Host long non-coding RNAs (lncRNAs) are emerging as critical regulators of influenza A virus (IAV) pathogenesis. Still, the functional landscape of avian host lncRNAs remains largely unexplored. In this study, we identified a novel H9N2induced transcript lncRNA, lncGVRP1, which serves as a conserved positive regulator of IAV replication across H9N2, H1N1, and H3N2 subtypes. In addition, we discovered that lncGVRP1 acts as a functional lncRNA containing a hidden open reading frame (ORF). This ORF encodes a novel 74-amino acid micropeptide, named GVRP1-ORF. Functional rescue experiments demonstrated that the enhancement of viral activity by the lncGVRP1 is strictly dependent on the peptide it encodes. Overexpression of the GVRP1-ORF recapitulated the pro-viral effect achieved by overexpressing the lncRNA. In contrast, an ORF-deleted mutant entirely failed to promote viral replication. Mechanistically, lncGVRP1 facilitates viral propagation by significantly suppressing the host type I interferon (IFN) response and downstream interferon-stimulated genes (ISGs). Furthermore, transcriptome-wide analysis indicated that lncGVRP1 modulates critical cellular machineries, including FoxO signaling and lysosomal trafficking. Collectively, our findings reveal a unique mechanism in which a lncRNA-derived micro-peptide hijacks host immunity to support viral persistence. Moreover, this micro-peptide may serve as a potential therapeutic target against influenza infection.
Dairy farms waste carries multiple bacterial species particularly Escherichia coli, a potential reservoir and disseminator of antimicrobial resistance genes (ARGs) and virulence genes (VGs) through mobilomes. We performed whole-genome sequencing and bioinformatics analysis on 64 multidrug-resistant E. coli isolates from dairy farm waste in Gansu, China, to characterize and predict the co-occurrence of ARGs and VGs on mobilomes. Bioinformatics analysis revealed that key ARGs (sul2, rmtB, blaTEM-1) and VGs (traT, iutA) were predominantly plasmidassociated. Notably, a single Tn2 transposon carrying co-localized blaTEM-1 (ARG) and clpK (VG) is direct evidence of convergence on mobile element. Prophage regions and genomic islands were also rich reservoirs, with ompT (VG) and emrE (ARG) found in 62.5% and 45.3% of isolates, respectively, within the prophage regions. Similarly, fimH (VG) and rsmA (ARG) genes were predominantly identified in 64.1% and 48.4% isolates within the genomic island regions. The convergence of ARGs and VGs associated on diverse mobilomes indicate their potential of dissemination within and between the bacterial pathogens of animal and human significance. Further, our findings highlight the urgent application of integrated One Health policies to mitigate the potential risks.
With the shift toward more ethical and sustainable egg production, the poultry industry is now increasingly shifting towards extended laying cycle. However, extending the production period raises critical concern about the health and welfare of aging laying hens (ALH). A primary challenge is progressive intestinal inflammaging, characterized by compromised barrier integrity, immune dysregulation, and gut microbiota dysbiosis. These changes collectively impair nutrient absorption, reproductive performance, and egg quality. Despite advances in genetics and management, extending production beyond 72 weeks remains challenging. This review concludes that inflammaging in ALH is driven by an agerelated shift in gut homeostasis involving intestinal barrier, immune function, microbiota dysbiosis and short chain fatty acids, and emphasized the mechanisms that promote intestinal inflammaging. Secondly, it also evaluated emerging nutritional interventions over the past decade including polyphenols, probiotics, fermented feed stuff, and enzyme supplementation, which showed promising results in restoring gut health and enhancing production performance. Based on this review we propose that substantial research gaps persist, particularly concerning the temporal dynamics of gut aging beyond 72 weeks and the long-term efficacy of nutritional interventions. Longitudinal studies are essential to clarify these aspects. Furthermore, optimizing nutritional strategies based on a deeper understanding of gut aging mechanisms offers a pathway to extend laying cycles and achieve sustainable, high-quality egg production.
Ostrich leg and toe disease causes substantial morbidity and economic losses; however, its etiology and pathological progression remain poorly understood and inadequately controlled. This study aimed to investigate pathological changes in the cartilage and subchondral bone of the metatarsophalangeal joint in ostriches with leg and toe disease, with particular emphasis on the expression of key inflammatory mediators. Eighteen 30-day-old male ostriches were divided into three groups (n=6 each): H (healthy), D1 (mild disease), and D2 (severe disease). Pathological changes were evaluated using hematoxylin-eosin (HE) and Masson's trichrome staining. The expression of IL-1 beta, MMP-7, LOXL2, and COL2A1 was analyzed at the mRNA level by quantitative reverse-transcription PCR (qRT-PCR) and at the protein level by immunofluorescence (IF) and enzyme-linked immunosorbent assay (ELISA). Joint effusion volume and metatarsophalangeal circumference were also measured. Compared with the H group, the D1 and D2 groups showed significant increases in joint fluid volume (D1 vs H, P<0.01; D2 vs D1, P<0.01) and joint circumference (D1 vs H, P<0.01; D2 vs D1, P <0.05). Histological examination revealed fragmentation of collagen fibers in the superficial cartilage, a reduction in chondrocytes in the middle layer, and loosely arranged trabeculae in the subchondral bone. IL-1 beta and MMP-7 were progressively upregulated at both mRNA and protein levels with increasing lesion severity (IL-1 beta: P <0.01; MMP-7: P <0.01). In contrast, LOXL2 and COL2A1 were progressively downregulated (LOXL2: P<0.01; COL2A1: D1 vs H, P<0.01; D2 vs D1, P<0.05). These findings demonstrate that the progressive upregulation of IL-1 beta and MMP-7, together with the downregulation of LOXL2 and COL2A1, is closely associated with lesion severity, suggesting that these mediators may play key roles in the pathogenesis of ostrich leg and toe disease and may serve as potential targets for therapeutic intervention.
There are ongoing debates regarding the taxonomy of the E. granulosus Echinococcus granulosus sensu lato (s.l.) complex, with the species status of genotypes G1/G3 (E. granulosus s.s.) and the G6-G10 cluster (E. canadensis) being particularly contested. To solve this challenge, we develop a machine learning (ML)-based k-mer method to predict genotypes of E. granulosus s.l. via the mitochondrial DNA sequence data available in GenBank. We used 7-mer sequences that represent mtDNA to reveal untapped diversity across 948 sequences of seven known genotypes of E. granulosus s.l. We evaluated the utility of varying k-mer lengths, including fixed vs adaptive lengths, for sequence comparisons. Principal component analysis (PCA) identified the most discriminative patterns, addressed the computational challenges posed by high-dimensional features and fed them into 5 distinct ML algorithms, including both conventional (LR, RF and SVM) and DL (1D-CNN and the LSTM) methods. Moreover, crucial insights into performance implications and efficiency improvements have been emphasized via 10-fold cross-validation. Our method attains approximately 95% accuracy in predicting E. granulosus s.l. genotypes. High genetic diversity within G6, G8 and G3 contributes significantly to the controversial taxonomy of the E. canadensis cluster and E. granulosus s.s., respectively. Moreover, ML can potentially compete with BLAST (the NCBI sequence alignment tool) when the BLAST-Similarity-KNN classifier is implemented on the E. granulosus mtDNA data. This study provides a novel approach for classifying E. granulosus s.l. at the species level, thereby supporting disease control activities.
Probiotics have emerged as a natural and effective alternative to antibiotics due to their relevancy to gut health and immune booster functions both in animals and humans. In this study, five indigenous Streptomyces strains-Streptomyces rochie, Streptomyces fimbriatus, Streptomyces WSN2, Streptomyces globiosporus, and Streptomyces toxytricin-were selected for molecular characterization and in vitro evaluation of their probiotic potential. These strains were isolated from native soil samples and identified using 16S rRNA gene sequencing. Each strain was screened for key probiotic traits, including resistance to pH, antimicrobial activity against common pathogens, and compatibility with standard antibiotics. The results indicated that all five strains exhibited notable tolerance to gastrointestinal-like conditions, with S. WSN2 and S. fimbriatus showing the most robust antimicrobial effects. In conclusion, the selected indigenous Streptomyces species possess promising traits to be considered as probiotic candidates, with possible applications for the improvement of human and animal health.
The uterus has a strong self-regulatory capacity that maintains immune homeostasis and self-tolerance by balancing antimicrobial defense with protection against excessive inflammation. Escherichia coli (E. coli) is the major etiological agent of bovine endometritis, which severely impairs reproductive performance and causes substantial economic losses. The alpha 7 nicotinic acetylcholine receptor (alpha 7nAChR) has been implicated in the regulation of infectious and inflammatory diseases; however, its role in E. coli-induced bovine endometritis remains incompletely understood. In this study, E. coli-infected bovine neutrophils and endometrial tissue models were established to investigate the potential regulatory role of alpha 7nAChR in infectioninduced uterine inflammation. alpha 7nAChR was highly expressed in normal bovine neutrophils and endometrial tissues, but was markedly reduced following E. coli pretreatment with PNU-282987 (an alpha 7nAChR agonist) reduced these levels, while pretreatment with MLA (an alpha 7nAChR antagonist) further enhanced them. Western blot analysis showed that alpha 7nAChR activation was associated with increased phosphorylation of JAK2 and STAT3 and reduced phosphorylation of ERK, p38, and p65. In addition, activation of alpha 7nAChR alleviated E. coli-induced bovine endometrial injury, whereas its blockade aggravated inflammatory damage. Collectively, these findings suggest that alpha 7nAChR may regulate inflammatory responses and exert protective effects in E. coli-induced bovine endometritis, possibly through the JAK2/STAT3, MAPK, and NF-kappa B pathways.
Copper (Cu), an environmental metallic pollutant, is closely associated with hepatic injury and inflammation, yet its molecular mechanisms remain incompletely understood. Although kaempferol (KAE) exhibits anti-inflammatory properties, its role in Cu exposure-induced hepatotoxicity, which is not fully clarified. In this study, we integrated animal experiments with network pharmacology and molecular docking to explore KAE's protective effects in a chicken model of Cu exposureinduced liver injury. Histopathological analysis revealed that Cu exposure induced inflammatory infiltration, hemorrhage, and collagen deposition, which were alleviated by KAE. Network pharmacology identified insulin (INS) as a key target, supported by strong binding affinity in molecular docking. Consequently, Cu exposure elevated blood glucose and upregulated INS and p38-MAPK expression, effects reversed by KAE. Further, the results showed that copper activated the p38MAPK/NF-kappa B pathway, promoting NLRP3 inflammasome assembly and caspase-1dependent pyroptosis, as evidenced by increased levels of NLRP3, ASC, caspase-1, GSDMD, N-GSDMD, IL-1 beta, and IL-18. Immunostaining confirmed upregulation of NLRP3 and N-GSDMD after Cu exposure, which KAE treatment suppressed. These findings elucidate a novel mechanism of Cu exposure-induced liver injury and highlight KAE's potential as a therapeutic agent for metal overload-related hepatotoxicity.
Given the increasing resistance to conventional acaricides, the search for plantbased therapeutic alternatives to control the cattle tick Rhipicephalus (R.) microplus has been driven. This tick is responsible for transmitting zoonotic hemoparasites and causing significant economic losses. This study aimed to validate ethnopharmacological reports on the acaricidal effects of plants traditionally used in the state of Meta, Colombia, on R. microplus teleogines and larvae, and to identify groups of secondary metabolites. Ethnopharmacological surveys were conducted, finding seventeen plant species. Eighteen ethanolic extracts were prepared from these species for in vitro evaluation of their acaricidal effect at concentrations of 10, 20, 40, 80, and 160mg/mL. In addition to performing a preliminary phytochemical analysis, adult immersion tests (AIT) and larval immersion tests (LIT) were performed observing mortality and impact on the reproductive cycle. Probit analysis was used to determine LC50 and LC90 values, and ANOVA and Tukey's post-hoc test were used to determine differences between the concentrations of the extracts and the negative (Tween-80 2%) and positive (Cypermethrin 0.015%) controls (P<0.05). When mortality in AIT and LIT was compared between the highest concentration (160mg/mL) of the extracts and cypermethrin, five and three extracts, respectively, showed higher mortality rates (P<0.05). Similarly, eight extracts had significant adverse effects on the reproductive cycle in AIT (P<0.05). The best LC50 and LC90 values were obtained in Annona muricata (AIT=20.74mg/mL and 106.04mg/mL) and Dioclea virgata (LIT=26.80mg/mL and 116.78mg/mL). Of the twelve groups of secondary metabolites evaluated in the phytochemical analysis, alkaloids, coumarins, cardiotonic glycosides, and sapogenins were the most prevalent. Seven promising plants were found for the control of R. microplus. These results confirm the value of traditional ethnopharmacological knowledge and provide evidence of the potential of plant extracts as a sustainable alternative for tick management in cattle.
Bovine endometritis is characterized by endometrial inflammation due to bacterial infection, and the endometrial epithelium is the predominant site of bacterial infiltration. YiMu-QingGong San (YMQGS) has been validated for treating bovine endometritis, but the mechanisms regarding the regulation of endometrial epithelial cell inflammation remain unclear. This study aims to elucidate the protective effects of YMQGS against inflammatory damage in bovine endometrial epithelial cells (BEND). BEND was acted on using 10 & micro;g/mL of LPS to induce cell damage. Network pharmacology suggested that YMQGS may alleviate endometritis via the TNF, NF kappa B pathway, by targeting genes such as TNF, IL-6, and IL-1(3. Experimental validation demonstrated that YMQGS suppressed LPS-induced inflammation in BEND cells, with YMQGS-H displaying a potent effect by downregulating IL-6, TNF-alpha, and IL-1(3 mRNA expression by 66.67, 60.93, and 65.83%, respectively. Furthermore, YMQGS reversed LPS-induced oxidative stress and the disruption of the cellular cycle in BEND cells. We also found that LPS activated the TGF-(31 signaling pathway and promoted EMT in BEND cells, while YMQGS-H downregulated TGF-(31 protein expression by 33.93%, effectively rescuing the cells from this injury. Collectively, YMQGS mitigates LPS-induced BEND cell damage by suppressing inflammatory responses, oxidative stress, and EMT, thereby providing novel insights into its mechanisms for treating bovine endometritis.
Chinese indigenous pig breeds are generally more susceptible to Mycoplasma hyopneumoniae (Mhp), however, the underlying molecular mechanisms remain poorly understood. At slaughter, Ningxiang pigs (n=77) exhibited a 2.6-fold higher Mhp detection rate and a 1.5-fold higher average Goodwin (GW) score than Duroc & times;Landrace & times;Yorkshire (DLY) pigs (n=83), (P<0.01). Here, we demonstrated that genetic variation at position 2906 of the NOD-like receptor family pyrin domain containing 3 (Nlrp3) gene in Ningxiang pigs (as compared to DLY pigs) was associated with reduced phagocytic efficiency of Porcine Alveolar Macrophages (PAMs) against Mhp, primarily by suppressing the expression of Nlrp3 and interleukin-1/beta (Il-1/beta), thereby compromising host immune defense and enhancing susceptibility to Mhp infection in Ningxiang pigs. Genetic sequencing revealed that all PAMs derived from Ningxiang pigs harbored a Guanine (G) allele at position 2906 of the Nlrp3 gene, whereas 88.64% of DLY pig-derived PAMs carried an Adenine (A) allele at this locus. Site-directed mutagenesis of Nlrp3-2906G PAMs to Nlrp3-2906A PAMs by CRISPR-Cas9 was performed to investigate the functional consequences. Transcriptomic analysis revealed that the p.Q969 (A allele) mutation significantly upregulates Nlrp3 mRNA expression. Transcriptomic analysis and ELISA validated that this improvement was mediated by upregulated expression of Nlrp3 and Il-1/beta. Furthermore, functional assays demonstrated that the A allele enhanced phagocytic efficiency against pathogens by an average of 1.8-to 3.8-fold (P<0.01). Mhp Real-time fluorescence quantitative PCR (qPCR) and fluorescence tracing assays demonstrated the phagocytic efficiency of PAMs derived from Ningxiang pigs against Mhp was approximately 2.65-fold lower than that in DLY pigs (P<0.01), indicating a significant impairment in Mhp clearance. Collectively, these results suggest that the Nlrp3-2906G variant enhances Mhp susceptibility in Ningxiang pigs by impairing PAM-mediated microbial clearance, revealing a novel genetic mechanism that sheds light on the specific susceptibility of pigs to Mhp.
Mesenchymal stem cells (MSCs), especially those derived from gonadal tissues that are easy to collect during routine sterilization procedures, provide an ethically viable source of feline MSCs. Considering MSCs are potential candidates for regenerative medicine, reporting therapeutic effects suggests that further research to develop more effective therapy using feline gonadal MSCs is needed for clinical application. Gene expression analysis and therapeutic potential of MSCs can be analyzed using quantitative real-time polymerase chain reaction (qRT-PCR). Reliable interpretation of results requires accurate normalization. Hence, this study focused on determining the highest stability of candidate reference genes and accurate gene expression determination in ovary-derived MSCs (O-MSCs) and testis-derived MSCs (T-MSCs). O-MSCs and T-MSCs were retrieved from three female and three male felines, and then their stemness properties, which included spindle-like morphology, specific surface markers, and trilineage differentiation capacity, were characterized. To rank the nine candidate reference gene stability and pairwise variation stability, geNorm and NormFinder were used. In terms of overall analysis, TBP and HPRT1 had the lowest stability values (<0.03), whereas GUSB, RPL7, and ACTB had higher stability values (>0.04). When stable reference genes were utilized for normalization, statistically significant differences in OCT4 expression were observed between O-MSCs and T-MSCs (P<0.05), which were not observed with unstable reference genes. This provides a methodological framework by which future qRT-PCR-based studies can be conducted on feline gonadal MSCs. The potential therapeutic applications of these cells can also be studied.
Sexual development disorders in animals are congenital disorders and have significant genetic and clinical implications. This study reports the case of a sixmonth-old hornless Creole goat with true hermaphroditism. The clinical examination revealed an ambiguous phenotype, with the presence of a vulva, prominent clitoromegaly (4.5cm in length), a scrotum, and two testicles in normal position. The necropsy confirmed the presence of the testicles along with a complete M & uuml;llerian tract (uterus and vagina). Male gonads measured 3.5 and 3.6cm in length for the right and the left, respectively. The right and left ovaries measured 1.6 and 2.5cm, respectively; they did not contain follicles or corpus luteum. Pyometra was present. Hematology revealed mild leukocytosis (17.41 & times;103/& micro;L), supporting the presence of a localized chronic inflammatory condition. This case highlights the complexity of this reproductive developmental disorder. Blood testosterone level of 0.068ng/mL confirmed the endocrine functionality of the testes, while baseline progesterone levels (0.566ng/mL) ruled out the presence of cyclic ovarian tissue. It was concluded that the case was a true hermaphroditism.
While molybdenum (Mo) is essential for biological processes, overexposure can result in liver failure. Cadmium (Cd) is widely present in the environment, which interferes with the normal metabolic processes within cells. Mitochondria are the main target organ of heavy metal toxicity, and the coping mechanism of mitochondria under Mo and Cd co-exposure is still worthy of further study. This study aimed to decipher the mitochondrial quality control response in sheep hepatocytes under Mo and Cd co-exposure. Using an In vitro model, we found that Mo/Cd induced mitochondrial ultrastructural damage, dysfunction (elevated reactive oxygen species (ROS), reduced mitochondrial membrane potential (MMP) and adenosine triphosphate (ATP)), and disrupted dynamics by promoting fission proteins dynaminrelated protein 1 (Drp1) and fission 1 (Fis1) while suppressing fusion proteins optic atrophy 1 (OPA1) and mitofusin 2 (Mfn2), thereby triggering excessive mitophagy. Interestingly, inhibiting mitophagy with cyclosporin A (CsA) exacerbated the injury, whereas targeting the mitochondrial porin Voltage-dependent anion channel 1(VDAC1) with VBIT-4 (a small-molecule VDAC1 inhibitor) attenuated both mitophagy and dysfunction. VBIT-4 restored membrane potential, rebalanced dynamics, and improved energy production, indicating that VDAC1-mediated mitophagy acts as a pivotal adaptive mechanism. Our findings propose VDAC1 inhibition as a promising strategy to alleviate Mo/Cd-induced hepatotoxicity by restoring mitochondrial homeostasis.
Severe fever with thrombocytopenia syndrome virus (SFTSV), a novel Phlebovirus within the family Phenuiviridae, is the causative agent of severe fever with thrombocytopenia syndrome (SFTS), a tick-borne zoonotic disease. Raccoon dogs (Nyctereutes procyonoides) have been identified as potential reservoirs of SFTSV. Interferon-induced transmembrane protein 3 (IFITM3) plays a critical role in the host antiviral response and has been implicated in restricting SFTSV infection. This study investigated the association between IFITM3 gene polymorphisms and susceptibility to SFTSV infection in raccoon dogs. Genotype, allele, and haplotype frequencies were compared between healthy and SFTSV-infected animals. In addition, in silico programs were used to evaluate the functional impact of a 3 ' untranslated region (UTR) single-nucleotide polymorphism (SNP) (c.447+34G>A) and a nonsynonymous SNP (c.52C>T, P18S). Furthermore, the 3D structure modeling was performed to assess structural alterations associated with the P18S variant. A significant difference in genotype frequency of the c.447+34G>A SNP was observed between healthy and SFTSV-infected raccoon dogs. RNAfold and CentroidFold predict that this SNP (c.447+34G>A) affects RNA structure and energy. Functional predictions from PolyPhen-2 and SIFT indicated that the c.52C>T (P18S) substitution may be deleterious, although E-SNPs & GO classified it as benign. Structural modeling suggested that the P18S variant alters local hydrogen bonding, potentially affecting protein stability and flexibility. This study presents the first genetic association analysis of IFITM3 polymorphisms in raccoon dogs. Our findings suggest a potential link to SFTSV susceptibility, emphasizing the potential role of host genetic variation in modulating SFTSV susceptibility.
Giardiasis, caused by the protozoan Giardia lamblia, is a prevalent global diarrheal disease associated with significant intestinal pathology, malabsorption, and systemic complications. Current treatments, such as metronidazole (MZ), are limited by side effects and potential resistance, underscoring the need for novel, safe, and effective therapeutic agents. Purslane (Portulaca oleracea L.) is a medicinal plant rich in bioactive compounds with documented antioxidant, antimicrobial, and immunomodulatory properties. This study evaluated the therapeutic potential of an aqueous purslane extract (PE), both alone and in combination with MZ, against giardiasis using a comprehensive in vitro and in vivo approach. Antigiardial efficacy was evaluated in vitro (MTT assay on trophozoites/cysts) and in vivo using Giardiainfected mice. Albino mice were divided into seven groups: uninfected control, infected control, PE-treated (50, 100, 200mg/kg diet), MZ-treated (200mg/kg), and a PE+MZ combination (200 mg/kg each). LC-MS identified major flavonoids (luteolin-7-O-glucoside, apigenin-7-O-glucoside), phenolic acids, and betalains in PE. PE exhibited substantial DPPH scavenging activity (IC50 = 58 & micro;g/mL) and notable antimicrobial effects. In vitro, PE showed potent antigiardial activity (trophozoite IC50 = 38.2 & micro;g/mL), surpassing MZ (IC50 = 86.5 & micro;g/mL), and induced a synergistic effect in combination. PE also triggered a severe oxidative stress response in the parasite, upregulating antioxidant genes more intensely than MZ. In vivo, PE treatment caused a dose-dependent reduction in cyst shedding, with the highest dose (200mg/kg) achieving 98.7% clearance. It fully restored growth performance in infected mice (body weight, ADG, FER), normalized serum biochemical and inflammatory markers, and enhanced local immunity. PE significantly downregulated intestinal apoptotic caspases and, in combination with MZ, provided the most complete histopathological recovery of duodenal architecture, reversing villous atrophy, brush border disruption, and mucosal inflammation while supporting immune integrity. Purslane extract demonstrates potent, multi-faceted efficacy against Giardia lamblia. Its mechanism involves direct anti-parasitic activity, induction of lethal oxidative stress in the parasite. The synergistic interaction with metronidazole highlights PE's potential as a powerful adjunct therapy, offering a strategy to enhance parasite clearance, accelerate mucosal healing, improve host defense, and optimize clinical outcomes in giardiasis.
This study investigated the effects of dietary supplementation with different levels of Broussonetia papyrifera (BP) leave on growth performance, serum antioxidant status, and serum metabolites in broilers. A total of 240 one-day-old broilers were randomly allocated to four treatments with three replicates of 20 birds each in a single-factor design. Birds in the control group were fed a basal diet, while those in groups B1, B2, and B3 were fed diets in which soybean meal was replaced with 4, 8 and 12% BP leaves, respectively. After a 42-day feeding trial, blood samples were collected from the subclavian vein for serum analysis. Results showed that 4% BP leaves had no adverse effect on broiler growth performance and improved immune organ indices compared with the control, whereas higher inclusion levels reduced growth performance. The 4% BP leaf group exhibited significantly increased serum T-AOC, GSH-PX, and CAT levels. Both 4% and 8% BP leaf groups presented higher SOD activity and lower MDA content than the control. Overall, 4% and 8% BP leaf supplementation (B1, B2) exerted favorable antioxidant and anti-damage effects and were considered optimal levels. LC-MS/MS-based untargeted metabolomics revealed that alpha-ketoglutarate, L-lysine and other key differential metabolites were notably enriched in pathways including alanine, aspartate and glutamate metabolism, and the tricarboxylic acid cycle. Metabolic regulation was relatively weak in the 4% BP leaf group, whereas the 8% group showed the most significant pathway enrichment and optimal metabolic balance among energy generation, amino acid utilization, and antioxidant capacity. Conversely, metabolic pathway disturbance was observed in the 12% group.
Natural zeolite has been proposed as a dietary additive to enhance broiler performance through modulation of the gastrointestinal environment, detoxification, and improved nutrient utilization. This study evaluated the impact of graduated addition of natural zeolite on growing performance, digestive enzyme activity, serum biochemistry, antioxidant status, hematological parameters, humoral immunity, and detection of NRF2 and TGF-beta 1 genes in broiler chickens. Three hundred one-day-old unsexed broiler chicks were divided into four food categories at random for 40 days: a control group and three groups supplemented with natural zeolite at 5, 10, and 15 g/kg diet. Zeolite supplementation significantly improved growth performance in a dose-dependent manner, as evidenced by increased live weight and body weight growth, reduced feed consumption, improved conversion of feed rate, and enhanced European Production Efficiency Factor (P<0.05). Digestive enzyme activities (amylase, lipase, and trypsin) were significantly increased (P<0.05). Serum biochemical analysis revealed improved protein metabolism (increased total protein and albumin) and reduced metabolic waste indicators (uric acid and bilirubin) (P<0.05). Antioxidant status was enhanced, with increased total antioxidant capacity and antioxidant enzyme activities (SOD, GSH-Px, and catalase), alongside reduced lipid peroxidation (MDA) (P<0.05). Moreover, the presence of NRF2 and TGF-beta 1 genes was confirmed in all experimental groups, indicating their potential involvement in antioxidant defense and immune regulation. Hematological parameters showed significant increases in hemoglobin, red blood cell count, and packed cell volume (P<0.05), while white blood cell counts remained stable. Humoral immunity was also improved, as indicated by increased IgM and IgA levels, with IgY showing optimal response at moderate inclusion levels (P<0.05). In conclusion, graded dietary supplementation of natural zeolite improves growth performance, antioxidant capacity, hematological status, and immune function in broiler chickens, potentially through enhancement of intestinal health, nutrient utilization, and modulation of antioxidant and immune-related pathways.
To elucidate the specific role and mechanism of IGF2 (a strongly selected gene in egg-production traits identified in our previous genome resequencing of Chinese Jinding ducks) in duck ovaries, this study investigated the expression and distribution of IGF2, assessed its impact on the proliferation of duck ovarian granulosa cells (GC), and elucidated the underlying regulatory mechanisms. The expression of IGF2 mRNA in the ovary was significantly higher than in all other tissues (P<0.05), being 5.69 times that in the liver the tissue with the second-highest expression. Its protein was mainly located in GC, oocyte cytoplasm and vascular endothelium of duck follicles. In vitro experiments demonstrated that IGF2 enhanced the proliferation of duck GC. Further mechanistic analysis showed that IGF2 promoted the phosphorylation of PI3K and AKT, as well as the expression of downstream CCND3 gene, a cell cycle regulation gene. These findings suggested that IGF2 regulated duck GC proliferation through the PI3K/AKT/CCND3 pathway, thereby affecting the laying traits of ducks. These results revealed a regulatory role of IGF2 in duck follicular development via the PI3K-AKT pathway and extended the existing understanding of IGF2 function in ducks.
Canine mammary tumors (CMTs) are the most prevalent neoplasms in female dogs; however, the diagnosis and prognosis of CMTs show a significant challenge in veterinary oncology. This study evaluates alpha-fetoprotein (AFP) and phosphodiesterase 4D (PDE4D) as possible biomarkers for the progression of CMTs, in correlation with the proliferation marker Ki-67. The protein expression was studied across the CMTs spectrum which were normal, adjacent, benign, and malignant tissues by using immunohistochemistry (IHC) and western blotting (WB). Quantitative IHC demonstrated that both AFP and PDE4D levels increased in a stage-dependent manner. AFP levels increased gradually from normal to benign and highest in malignant CMTs, indicating that it is an early and persistent marker of neoplastic transformation. PDE4D exhibited a comparable pattern, indicating its role in sustaining cancer cell viability. WB demonstrated that both proteins exhibited similar higher expression in benign and malignant CMTs, revealing differences between total protein and in situ localization. Correlation analyses showed a significant positive relationship between AFP and Ki-67 only in benign cases, while it disappeared in malignant CMTs. This uncoupling mechanism in malignant CMTs suggested that growth pathways beyond initial cell proliferation were involved. In conclusion, this study demonstrates a new perspective on the novel biomarkers for CMTs. While AFP acts as an early sensitive marker of dedifferentiation of mammary tissue, PDE4D serves as a definitive protein that flips during the transition to aggressive malignant CMTs. These findings establish an integration of AFP-and PDE4D into future precision diagnosis for the development of targeted therapy in veterinary oncology.
