive efficiency. Therefore, it is imperative to comprehend the regulatory mechanisms underlying sperm development. In this study, we established transcriptome profiles of lncRNAs, miRNAs, and mRNAs in 3 testis tissues and 3 epididymis tissues from “Jing Hong No.1” roosters at 24, 35, and 64 weeks of age. Using the data, we conducted whole transcriptome analysis and constructed a ceRNA network. We detected 10 differentially expressed mRNAs (DEmRNAs), 33 differentially expressed lncRNAs (DElncRNAs), and 10 differentially expressed miRNAs (DEmiRNAs) in the testis, as well as 149 DEmRNAs, 12 DElncRNAs, and 10 DEmiRNAs in the epididymis. These genes were found to be involved in cell differentiation and development, as well as various signaling pathways such as GnRH, MAPK, TGF-β, mTOR, VEGF, and calcium ion pathways. Subsequently, we constructed two competing endogenous RNA (ceRNA) networks comprising DEmRNAs, DElncRNAs, and DEmiRNAs. Furthermore, we identified four crucial lncRNA-mRNA-miRNA interactions that govern specific biological processes in the chicken reproductive system: MSTRG.2423.1-gga-miR-1563-PPP3CA and MSTRG.10064.2-gga-miR-32-5p-GPR12 regulating sperm motility in the testis; MSTRG.152556.1-gga-miR-9-3p-GREM1/THYN1 governing immunomodulation in the epididymis; and MSTRG.124708.1-gga-miR-375-NDUFB9/YBX1 controlling epididymal sperm maturation and motility. Whole transcriptome sequencing of chicken testis and epididymis screened several key genes and ceRNA regulatory networks, which may be involved in the regulation of epididymal immunity, spermatogenesis and sperm viability through the pathways of MAPK, TGF-β, mTOR, and calcium ion. These findings contribute to our comprehensive understanding of the intricate molecular processes underlying rooster spermatogenesis, maturation and motility.

Background: Reproductive performance is a crucial aspect of poultry production and is carefully controlled by endocrine, paracrine, and autocrine factors. This study aimed to investigate the effect of lycopene on testosterone synthesis in Leydig cells of laying breeder roosters, clarify the mechanism of lycopene improving Leydig cells function and promoting testosterone production, and explore the role of related signal transduction pathways in testosterone synthesi Results: A total of 96 healthy 55-week-old breeding roosters were randomly assigned to one of five dietary treatments. They were provided with a corn-soybean meal-based diet containing different levels of lycopene: 0 mg/kg (control), 50 mg/kg, 100 mg/kg, or 200 mg/kg. The experiment lasted for 6 weeks. With the increase in lycopene levels, the testosterone content in the plasma was significantly higher than in the control group. Testicular Leydig cells were isolated and cultured from fresh testicular tissue of 45-wk-old to 60-wk-old breeding roosters. Various doses of lycopene were administered to Leydig cells, and subsequently, cells were collected for the detection of cell viability and testosterone content. The optimal concentration of lycopene to be added was determined, and changes in mRNA expression and protein levels of key proteins involved in testosterone synthesis were investigated. The results showed that lycopene treatment significantly increased testosterone secretion, mRNA expression, and protein levels of steroid-producing enzymes. Cells were collected to measure the activity of antioxidant enzymes, the mRNA transcription level of apoptotic factors, and the protein expression of apoptotic factors after treatment with lycopene. The results showed that lycopene significantly increased the activities of antioxidant enzymes, and the ability to inhibit oxygen radicals, and decreased the content of malondialdehyde. Apoptosis was inhibited by regulating the expression of apoptosis-inducing and anti-apoptosis factors. After that, the MAPK signaling pathway and downstream SF-1, Nrf2 gene, and protein expression levels were detected. The results showed that lycopene treatment significantly increased the gene and protein expression of JNK, , SF-1 , and Nrf2, , and significantly decreased the gene and protein expression of p38. . Conclusions: Lycopene treatment could promote testosterone synthesis of testicular Leydig cells by activating MAPK-SF-1 (increasing steroid-producing enzyme level) and MAPK-Nrf2 pathways (resisting oxidative damage).

This study evaluated the effects of selenium-enriched yeast (SY) supplementation at various levels on health and production parameters in laying hens, including egg production, egg quality, selenium (Se) concentrations in eggs, liver health, serum biochemical markers, antioxidant function, and immune responses. A total of 360 Hy-Line Brown hens (28 weeks old) were randomly assigned to four dietary groups with six replicates of 15 birds each, monitored over a 12-week feeding trial after a two-week acclimatization period. The dietary groups included a control (basal diet without selenium) and three SY-supplemented groups with Se levels of 0.3 mg/kg (SY03), 1.5 mg/kg (SY15), and 6.0 mg/kg (SY60). The results showed no significant effects of dietary SY on laying performance or feed efficiency (P > 0.05). However, the SY15 group showed significant improvements in egg quality, particularly in albumen height, Haugh Unit and yolk color (P < 0.05). Selenium concentrations in eggs, albumen, and yolk increased dose-dependently, with significant differences in the SY-supplemented groups (P < 0.001). Increased activities of liver enzymes including alanine transaminase, alkaline phosphatase, and aspartate transaminase, alongside elevated levels of uric acid were notable in the SY60 group (P < 0.05). In addition, histological analysis revealed significant hepatocyte degeneration and a higher liver organ index (P < 0.05), in the SY60 group. All of which suggests potential liver toxicity at higher selenium levels. Antioxidant capacity of the birds were significantly enhanced due to dietary supplementation of SY as indicated by increased serum levels of total antioxidant capacity, and activities of catalase, glutathione peroxidase, and superoxide dismutase (P < 0.05). Analysis of hepatic genes expression revealed that SY15 supplementation significantly upregulated key antioxidant-related genes (Nrf2, HO-1, CAT, and NQO1) and downregulated Keap1 expression (P < 0.05), suggesting strong activation of the antioxidant defense system. In conclusion, SY supplementation at 1.5 mg/kg improved egg quality, increased Se concentrations in eggs, and enhanced antioxidant capacity without affecting laying performance or liver health. This makes it a balanced approach to improving egg quality and poultry health. However, higher supplementation levels (6.0 mg/kg) resulted in liver damage, underscoring the importance of careful dosage consideration.

Cryopreservation causes higher reactive oxygen species (ROS) concentrations, leading to oxidative stress and lipid peroxidation damaging sperm, and using antioxidants can improve semen quality after freeze-thaw. Natural astaxanthin (ASTA) can be inserted into cell membranes and its antioxidant properties are stronger than other antioxidants. We aimed to investigate the effects of ASTA supplementation in the Beltsville Poultry Semen Extender (BPSE) on post-thaw rooster semen quality and to explore the potential mechanism of rooster semen quality change. The qualifying semen ejaculates collected from 30 adult male Jinghong No. 1 laying hen breeder roosters (65 wk old) were pooled, divided into four aliquots, and diluted with BPSE having different levels of ASTA (0, 0.5, 1, or 2 μg/mL). Treated semen was cryopreserved and kept in liquid nitrogen. The entire experiment was replicated three times independently. Sperm viability, motility, curvilinear velocity, amplitude of lateral head displacement, straightness, plasma membrane integrity, and acrosome integrity were observed to be highest (P < 0.05) with 1 μg/mL ASTA at freeze-thawing. Higher (P < 0.05) antioxidant enzyme (CAT-like, SOD) activities and free radical (·OH, O2.-) scavenging ability, less ROS and malondialdehyde (MDA) concentrations were recorded with the addition of appropriate concentrations of ASTA compared to control. In addition, the levels of mitochondrial membrane potential (MMP), adenosine triphosphate (ATP), and lactate dehydrogenase (LDH) in the 1 μg/mL ASTA group improved compared to the control group, and decreased the amount of AIF protein level but increased the Bcl-2 protein level (P < 0:05). Collectively, these results demonstrate that adding ASTA in the BPSE promoted rooster freeze-thaw sperm quality, which may be related to reducing ROS levels, protecting the antioxidant defense system, preventing lipid peroxidation, improving mitochondrial structural and functional integrity, and inhibiting sperm apoptosis.