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Background: The increasing prevalence of antifungal resistance among Candida albicans isolates necessitates the exploration of alternative antifungal strategies and novel molecular targets. Plant-derived bioactive compounds represent a promising source of antifungal agents; however, their chemical composition and mechanisms of action remain insufficiently characterized. Methods: Ethanolic extracts of Azadirachta indica leaves and Ficus benghalensis aerial roots were subjected to qualitative phytochemical screening, gas chromatography–mass spectrometry (GC–MS), and Fourier transform infrared (FTIR) analysis to characterize their bioactive constituents. Molecular docking studies were performed to evaluate the interaction of selected phytochemicals with C. albicans N-myristoyltransferase (PDB ID: 1IYK), a validated antifungal drug target. Results: Phytochemical screening revealed the presence of alkaloids, carbohydrates, and phenolic compounds in both extracts, while saponins were detected only in A. indica. GC–MS analysis demonstrated greater chemical diversity in A. indica, including fatty acids and phenolic derivatives, compared with F. benghalensis, which was dominated by terpenoids and long-chain hydrocarbons. FTIR analysis confirmed functional groups relevant to protein–ligand interactions. Molecular docking showed favorable binding interactions between several A. indica–derived compounds and N-myristoyltransferase, whereas compounds from F. benghalensis did not exhibit detectable binding under the conditions tested. Conclusions: The integrated phytochemical and in silico analyses provide insight into the antifungal potential of A. indica, suggesting possible interactions between identified phytochemicals and the selected drug target protein. This study highlights the value of combining chemical characterization with target-based computational screening in the rational exploration of plant-derived antifungal agents. Full article

Triploid fish have attracted considerable attention in aquaculture because of their sterility and rapid growth. In this study, a novel allotriploid fish (WA, 3n = 150) was obtained through distant hybridization of a female diploid hybrid fish (WR-II, 2n = 100) with a male allotetraploid fish (4nAT, 4n = 200). The measurable traits of WA exhibit significant differences from WR-II and its original parents (white crucian carp (WCC) and red crucian carp (RCC)). WA muscles exhibited higher protein, fat, and essential amino acid levels but lower moisture. Furthermore, WA enhanced growth performance and muscle hypertrophy compared to WCC and RCC. The transcriptomes and metabolomics of the muscle tissues of WA, WCC, and RCC were analyzed. In total, 3889 and 1863 differential expressed genes (DEGs) were identified in WA vs. WCC and WA vs. RCC, respectively. These DEGs were mainly enriched in pathways related to amino acid biosynthesis and oxidative phosphorylation. In total, 421 and 304 differential metabolites (DMs) were identified in WA vs. WCC and WA vs. RCC, respectively. These DMs were mainly enriched in the pathways related to glycerophospholipid and fatty acid metabolism. Integrated transcriptomic and metabolomic analyses revealed that the expression patterns of acta1, myh, myog, and aldoa were associated with changes in the abundance of oleoyl ethanolamide, betaine, inosine, docosahexaenoic acid (DHA), and PC (38:6), suggesting their potential involvement in the enhanced growth performance of WA. This study provides a potential genetic resource for aquaculture and identifies molecular features associated with enhanced growth performance in this allotriploid fish. Full article

The growing demand for high-quality beef has driven the selection of bovine genotypes offering superior marbling and tenderness. This study evaluated meat quality parameters in heifers from four Nellore-based genetic groups: ½ Aberdeen Angus × ½ Nellore (AN), ½ Japanese Brown × ½ Nellore (BN), ½ Japanese Brown × ¼ Aberdeen Angus × ¼ Nellore (TriBAN), and ½ Japanese Black × ¼ Aberdeen Angus × ¼ Nellore (TriJAN). Thirty-two heifers (eight/group) were finished in commercial feedlots. Samples of the longissimus thoracis muscle were analyzed for physical attributes (pH, cooking loss, shear force, marbling, water retention capacity, and colour), chemical composition, and fatty acid profile. In addition, animal performance (daily gain and final weight) was determined. A completely randomized design was used with analysis of variance followed by Tukey’s test (α = 0.05). The AN group showed the greatest final weight and daily gain (p < 0.01). There were no significant differences among genetic groups for pH, cooking loss, shear force, marbling, water-holding capacity, or colour (p > 0.05). Across all groups, the mean ultimate pH was 5.59, shear force (WBSF) averaged 41.11 N, and intramuscular fat (ether extract) content averaged 13.15 g 100 g⁻¹, indicating desirable technological and eating-quality characteristics. Most fatty acids did not differ among treatments (p > 0.05), except for arachidonic acid (C20:4 n-6), which was present at higher concentrations (p < 0.05) in the TriBAN group than in the other genetic groups. In conclusion, despite differences in productive performance, all genotypes produced meat with consistent quality traits, including tenderness, marbling, and fatty acid profiles associated with high-quality beef. Full article

Sex-related differences in lipid metabolism and oxidative stress are recognized as important modulators of cardiovascular physiology, but they are still insufficiently explored in large animal models. This study examined the influence of sex and gonadal status on oxidative stress, lipid metabolism, and hematological parameters in clinically healthy domestic pigs. Twenty sexually mature pigs (10 months old) were divided into four groups: boars, barrows, gilts, and ovariectomized females (n = 5 per group). In serum, the activities of glutathione peroxidase (GSH-Px), glutathione reductase (GSH-RD), total superoxide dismutase (TSOD), gamma-glutamyl transferase (GGT), and lactate dehydrogenase (LDH), together with concentrations of triglycerides, phospholipids, total cholesterol, high-density lipoprotein cholesterol (HDL cholesterol), low-density lipoprotein cholesterol (LDL cholesterol), non-esterified fatty acids (NEFAs), and malondialdehyde (MDA), were determined. In myocardial and liver tissues, antioxidant enzymes, lipid parameters, and MDA were analyzed. Spectrophotometric methods were used for enzyme activities and lipid parameters, while MDA was quantified by high-performance liquid chromatography. Hematological parameters were determined using an automated hematology analyzer, and serum hormone concentrations were measured by chemiluminescent immunoassays. Group-dependent differences were observed, although the relatively small sample size warrants cautious interpretation. In serum, gilts had higher phospholipid, total cholesterol, and HDL cholesterol concentrations, along with the most favorable LDL: HDL ratio, whereas boars showed the least favorable ratio. Lipid peroxidation, assessed by MDA, was lowest in gilts and highest in barrows. In myocardial tissue, gilts showed higher GSH-RD and TSOD activities and lower MDA concentrations than barrows, suggesting potentially stronger antioxidant protection. In the liver, differences were found in cholesterol, triglycerides, and MDA concentrations, with higher cholesterol in barrows, higher triglycerides in boars, and higher MDA in gilts and ovariectomized females. Hematological differences were also evident, with lower leukocyte and lymphocyte counts in gilts and higher platelet count and plateletcrit in barrows and ovariectomized females. Overall, sex and gonadal status were associated with distinct patterns of metabolic and oxidative responses across serum and tissues. Given the small sample size and cross-sectional design, these findings should be interpreted cautiously and as associative rather than causal. Full article

Fructooligosaccharides (FOSs) are a class of functional prebiotics that effectively modulate the composition of the intestinal microbiota and regulate metabolic processes in animals. This study aimed to determine how early FOS supplementation affects jejunal development, which is vital for the rapid growth of suckling piglets. Sixty newborn piglets (Duroc × Landrace × Large White) from six litters were randomly assigned to either a control (CON) or an FOS group (n = 5 per litter per group). From days 2 to 21, piglets in the FOS group received 5 mL of an FOS solution orally (3 g/day/piglet), while those in the CON group received an equal volume of saline. Piglets were weighed on days 1, 7, 14, and 21, and samples were collected on days 7, 14, and 21. Early FOS intervention can enhance the growth performance of suckling piglets (p < 0.01), which is related to the development of the jejunum. Specifically, it improved intestinal morphology, stem cell function, and nutrient transport by increasing mRNA and protein expression of key genes in the Wnt/β-catenin signaling pathway. This may be related to increased abundance of jejunal Lactobacillus and the concentrations of metabolites such as lactate and short-chain fatty acids (SCFAs). Early FOS intervention drove jejunal development through Wnt/β-catenin activation, a process mediated by microbiota-metabolite shifts that augmented intestinal stem cell renewal. Full article

Despite the pivotal importance of soil microbial communities in regulating the terrestrial carbon (C) cycle and ecosystem functioning, their vertical patterns and their responses to long-term nitrogen (N) deposition, in terms of microbial biomass and relative abundance, still remain unclear, particularly for fungi versus (vs.) bacteria and Gram-positive (GP) vs. -negative (GN) bacteria, where they traditionally have contrasting biological characteristics. In this study, we investigated the responses of GP and GN bacteria, total bacteria (TB), and fungi (TF) and their relationships with four nutrient ratios to 10 years of N addition with three N addition rates (control, N2, and N10 representing 0, 2, and 10 g m⁻²yr⁻¹, respectively) across a 100 cm soil profile (0-10-20-30-40-70-100 cm) in a semiarid grassland, both in terms of absolute biomass and relative abundance represented by the amount and percentage of phospholipid fatty acids (PLFAs) (e.g., GP bacterial PLFAs: total PLFAS) of specific microbial groups, respectively. Our results showed that N addition has significant negative effects on the biomass of these four microbial groups across the whole soil profile, while N addition significantly decreased the relative abundance of GN bacteria and increased those of TF and GP bacteria. Additionally, N addition significantly decreased the biomass of TF and GP bacteria in 0–10 cm soil, and of TB and GN bacteria in 0–10, 40–70, and 70–100 cm soils, with the significant increase or decrease mainly occurring at the N10 rate. Our results also showed that the biomass of these four microbial groups decreased with soil depth regardless of N addition, but that of TB and GN bacteria decreased faster than that of TF and GP bacteria did, and further caused the relative abundance of TF and GP bacteria to increase with soil depth. Significant Pearson correlations among the biomass and relative abundance of these microbial taxa with soil pH, total phosphorus, NH₄⁺-N, and NO₃⁻-N were mainly found in 0–10 cm soil. C:N and NH₄⁺-N: NO₃⁻-N ratios increased with increasing soil depth, while C: phosphorus (P) and N:P ratios decreased. N addition significantly increased C:P and NH₄⁺-N: NO₃⁻-N ratios and decreased N:P ratios, especially under the N10 rate. Among these four nutrient ratios, N:P significantly explained the highest variations in F:B (76%) and GP:GN (38%) across the whole soil profile. Together, these results underline the different responses of soil microbial relative abundance and biomass along the soil profile under long-term N addition. They also suggest a relationship between soil microbial diversity and biomass relationships, and how this links to nutrient ratios in the subsoil needs to be investigated in future. Full article

Chronic cyclic heat stress impairs thermoregulation, growth, tissue integrity, microbial metabolism, and stress-related molecular responses in broiler chickens. N-acetyl-L-cysteine, a cysteine donor involved in glutathione synthesis, has been proposed as a nutritional strategy to support stress adaptation. Therefore, this study evaluated NAC in 324 Ross 308 broilers allocated to thermoneutral conditions (TN, 7 pens), HS (HS, 10 pens), or HS plus 2000 mg/kg NAC (HS + NAC, 10 pens). From day 20 to 35, HS birds were exposed to 34 °C for 7 h/day and 26 °C otherwise, while TN birds were kept at 22 °C. Heat stress increased rectal temperature and respiratory rate (p < 0.001), reduced average daily gain, and increased drinking and gasping behavior (p < 0.05). Heat stress also increased hepatic cord disorganization, cerebral vacuolisation–necrocytosis, hepatic and cerebral HSP70 expression, and caecal propionate, while reducing cerebral apelin expression (p < 0.05). Jejunal villus morphology and microbiota α-diversity were unchanged, although caecal profiles indicated treatment-related shifts and potentially higher Bilophila spp. in heat-stressed birds. NAC did not improve growth, thermoregulation, oxidative status, caecal fermentation, microbial structure, or HSP70 expression. It increased jejunal villus fusion and hepatocellular vacuolation scores and reduced cerebral ghrelin expression (p < 0.05). In conclusion, heat stress impaired growth and induced tissue, microbial, fermentative, and cerebral gene-expression changes, whereas 2000 mg/kg NAC did not provide measurable protection under the chronic cyclic HS in the present study. Full article

Frozen mysid shrimp is the core feed for commercial seahorse aquaculture, yet quality deterioration caused by long-term cold storage severely constrains the survival and reproductive performance of seahorses. By integrating targeted metabolomics, physicochemical spoilage indicators, and 16S rRNA high-throughput sequencing, this study multidimensionally analyzed the stage-specific mechanisms of nutritional degradation and microecological deterioration of mysid feed at −20 °C under fresh (0 months), short-term frozen (2 months), and long-term frozen (10 months) conditions. The results demonstrate that short-term storage (2 months) effectively suppresses microbial-induced spoilage of mysids; however, freeze–thaw stress triggers cellular lysis, leading to a significant depletion of key water-soluble feeding-attractant amino acids, notably glycine and aspartate. In contrast, long-term frozen storage (10 months) induces severe lipid peroxidation and a massive accumulation of total volatile basic nitrogen (TVB-N). Microbiome analysis confirmed that the fundamental trigger for feed deterioration during the late storage stage is the explosive community succession of psychrotrophic specific spoilage organisms (SSOs), with Shewanella, Photobacterium, and Pseudoalteromonas emerging as the absolute dominant taxa. The highly active extracellular lipases and proteases secreted by these microbial communities extensively degrade the structural lipids of the feed, ultimately resulting in a paradoxical rebound in free fatty acid content during long-term storage. In summary, our research comprehensively details how freezing compromises diet quality through specific biological and chemical pathways. This establishes essential scientific groundwork aimed at refining commercial preservation techniques, executing precise dietary enrichments, and ultimately securing long-term viability across the captive Hippocampus breeding sector. Full article

Integrated rice–fish farming is increasingly promoted as a sustainable aquaculture model, yet its effects on flesh quality and gut microbial assembly in Pelteobagrus fulvidraco remain insufficiently understood. Here, we compared pond-cultured (PP) and rice-field-cultured (PR) individuals using growth measurements, muscle amino acid and fatty acid profiling, untargeted LC-MS/MS metabolomics and 16S rRNA gene sequencing. Body weight and length did not differ significantly between groups, whereas PP fish showed greater body width and height. PR fish contained higher levels of Ser, Val, Ile and total essential amino acids, together with amino acid and energy-related metabolites. In contrast, PP fish showed higher concentrations of saturated fatty acids, monounsaturated fatty acids, n-3 polyunsaturated fatty acids, total fatty acids and lipid-associated metabolites. The gut microbiota also differed markedly between culture systems. PP fish exhibited higher α-diversity and enrichment of Proteobacteria, Firmicutes, Pseudomonas and Ralstonia, whereas PR fish were characterized by Verrucomicrobiota, Fusobacteriota, Akkermansia and Cetobacterium. Predicted functions suggested enhanced carbohydrate and energy metabolism in PR fish and stronger environmental-response-related functions in PP fish. These findings indicate that pond and rice-field culture promote distinct quality-forming pathways in yellow catfish, providing a basis for optimizing rice–yellow catfish co-culture toward quality-oriented production. Full article

This study evaluated the use of finishing diets to reduce fish oil (FO) inclusion in European sea bass feeds while preserving fillet quality and n-3 fatty acid content. Fish (197 g) were fed for 25 weeks with a control FO diet, based on 9% FO and 6% plant oil, or diets where 6% FO was replaced by rapeseed oil (RO) or microalgae (AO; Nannochloropsis sp. and Schizochytrium sp.). After 13 weeks, 2 groups of fish fed the RO diet were switched to the FO or AO finishing diets for the final 12 weeks. Growth, feed efficiency, body composition, and health were unaffected by dietary treatments. However, long-term RO feeding reduced eicosapentaenoic acid (EPA) and docosahexanoic acid (DHA) and increased monounsaturated and n-6 fatty acids compared to fish fed AO or FO. A 12-wk FO finishing diets shifted morphometric traits, fatty acid profiles, liver histology, and overall fish quality of fish previously fed with RO toward those of fish continuously fed FO, whereas the AO finishing diet produced a smaller shift. Longer finishing periods may further enhance fillet quality when using microalgae-based oils. Full article

The peripartum period in prolific ewes is associated with increased metabolic demands, and dietary lipid supplementation may increase the energy density of the prepartum diet. This study evaluated the effects of prepartum supplementation with alternative lipid matrices on the physicochemical characteristics and comprehensive fatty acid (FA) profiles of mature and transitional sheep milk. Thirty-two pregnant Olkuska ewes were randomly assigned to four cohorts (n = 8/group): Control (basal diet), flaxseed oil (low n-3/high n-6 variety; FO), black soldier fly larvae oil (BSFL oil), and Mix oil (2:1 ratio of FO to BSFL oil). Dietary treatments exerted no significant effects (p > 0.05) on transitional milk composition or lipid quality indices. In contrast, in mature milk, BSFL oil supplementation significantly increased milk fat content (6.09%) compared to the Control group (4.70%; p < 0.01). Furthermore, all oil treatments significantly reduced total saturated FAs (p < 0.01), while the BSFL oil and Mix oil supplementation significantly increased the monounsaturated FA fraction (p < 0.01). The differences, particularly the increase in cis-9, trans-11 CLA concentration and the significant reduction in both the atherogenic and thrombogenic indices (p < 0.01), were observed exclusively in mature milk. These findings suggest that the combination of sustainable insect lipids with high-linoleic plant oils may represent a viable strategy to transform sheep milk into a functional food with potential cardiovascular health benefits for consumers. These findings additionally indicate that prepartum lipid supplementation may exert a persistent carry-over effect into early lactation, influencing the fatty acid composition of mature sheep milk. Full article

This study developed an ABTS-targeted sequential fermentation strategy for fermented apple beverages using Metschnikowia sp. Mp58 belonging to the M. pulcherrima clade (Mp) and Lactiplantibacillus plantarum Cro3-2 (Lp). Thirteen Metschnikowia isolates and ten Lactiplantibacillus isolates were initially screened according to their effects on ABTS radical-scavenging activity, after which Mp58 and Cro3-2 were selected for further investigation. Different fermentation strategies were compared, and sequential inoculation of Mp followed by Lp produced the highest ABTS radical-scavenging activity. Single-factor experiments, an orthogonal design, and response surface methodology were subsequently used to optimize the inoculation interval, fermentation temperature, and total fermentation time, with ABTS radical-scavenging activity as the sole response variable. The selected conditions consisted of Mp inoculation at 0 h, Lp inoculation after 25 h, and fermentation for a total of 46 h at 21.50 °C. The experimentally obtained ABTS radical-scavenging activity was 78.17 ± 0.49%, which was close to the model-predicted value. Under the selected conditions, Mp–Lp sequential fermentation significantly altered the physicochemical, bioactive, in vitro functional, and volatile characteristics of the apple beverage. Compared with the uninoculated control, total phenolic content, total flavonoid content, and ascorbic acid content increased by 73.08%, 32.97%, and 7.14%, respectively. These changes were accompanied by increases of 41.00%, 39.77%, and 37.86% in ABTS radical-scavenging activity, DPPH radical-scavenging activity, and ferric reducing antioxidant power, respectively. The Mp–Lp beverage also exhibited the highest α-glucosidase and α-amylase inhibitory activities, reaching 85.97% and 78.35%, respectively, as well as bile salt-binding capacities of 51.57% for sodium glycocholate and 54.97% for sodium taurocholate. Ethanol accumulation remained limited under the tested conditions, although concentrations below the measurement capability of the distillation–hydrometer method could not be excluded. A total of 95 volatile compounds were tentatively identified by HS-SPME-GC-MS. The sequentially fermented beverage contained a larger number of detected volatile compounds than the single-strain fermented beverages and showed treatment-associated changes in the relative distribution of esters, alcohols, and fatty acids. Exploratory PCA and OPLS-DA indicated differences in volatile composition among treatments, while nonanoic acid, ethyl hexanoate, n-decanoic acid, isophorone, and (E)-2-hexen-1-ol were identified as candidate compounds associated with group separation. These chemical changes should not be interpreted as direct evidence of improved aroma or sensory quality in the absence of sensory evaluation. Overall, Mp–Lp sequential fermentation represents a potentially useful bioprocessing strategy for modifying the bioactive properties, in vitro functional indicators, and volatile composition of fermented apple beverages. Full article

Aim: To establish and validate a quantitative transporter-mediated imaging framework based on gadoxetic acid-enhanced T1 mapping for assessing liver functional reserve (LFR) and to investigate the physiological significance of the relative change in longitudinal relaxation rate (ΔR1%) as a quantitative imaging biomarker. Methods: Female C57BL/6J mice (6–8 weeks old) representing five experimental liver conditions (control, transporter-deficient Slco1b2/Slco1a5 double-knockout, carbon tetrachloride-induced fibrosis, methionine–choline-deficient diet-induced steatohepatitis, and alcohol-associated fatty liver disease; n = 6 per group) underwent serial Gd-EOB-DTPA-enhanced T1 mapping. Quantitative ΔR1% was calculated to characterize hepatobiliary enhancement kinetics. Liver functional reserve was independently evaluated using multispectral optoacoustic tomography of indocyanine green (ICG) pharmacokinetics and serum ICG retention assays, with histopathological and hepatocellular transporter analyses performed for mechanistic validation. Longitudinal data were analyzed using restricted maximum likelihood (REML)-based mixed-effects models. Intergroup comparisons were performed using one-way ANOVA or Kruskal–Wallis tests with appropriate post hoc analyses, and associations between imaging and functional parameters were evaluated using Spearman rank correlation analysis. A two-sided p < 0.05 was considered statistically significant. Results: Five experimental liver models exhibited distinct transporter-dependent hepatobiliary enhancement patterns. The transporter-deficient knockout mice showed minimal enhancement, whereas fibrosis and steatotic liver injury models demonstrated intermediate but clearly distinguishable functional profiles. Longitudinal mixed-effects analysis identified significant effects of time, experimental group, and time-by-group interaction on ΔR1% dynamics (all p < 0.0001). Although MRI-derived ΔR1% parameters were not significantly correlated with regional optoacoustic ICG kinetics, ΔR1% area under the curve showed a strong inverse correlation with serum ICG retention at 600 s (r = −0.729, p < 0.0001), indicating that MRI-derived ΔR1% and ICG-based measurements provide complementary rather than interchangeable assessments of liver function. Histological and molecular analyses further demonstrated marked heterogeneity in fibrosis, steatosis, and hepatobiliary transporter expression across models, whereas transporter abundance alone did not consistently predict imaging-derived functional performance. Conclusions: Quantitative Gd-EOB-DTPA-enhanced T1 mapping provides a transporter-mediated imaging framework for evaluating hepatic functional reserve across mechanistically distinct liver injury models. As a normalized quantitative imaging biomarker, ΔR1% captures the integrated functional consequences of hepatobiliary transport dysfunction and complements established liver function tests. These findings support the translational potential of quantitative T1 mapping as a standardized, noninvasive approach for assessing liver functional reserve. Full article

Myocarditis is a clinically challenging form of inflammatory heart disease with heterogeneous etiologies, limited diagnostic tools, no targeted therapies, and a substantial risk of progression to heart failure or sudden cardiac death, particularly in young adults. Emerging evidence has increasingly associated myocarditis with gut microbiota dysbiosis. This review explores the gut–myocarditis axis, highlighting key mechanisms and therapeutic strategies. Significant alterations in gut microbial composition are observed in myocarditis patients and animal models. Gut microbiota influences disease development through multiple pathways: compromised intestinal barrier integrity leading to bacterial translocation and systemic inflammation via MAMP/PRR signaling (e.g., TLRs, NLRs); production of metabolites—including pro-inflammatory trimethylamine N-oxide (TMAO), anti-inflammatory short-chain fatty acids (SCFAs), and immunomodulatory bile acids—that regulate host inflammatory responses, immune cell differentiation, oxidative stress, and fibrotic remodeling; and molecular mimicry, where microbial peptides (e.g., from Bacteroides thetaiotaomicron) trigger cross-reactive autoimmune responses against cardiac proteins. Regarding therapeutic strategies, this review discusses fecal microbiota transplantation (FMT), probiotics, prebiotics, dietary modulation, and emerging approaches including engineered bacteria and oral nanomedicines. Although these strategies hold promise, their efficacy and safety remain to be validated in large-scale clinical trials, and further investigation is warranted. Full article

Background/Objectives: Circulating insulin-like peptide 3 (INSL3) is a constitutively secreted product of mature Leydig cells and is considered a stable biomarker of Leydig cell functional capacity, a key determinant of androgenic status across the male lifespan. Since the adult Leydig cell population is established during late childhood and puberty, nutritional and metabolic exposures during these periods may contribute to later variation in young adult INSL3. To investigate whether childhood and adolescent nutritional/metabolic markers, particularly vitamin D, fatty acids, amino acids, and dietary trace-element intake, are associated with circulating INSL3 concentrations in young adulthood. Methods: Data were analysed from the Avon Longitudinal Study of Parents and Children (ALSPAC), a population-based UK birth cohort. Nutritional and metabolic markers measured during childhood and adolescence included vitamin D, fatty acid-related traits, glutamine, leucine, dietary zinc intake, and dietary selenium intake. Associations with circulating INSL3 at 17 and 24 years were first explored using bivariate correlation analysis. Variables showing signals of interest were then examined in multiple linear regression models using log-transformed data and relevant covariates, including BMI at 8 years and log-transformed exact age at the age-24 clinic visit. Results: Among the variables examined, vitamin D at 9 years showed a small positive association with INSL3 at 24 years (r = 0.098, n = 735, p = 0.008), remaining below the corrected threshold after both Benjamini–Hochberg and Bonferroni correction (q = 0.032 and Bonferroni-adjusted p = 0.032). This relationship remained evident after adjustment for BMI at 8 years and after further adjustment for log-transformed exact age at the age-24 clinic visit. In these multivariable models, BMI at 8 years showed little evidence of association with INSL3 at 24 years, whereas log-transformed exact age at the age-24 clinic visit showed a negative association with INSL3. In contrast, no meaningful associations were observed for fatty-acid-related measures. Among amino acids and dietary trace-element measures, glutamine at 17 years showed a weak nominal positive association with INSL3 at 24 years, but this did not remain below the threshold after multiple-testing correction. Conclusions: Childhood vitamin D status, particularly at 9 years of age, may represent an early-life factor associated with variation in adult Leydig cell functional capacity. Fatty acid-related measures showed no clear associations, while glutamine yielded only a weak exploratory signal. These findings support the concept that the childhood metabolic environment may influence the developmental establishment of adult Leydig cell capacity. Full article