Dose-dependent effects of Cannabidiol in drinking water on yolk fatty acid profile and hepatic expression of PUFA-related genes in laying hens: insights into the CBD systemic and molecular mechanisms of action

Dose-dependent effects of Cannabidiol in drinking water on yolk fatty acid profile and hepatic expression of PUFA-related genes in laying hens: insights into the CBD systemic and molecular mechanisms of action | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Dose-dependent effects of Cannabidiol in drinking water on yolk fatty acid profile and hepatic expression of PUFA-related genes in laying hens: insights into the CBD systemic and molecular mechanisms of action Sepideh Fallahi, Agnieszka Śmieszek, Łukasz Bobak, Anna Lipińska, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7387031/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 12 Nov, 2025 Read the published version in BMC Veterinary Research → Version 1 posted 10 You are reading this latest preprint version Abstract The present research evaluated the effect of water-soluble cannabidiol (CBD) emulsion on egg yolk fatty acid profile, liver health, and gene expression in laying hens. A total of 180 Lohmann Brown Classic hens were divided into experimental groups, including a control group, a blank group (emulsifier only), and three groups receiving CBD. Cannabidiol was administered in drinking water for 15 weeks at doses of 20, 40, and 80 mg/kg body weight in GI, GII, and GIII, respectively. The analysis indicated that the highest-dose CBD significantly increased yolk polyunsaturated fatty acids (PUFAs), particularly omega-3 and omega-6, as well as the serum HDL level. However, this was accompanied by downregulation of PPAR-γ expression. The histopathology of the liver did not show any differences among the groups. The expression of fatty acid desaturation genes, such as FADS1 and FADS2 , remained stable, indicating preserved desaturation function and PUFAs biosynthesis. These results suggest that while the highest dose of CBD enhances lipid mobilization and yolk deposition, it simultaneously may affect PPAR-γ -mediated lipid pathways (e.g., adipogenic programming or lipid storage/transport) despite intact desaturation pathways. This is the first study to investigate the molecular impact of purified CBD on lipid metabolism and liver function in laying hens, explaining its potential as a functional feed additive in poultry nutrition. CBD Laying hens Yolk deposition Lipid metabolism Gene expression Figures Figure 1 Figure 2 Figure 3 Introduction Poultry health is a significant concern in the poultry industry due to concerns about animal welfare, longevity, and the quality of derived products. This is particularly relevant in terms of laying hens which are maintained for a long period due to their role in producing eggs. Eggs are a highly valued food in the diets of many people, and their quality leads to both direct and indirect effects on human health. Therefore, customers have become more conscious and selective about the products they purchase. Eggs, as a good source of essential fatty acids, are important for the proper functioning of the human body. The yolk is composed of approximately 30% lipids, making it a primary source of dietary fats [ 1 ]. Eggs were long considered as a health risk due to their high yolk cholesterol content, although more recent studies have questioned this association with cardiovascular disease, obesity, type 2 diabetes, and higher mortality rates in diabetic patients [ 2 , 3 ]. Beyond cholesterol levels, the fatty acid composition of eggs plays a crucial role in determining their overall impact on human health. Raising awareness of the health benefits of specific fatty acids in the diet has led to intensive research into options for changing the fat composition of animal products. The amount of saturated fatty acids (SFAs), monounsaturated fatty acids (MUFAs), and polyunsaturated fatty acids (PUFAs) is an important factor in lipid metabolism and cardiovascular risk [ 1 ]. Higher PUFA and MUFA, and lower SFA, in diets have been reported to mitigate the adverse effects of dietary cholesterol [ 4 , 5 ]. Furthermore, some evidence suggests that high egg consumption (i.e., one egg/day) is associated with less severe coronary atherosclerosis [ 6 ]. The egg lipid profile is dynamic and varies according to the bird’s genetics, age, feeding, and the nature and amount of fat in its diet [ 7 ]. The PUFAs are widely reported for their beneficial effects on the heart, brain, and general health; specifically, omega-3 (n-3), omega-6 (n-6), and omega-9 (n-9) [ 8 , 9 ]. The poultry industry has responded to increased consumer interest in healthier and functional foods by enhancing the fatty acid profile of eggs, particularly by increasing the content of health-promoting polyunsaturated fatty acids [ 10 ]. For this reason, eggs are now being considered not only as a source of high-quality protein but also as a functional food that possesses additional health benefits. In recent years, considerable attention has been directed towards plant additives in poultry feeding to improve poultry health, production, and welfare. These phytogenic compounds could be a natural alternative to synthetic additives and antibiotics. Cannabidiol (CBD), a non-psychoactive compound that is isolated from the hemp plant ( Cannabis sativa ), has attracted attention for its potential therapeutic effects. Research in mammals indicates that CBD exhibits anti-inflammatory, antioxidant, anxiolytic, and immunomodulatory effects [ 11 , 12 ]. This has led to investigations on the use of CBD in poultry to determine its effects on stress relief, immunity, and performance. In addition, the endocannabinoid system (ECS) is a signaling system responsible for regulating numerous physiological functions, including pain, inflammation, appetite, immune response, as well as oxidative stress reduction [ 13 ]. This system has been identified in both invertebrates [ 14 ] and vertebrates, including poultry (chicken) [ 15 ], thus highlighting its evolutionary conservation and fundamental role in maintaining physiological balance across vertebrates. The ECS consists of endocannabinoids, cannabinoid receptors (CB1 and CB2), and metabolic enzymes [ 16 , 17 ]. Endocannabinoids are bioactive lipids produced from long-chain polyunsaturated fatty acids and consist of ethers, amides, and esters [ 18 ]. CB1 receptors are localized in the central nervous system; however, they are also found in the peripheral nervous system, including the liver and adipose tissue. In contrast, CB2 receptors are found predominantly in the peripheral nervous system, e.g., in the liver, kidney, and adipose tissue. The enzymes involved in the ECS regulate the balance of endocannabinoid levels via biosynthetic and catabolic pathways [ 19 ]. The liver has a significant role in both human and animal health. In laying hens, the liver is essential for metabolic regulation as well as the synthesis of egg-yolk precursors, such as vitellogenin and yolk lipoproteins. Any changes in liver function can cause lipid metabolism disorders, damage nutrient transport, affect reproduction performance, and ultimately influence egg production and quality. Moreover, many metabolic and inflammatory responses are controlled at the cell level by gene expression and the corresponding proteins. Regulation of genes involved in inflammatory and lipid metabolism pathways is key to understanding how the body responds to dietary interventions and environmental challenges. Among these, the FADS1 and FADS2 genes are of particular interest. These genes encode delta-5 desaturase and delta-6 desaturase enzymes, respectively, which play a central role in the conversion of linoleic acid (LA) and alpha-linolenic acid (ALA) into longer-chain PUFAs, such as arachidonic acid (AA) and eicosapentaenoic acid (EPA). These products, in turn, are key players in immune and inflammatory reactions [ 20 ]. In addition, peroxisome proliferator-activated receptor gamma ( PPAR-γ ) is a nuclear receptor involved in adipogenesis, glucose metabolism, and anti-inflammatory responses. Activation of PPAR-γ has been linked to reduced inflammation and enhanced lipid metabolism, and its expression in the liver and adipose tissues plays a crucial role in maintaining metabolic homeostasis [ 21 , 22 ]. Therefore, the expression of these genes provides valuable insights into the mechanisms of liver function in response to various physiological and dietary factors, including the modulation of inflammation and lipid metabolism in laying hens. The present study investigates, for the first time, the influence of the water-soluble emulsion of cannabidiol (CBD) on the egg yolk fatty acid profile, liver health, and the gene expression associated with lipid metabolism in laying hens. This represents a novel approach in poultry research, as the potential of CBD as a dietary modulator of lipid metabolism in avian species remains largely unexplored. Materials and Methods Experiment Design The research was conducted on 180 Lohmann Brown Classic laying hens (26-week-old, purchased from a commercial farm) at the Research and Education Station in Swojczyce, belonging to Wrocław University of Environmental and Life Sciences (Wrocław, Poland). The animals were obtained from a commercial farm. The experimental design used in this investigation was a Completely Randomized Design, consisting of 5 treatments with 12 replications for each treatment, using 3 hens per replication (36 hens per group). Each replication was a separate cage. Hens were allocated into standard furnished cages (0.125 m² per bird) in a random order. The environmental conditions were maintained according to Lohmann’s recommendations i.e., average temperature of 18°C, humidity 65%, and a photoperiod of 14 hours of light and 10 hours of darkness (14L:10D) [ 23 ]. Water-soluble cannabidiol (CBD) emulsion was administered in the drinking water for 15 weeks. The birds were divided into five experimental groups: a control group receiving water without additives (C), a blank group receiving water with an emulsifier (B), and three CBD-treated groups, which received water supplemented with 20, 40, or 80 mg/kg BW of CBD along with an emulsifier (groups I, II, III, respectively). All experimental groups received the same basal diet ad libitum . Diets were formulated based on CVB [ 24 ] nutrient standards. Additional information about diets is presented in the supplementary material (Tables S1 and S2). CBD solution An emulsion (CBD) was prepared with a cannabidiol (CBD) isolate (Purity- 99.14%; A-Sense Ltd., Puławy, Poland) and rapeseed lecithin as the emulsifier (Bunge Poland Ltd., Brzeg, Poland). The CBD content was verified by chromatographic analysis, using an Agilent 1290 Infinity II system equipped with a thermostated Ascentis Express C8 column (2.7 µm, 150 × 3.0 mm; Merck 53853-U) maintained at 30°C. Isocratic elution was performed using a mobile phase composed of 27% solvent A (5 mM ammonium formate with 0.1% formic acid in water) and 73% solvent B (0.1% formic acid in acetonitrile). Calibration standards were prepared using a certified reference material (CRM, 1.0 mg/mL in methanol; MERCK C-045). A stock solution was made, composed of 2% rapeseed phospholipids and 100 mg/mL of CBD isolate. Pre-homogenization was done by means of the T 25 digital ULTRA-TURRAX homogenizer (IKA, Warsaw, Poland) with an S 25 EC-T-C-18 G ST probe running at 12,000 rpm for 10 min. The resulting suspension was subjected to high-pressure homogenization (M-110P Microfluidizer, processor) at 200 MPa for five passes through the system. The emulsion was diluted at the necessary concentrations according to the average body weight (BW) of each experimental group. To verify the actual CBD intake, working solutions were sampled and analysed at the start and after 13 weeks of administration. The actual CBD consumption per kilogram of body weight was calculated at the start of the treatment period and reassessed after 13 weeks. Additional information supporting this assessment is presented in the supplementary Excel spreadsheet (Tables S3, S4, and S5). Eggs collection and Fatty Acid Methyl Ester (FAME) analysis Eggs were collected at two time points during the experiment: days 0 and 90, with three eggs per cage, 180 eggs per collection in total. Egg yolks were separated from the albumen, weighed, and transferred to plates. Each plate contained a pooled sample of three yolks from a single replication (cage). The yolks were thoroughly stirred to achieve a homogenized consistency. Homogenized yolk samples from each group were lyophilized using a Labconco freeze dryer under the following conditions: initial shelf temperature of -40°C with a holding time of 1 hour, followed by heating at a rate of 1°C per minute to 25°C, where samples were held for 24 hours. For FAME preparation, 100 ± 1 mg of lyophilized yolk was weighed into screw-capped tubes. Each tube was supplemented with 4 mL of 0.5 M NaOH in methanol and 14% BF₃ in methanol. The tubes were tightly sealed and incubated in a water bath at 70°C for 30 minutes. After incubation, the tubes were rapidly cooled in ice water and 6 mL of hexane was added. The mixture was vortexed for 30 seconds, and the hexane layer was collected. The extracted hexane phase was dried using an anhydrous MgSO₄ filtration system (PP tube with PE frits, 20 µm porosity) and evaporated under reduced pressure. The residue was dissolved in 1.5 mL of hexane and subjected to chromatographic analysis. FAMEs were analyzed using an Agilent 6890N gas chromatograph (GC) equipped with a 5973 MSD detector and a split/splitless injector. Separation was performed using an HP-88 column ((88%-cyanopropyl) aryl-polysiloxane; length: 100 m, ID: 0.25 mm, film thickness: 0.25 µm). The oven temperature program was as follows: initial temperature of 60°C (2 min hold), increasing at 20°C/min to 180°C, followed by a ramp of 3°C/min to 220°C (15 min hold), and a final increase of 5°C/min to 250°C (8 min hold), with a total run time of 50.33 minutes. The injection volume was 2 µL, applied in a 10:1 split mode [ 25 ]. Blood and liver sampling At the end of the experiment, six hens per group (30 hens in total) were euthanized for the collection of blood and liver samples. Euthanasia, following Annex IV to Directive 2010/63/EU of the European Parliament and of the Council of 22 September 2010 on the protection of animals used for scientific purposes [ 26 ], was carried out by stunning the birds with carbon dioxide and completed by exsanguination. Blood from each hen was immediately transferred to the tubes (volume: 10ml) containing the anticoagulant substance. Liver samples were collected under strictly aseptic conditions. Tissue sections measuring approximately 1 × 1 × 0.5 cm were promptly immersed in 10% neutral buffered formalin (pH 7.2–7.4) at a minimum tissue-to-fixative volume ratio of 1:10. Liver histology Fixation was performed at ambient temperature for 24 hours. Following fixation, the samples were rinsed under running tap water for 12 hours to remove residual fixing solution. The tissues were subsequently dehydrated through a graded ethanol series (70%, 80%, 90%, 96%, and 100%), cleared in xylene, and infiltrated with paraffin wax (melting point 56–58°C). Serial sections of 5 µm thickness were cut using a rotary microtome and mounted on poly-L-lysine-coated glass slides. The sections were stained with hematoxylin and eosin (H&E; Merck, Darmstadt, Germany) according to standardized histological protocols, which included rehydration through decreasing ethanol concentrations, hematoxylin staining, differentiation in acid alcohol, counterstaining with eosin, dehydration, and permanent mounting. Histomorphological evaluation was performed using a Stemi 508 stereo microscope (Carl Zeiss, Jena, Germany) equipped with an Axiocam 208 high-resolution color camera. Digital images were acquired and processed using ZEN™ image analysis software (Carl Zeiss, Jena, Germany). Blood serum assessment Tubes containing blood samples were transferred to the laboratory. In order to separate the serum, samples were centrifuged at 6000 rpm for 10 minutes. The serum for each replication was transferred to the 1.5 ml tubes to analyze for AST, ALT, Total Cholesterol, HDL, LDL, and TG with ABX Pentra reagents (Horiba ABX). Serum biochemical assessment was conducted with a Pentra 400 spectrophotometric device (Horiba ABX- France). Analysis of gene expression in liver samples The total RNA was isolated from liver tissue collected after the experiment. The 200 mg of the specimen was homogenized using 1 mL of TRI Reagent® (Sigma-Aldrich/Merck, Poznan, Poland). The procedure was performed according to the manufacturer’s protocol, with a few modifications, including a 1-hour centrifugation at 12,000 × g during precipitation, and twice washing of RNA pellets with 80% ethanol to ensure the RNA purity. The extracted RNA was diluted in nuclease-free water (Sigma-Aldrich/Merck, Poznan, Poland), and its concentration and purity were assessed using a DS-11 Fx nanospectrophotometer (DeNovix, Wilmington, DE, USA). The samples with an A260/A280 ratio of 2.1 ± 0.2 were used for gene expression analysis. To ensure RNA integrity, gDNA digestion was performed using the DNase I, RNase-free enzyme (1 U/µL; Thermo Scientific, Warsaw, Poland). The obtained total RNA (1 ug) was reverse-transcribed using the Tetro cDNA Synthesis Kit (Bioline Reagents Limited, London, UK). Both reactions, gDNA digestion and reverse transcription) were performed in a T100 Thermal Cycler (Bio-Rad, Hercules, CA, USA) according to the manufacturer’s instructions. Quantitative real-time PCR (RT-qPCR) was performed using the SensiFAST SYBR® & Fluorescein Kit (Bioline Reagents Ltd., London, UK) on a CFX Opus 384 Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA). Each 10 µL reaction contained 1 µL of cDNA, 5 µL of Master Mix, and gene-specific primers at a final concentration of 400 nM. Thermal cycling conditions included initial denaturation at 95°C for 2 minutes, followed by 40 cycles of 95°C for 15 seconds, annealing at primer-specific temperature, i.e., 62°C for 15 seconds, and 72°C for 15 seconds of elongation. The expression of target genes was normalized against the expression of two reference genes, i.e., coding GAPDH and β-actin. Relative gene expression was determined using the 2 –ΔΔCq method, with normalization based on the gene exhibiting the lowest expression level (RQMAX approach) [ 27 ], and the data were presented in a logarithmic scale. The specificity of each product was determined based on its melting temperature. Primer sequences are described in detail in Supplementary Table S6. Statistical data analysis Data analysis for egg yolk fatty acid composition and liver parameters was performed using R software, version 4.4.0 [ 28 ]. Descriptive statistics, including the mean and standard deviation, were calculated. Group comparisons for these parameters were conducted using one-way analysis of variance (ANOVA), followed by Fisher’s Least Significant Difference (LSD) post-hoc test. For molecular assessments, data were derived from both biological and technical replicates within each experimental group. Statistical comparisons were made using one-way ANOVA, followed by Tukey’s post-hoc test where applicable. These analyses were performed using GraphPad Prism (version 10.3.1; GraphPad Software, CA, USA), with statistical significance set at p < 0.05. Results CBD enhances yolk PUFA profile and HDL levels, indicating improved lipid transport, with no significant hepatic alterations The fatty acid profile of egg yolks across the experimental groups is presented in Tables 1 and 2 . Except for the arachidonic acid (C20:4 n-6), all measured fatty acid levels showed significant differences between day 0 and day 90. 1) Saturated Fatty Acids (SFA) After 90 days of cannabidiol (CBD) supplementation, Group I exhibited the highest concentrations of myristic acid (C14:0) and palmitic acid (C16:0). The highest level of stearic acid (C18:0) was detected in Group B. Total SFA levels differed significantly among the groups both at the beginning of the experiment and after three months. Notably, Group III had the highest SFA concentration at the start but the lowest by day 90 (Table 1 ). Table 1 Yolk saturated fatty acids (% of total) on day 0 and 90 of CBD use Fatty Acid Group Day 0 (% ± SD) Day 90 (% ± SD) C 14:0 C 1 B I II III 0.33 ± 0.05 0.35 ± 0.09 0.37 ± 0.08 0.34 ± 0.05 0.32 ± 0.05 0.31 b ± 0.01 0.29 b ± 0.06 0.38 a ± 0.08 0.39 a ± 0.03 0.31 b ± 0.01 p- value 0.51 < 0.001** C 16:0 C B I II III 26.12 ± 1.24 25.52 ± 0.62 25.32 ± 0.9 25.93 ± 0.77 25.51 ± 0.53 26.43 a ± 0.98 25.49 b ± 0.77 26.96 a ± 1.61 25.11 b ± 0.56 25.11 b ± 0.56 p- value 0.14 < 0.001** C 18:0 C B I II III 21.7 ± 3.6 23.95 ± 1.68 22.14 ± 3.71 22.84 ± 2.41 24.32 ± 1.55 20.19 b ± 0.85 24.94 a ± 0.61 21.1 b ± 1.83 21.24 b ± 1.54 21.03 b ± 1.63 p- value 0.10 < 0.001** SFA 2 C B I II III 48.15 bc ± 2.54 49.82 ab ± 1.33 47.83 c ± 3 49.1 ab ± 1.82 50.15 a ± 1.3 46.93 c ± 1.05 50.72 a ± 0.94 48.44 b ± 0.95 46.74 c ± 1.28 46.44 c ± 1.46 p- value 0.04* < 0.001** 1 C (Control): water without additive; B (Blank): water + emulsifier; I: water + CBD 20 mg/kg BW + emulsifier; II: water + CBD 40 mg/kg BW + emulsifier; III: water + CBD 80 mg/kg BW + emulsifier. 2 Saturated Fatty Acids (SFA): Myristic (C14:0); Palmitic (C16:0); Stearic (C18:0); a,b,c Values within a column with different superscripts differ significantly at the level P < 0.05. 2) Monounsaturated Fatty Acids (MUFA) Group I and B demonstrated the highest levels of palmitoleic acid (C16:1 n-7). After 90 days, the overall MUFA content was the highest in Groups C and II (Table 2 ). 3) Polyunsaturated Fatty Acids (PUFA): Group III exhibited the highest concentrations of linoleic acid (C18:2 n-6), α-linolenic acid (C18:3 n-3), docosahexaenoic acid (C22:6 n-3), as well as total omega-3, omega-6, and overall PUFA levels (Table 2 ). At the beginning of the experiment, the n3/n6 ratio for all groups ranged from 0.22 to 0.23. After 90 days, it ranged from 0.20 to 0.24, with Group B showing the lowest and Groups I and III the highest values, respectively. Table 2 Yolk MUFA and PUFA levels (% total) at day 0 and day 90 with CBD Fatty Acid Group Day 0 (% ± SD) Day 90 (% ± SD) C 16:1 (n-7) C 1 B I II III 2.5 ± 0.37 2.4 ± 0.3 2.63 ± 0.29 2.52 ± 0.32 2.34 ± 0.25 2.47 b ± 0.09 3 a ± 0.45 2.86 a ± 0.3 2.61 b ± 0.14 2.47 b ± 0.09 p- value 0.19 < 0.001** C 18:1 (n-9) C B I II III 24.1 ± 3.14 23 ± 2.16 24.49 ± 3.55 23.63 ± 2.56 22.28 ± 1.27 25.57 a ± 0.96 20.1 c ± 1.12 24 b ± 0.68 25.81 a ± 1.33 23.99 b ± 1.18 p- value 0.28 < 0.001** MUFA 2 C B I II III 26.6 ± 3.42 25.4 ± 2.31 27.12 ± 3.48 26.15 ± 2.74 24.62 ± 1.32 28.04 a ± 1.03 23.1 c ± 1.07 26.85 b ± 0.97 28.42 a ± 1.33 26.47 b ± 1.22 p- value 0.21 < 0.001** C 18:2 (n-6) C B I II III 14.66 ± 0.74 13.98 ± 1.04 14.21 ± 0.6 14.13 ± 0.68 14.51 ± 1.04 14.73 b ± 0.4 15.6 a ± 0.96 13.27 c ± 0.98 14.32 b ± 0.51 15.64 a ± 0.64 p- value 0.28 < 0.001** C 18:3 (n-3) C B I II III 0.43 ± 0.01 0.42 ± 0.05 0.44 ± 0.05 0.43 ± 0.01 0.43 ± 0.01 0.45 c ± 0.03 0.48 ab ± 0.03 0.47 b ± 0.04 0.45 c ± 0.03 0.5 a ± 0.03 p- value 0.48 < 0.001** C 20:4 (n-6) C B I II III 6.07 ± 0.39 6.2 ± 0.53 6.3 ± 0.39 6.14 ± 0.52 6.08 ± 0.34 5.81 ± 0.85 6.24 ± 1.32 6.57 ± 0.47 5.86 ± 0.3 6.3 ± 0.18 p- value 0.69 0.09 C 22:6 (n-3) C B I II III 4.09 ± 0.41 4.17 ± 0.32 4.09 ± 0.39 4.04 ± 0.39 4.2 ± 0.29 4.05 b ± 0.28 3.86 c ± 0.34 4.4 a ± 0.41 4.21 b ± 0.34 4.65 a ± 0.35 p- value 0.80 < 0.001** n3 C B I II III 4.52 ± 0.41 4.59 ± 0.33 4.53 ± 0.38 4.48 ± 0.4 4.63 ± 0.29 4.49 c ± 0.28 4.34 c ± 0.34 4.86 b ± 0.42 4.66 b ± 0.35 5.16 a ± 0.35 p- value 0.83 < 0.001** n6 C B I II III 20.73 ± 0.88 20.18 ± 1.16 20.52 ± 0.73 20.27 ± 1.02 20.59 ± 1.07 20.54 b ± 1.08 21.84 a ± 1.26 19.84 b ± 1.26 20.18 b ± 0.61 21.93 a ± 0.63 p- value 0.64 < 0.001** PUFA 3 C B I II III 25.25 ± 1.04 24.77 ± 1.35 25.05 ± 0.94 24.75 ± 1.29 25.22 ± 1.2 25.03 c ± 1.02 26.19 b ± 1.08 24.7 c ± 1.58 24.84 c ± 0.74 27.09 a ± 0.8 p- value 0.74 < 0.001** 1 C (Control): water without additive; B (Blank): water + emulsifier; I: water + CBD 20 mg/kg BW + emulsifier; II: water + CBD 40 mg/kg BW + emulsifier; III: water + CBD 80 mg/kg BW + emulsifier. 2 Mono Unsaturated Fatty Acids (MUFA): Palmitoleic (C16:1 (n-7); Oleic (18:1 (n-9); 3 Poly Unsaturated Fatty Acids (PUFA): Linoleic (18:2 (n-6); Alpha-linolenic acid (ALA) (18:3 (n-3); Arachidonic (AA) (20:4 (n-6); Docosahexaenoic (DHA, Cervonic (22:6 (n-3). a,b,c Values within a column with different superscripts differ significantly at the level P < 0.05. The results of the serum biochemical parameters are presented in Table 3 . Aspartate aminotransferase (AST) levels were significantly rose in Group C compared to the other groups. Besides, high-density lipoprotein (HDL) levels were higher in Groups I and III. No statistically significant differences were observed for the remaining serum parameters. Table 3 Analysis of biochemical blood serum after 15 weeks Parameter Group Mean SD P- value AST (U/L) 1 C 2 98.68 a 19.18 0.033* B 77.69 b 19.83 I 76.24 b 13.76 II 79.34 b 15.94 III 81.40 b 21.47 ALT (U/L) C 10.37 5.19 0.502 B 11.90 6.95 I 9.74 6.49 II 9.07 3.06 III 8.15 3.75 Cholesterol (mmol/L) 3 C 1.75 0.87 0.336 B 2.32 0.97 I 2.05 0.70 II 2.17 0.80 III 2.40 0.64 HDL (mmol/L) C 0.61 b 0.15 0.024* B 0.69 b 0.16 I 0.76 ab 0.18 II 0.73 b 0.24 III 0.93 a 0.37 LDL (mmol/L) C 0.38 0.28 0.408 B 0.56 0.32 I 0.45 0.19 II 0.53 0.27 III 0.58 0.27 TG (mmol/L) C 5.84 4.54 0.392 B 9.48 6.23 I 6.64 4.44 II 7.24 4.21 III 6.58 3.38 1 Units per liter 2 C (Control): water without additive; B (Blank): water + emulsifier; I: water + CBD 20 mg/kg BW + emulsifier; II: water + CBD 40 mg/kg BW + emulsifier; III: water + CBD 80 mg/kg BW + emulsifier 3 Millimoles per liter a,b Values within a column with different superscripts differ significantly at the level P < 0.05. The liver histology was consistent across all experimental groups (Fig. 1 ), showing a homogeneous tissue structure without signs of disruption, degeneration, or hepatocyte necrosis. There was no evidence of hepatocyte vacuolization (cytoplasmic vacuoles appearing as empty spaces), cellular swelling, or nuclear displacement. Furthermore, no vacuolar degeneration characterized by a diffuse, foamy, or feathery appearance of hepatocytes was observed. Taken together, the histological assessment of the liver revealed no significant alterations in any of the examined groups. Hepatic levels of mRNA for PPAR-γ decrease in response to high-dose CBD, while FADS1 and FADS2 remain unchanged RT-qPCR analysis revealed that expression of PPAR-γ is modulated by the emulsion with the highest CBD concentration. The significant reduction in PPAR-γ expression in group III suggests that the treatment may have an inhibitory effect on lipid metabolic pathways or adipogenic differentiation processes. This decrease could also indicate a cellular stress response or the activation of pro-inflammatory pathways that are known to downregulate PPAR-γ transcription. On the other hand, the stable levels of FADS1 and FADS2 across all experimental groups suggest that the treatment did not adversely affect the genes coding major enzymes responsible for hepatic fatty acid desaturation. The heatmap illustration was also prepared to demonstrate that PPAR-γ exhibited the lowest expression in the hen’s liver among the tested genes, simultaneously showing the most dynamic regulation. The expression signature also indicated that PPAR-γ is responsive to CBD treatment and may represent a sensitive molecular target. The accumulation of FADS1 and FADS2 transcripts in the hen's liver was higher than PPAR-γ . Moreover, the stability in mRNA expression in FADS1 and FADS2 implies that core lipid metabolic pathways are not significantly altered by CBD supplementation (Figs. 2 and 3 ). Discussion The use of bioactive substances in poultry diets to promote animal welfare and enhance the safety and quality of poultry-derived products has recently garnered significant interest. Most recently, cannabidiol (CBD), a non-psychoactive phytochemical derived from Cannabis sativa , has gained recognition as a promising phytogenic additive with multiple potential benefits for systemic metabolism. Its biological activity is primarily mediated through interaction with the endocannabinoid system, influencing key processes such as lipid metabolism, as well as inflammatory signaling, oxidative stress responses, and energy homeostasis. These biological properties make CBD a compelling candidate for applications aimed at supporting the metabolic health of animals. The role of CBD is particularly notable in the context of its use as a functional nutrition strategy for livestock and poultry. In the context of liver physiology, CBD has exhibited a range of beneficial effects across in vitro , in vivo , and clinical models of metabolic dysfunction. These include the attenuation of hepatic inflammation, reduction of oxidative stress, and modulation of lipid accumulation, indicating its multifaceted role as a regulator of hepatic metabolism, cellular homeostasis, and disease progression. In the current study, we aimed to investigate the effects of dietary CBD-emulsion on lipid metabolism and hepatic function in laying hens, with a particular focus on yolk PUFA deposition, liver histopathology, and gene expression. Several studies have reported the beneficial effects of CBD in pets and rodents, including alleviation of stress and anxiety [ 29 , 30 ] and relief of pain [ 31 , 32 , 33 ]. Despite growing interest in phytogenic additives, research on purified CBD in poultry nutrition remains scarce, with most studies to date concentrating on hemp seeds or processing byproducts rather than isolated CBD. Gakhar et al. [ 34 ] reported that including 20% hemp seed in the diet of laying hens increased omega-3 fatty acids in egg yolk. Similarly, another study on broiler chicken showed that the addition of 7.5% of hemp seed meal decreased serum cholesterol concentration [ 35 ]. Halle and Schöne [ 36 ] reported that supplementation of diets containing 5%, 10% or 15% hemp seed cake in laying hens over six months decreased SFAs and MUFAs levels while linoleic and linolenic acid (PUFAs) levels increased. A 25% of hemp seed alone or combined with 2% of ginger or turmeric in a diet of laying hens increased omega-3 content in the egg yolk, improved the n-3/n-6 fatty acid, and decreased the proportion of SFAs [ 37 ]. Besides, an increased content of egg yolk PUFAs was reported by Kasula et al. [ 38 ] when laying hens were fed with diets containing 10, 20, or 30% of hemp seed cake over 19 weeks. In the present study, the level of egg yolk PUFAs. e.g., omega-3 and omega-6 raised significantly in group III (CBD 80 mg/kg BW) after 90 days of CBD administration. This finding suggests that CBD may facilitate the mobilization and deposition of PUFAs into the yolk probably via regulation of the lipid transport mechanisms. Elevation of high-density lipoprotein (HDL) serum level supports the idea of increased lipid delivery to the ovary. According to Neijat et al. [ 39 ], hempseed products enhanced egg yolk nutritional value and n-3 PUFA levels. Furthermore, a recent meta-analysis by Sopian et al. [ 40 ] demonstrated that hemp-derived products can perform as functional feed ingredients for laying hens, enhancing yolk pigmentation and enriching long-chain omega-3 fatty acids, particularly DHA, without impairing performance parameters when inclusion levels are well managed. Understanding the molecular mechanisms underlying CBD’s effects is crucial for explaining its influence on lipid metabolism and tissue-specific responses. CBD interacts with a variety of molecular targets, including cannabinoid receptors (CB1 and CB2), peroxisome proliferator-activated receptors (PPARs), G protein-coupled receptor 55 (GPR55), adenosine receptors, and transient receptor potential (TRP) channels, which collectively mediate all its biological effects [ 41 ]. Among these, PPAR-γ is of particular interest due to its key function in lipid metabolism and liver homeostasis. PPAR-γ is a nuclear receptor that plays a central role in regulating gene expression and is also involved in adipocyte differentiation and adipogenesis. It functions as a transcriptional regulator that promotes the development and maturation of adipose tissue, making it a critical factor in maintaining energy balance and tissue remodeling [ 42 ]. Chang et al. [ 43 ] indicated that CBD can bind and activate PPAR-γ ; therefore, it could function as a PPAR-γ agonist, which could improve adipogenesis. The interaction between CBD and PPAR-γ is complex; whereas it may act as an agonist and stimulate adipogenesis, chronic high-dose exposure may cause a feedback inhibition or stress-related suppression of PPAR-γ expression, contributing to hepatic lipid accumulation. Importantly, PPAR-γ may function as a metabolic sensor in the liver, responding to changes in cellular energy status and contributing to the regulation of pathways involved in lipid accumulation and early-stage metabolic disturbances that precede fibrogenesis [ 44 ]. Indeed, the highest CBD dose (80 mg/kg BW) in the present study positively influenced the fatty acid composition of egg yolk with no signs of hepatic pathological changes. The liver is a primary site for energy storage and possesses remarkable ability to regenerate and adapt. Its adaptation to various conditions may involve, for instance, enhancing functional efficiency to maintain the body’s homeostasis. This adaptability is particularly crucial in responding to physiological challenges, such as exposure to an aqueous cannabidiol solution. However, at the molecular level, we observed a significant downregulation of PPAR-γ expression. Such alterations may be attributed to the metabolic burden imposed by CBD biotransformation or to intensified lipid mobilization processes [ 21 ]. The synthesis of longer-chain PUFAs from linoleic acid (LA) and alpha-linolenic acid (ALA) mainly depends on the action of Δ5 and Δ6 desaturase enzymes that are encoded by FADS1 and FADS2 genes, respectively [ 45 ]. The Δ5 desaturase (encoded by FADS1 ) is particularly important in the biosynthesis of longer-chain PUFAs, including arachidonic acid (ARA) and eicosapentaenoic acid (EPA) [ 46 ]. In the current study, hepatic mRNA expression levels of both FADS1 and FADS2 were not significantly affected by the dietary levels of cannabidiol (CBD) administered to laying hens. The genes encode highly conserved enzymes, with an overall role in cellular homeostasis. Therefore, their stable expression across experimental conditions may reflect a tightly regulated mechanism necessary to maintain physiological balance, regardless of external dietary modulation. The signature of PPAR-γ and FASD1/2 gene expression may also indicate that the observed increase in yolk PUFAs is more likely due to enhanced mobilization and transfer of existing fatty acids rather than increased hepatic synthesis. Conclusion This study provides novel insights into the impact of dietary CBD supplementation on lipid metabolism in laying hens, focusing on hepatic gene expression and fatty acid deposition in egg yolks. To our knowledge, this is the first report assessing the expression of PPAR-γ , as well as FADS1 and FADS2 , in the context of CBD intake in poultry. We showed that the highest dose of CBD-emulsion in drinking water increased the egg yolk PUFAs content, possibly by boosting lipid mobilization and transport mechanisms. The liver did not show any pathological changes with the administration of different CBD doses. However, a decrease in PPAR-γ expression at the mRNA level indicates a complex interaction between CBD, the ECS, and lipid metabolism pathways. Nevertheless, the stable expression of FADS1 and FADS2 genes suggests that desaturation activity is maintained, which may confirm continued PUFA synthesis. Moreover, the elevated HDL levels may facilitate the transport of PUFAs from the liver to the developing yolk, providing a connection between liver lipid metabolism and yolk lipid deposition. Together, our findings contribute to the knowledge of the physiological and molecular effects of phytocannabinoids in animal production. We believe that such studies are essential for opening new avenues to optimize egg lipid profiles through non-traditional dietary strategies. Further research is required to clarify the precise role of the endocannabinoid system in poultry, to identify a safe CBD dose that enhances egg composition, and to expand future diagnostics to include assessments of enzyme activity in the liver. Declarations Ethics approval and consent to participate All experimental procedures were approved by the Local Ethical Committee for Animal Experimentation in Wrocław (Resolution No. 017/2023/P1 of 19 April 2023). All animals were maintained under conditions specified by Directive 2010/63/EU on the protection of animals used for scientific purposes [26]. The animal-handling research team included the Ph.D. or M.S. in Animal Science, as well as experienced veterinary practitioners. Consent for publication Not applicable Availability of data and materials The datasets used and analysed during the current study are available from the corresponding author on reasonable request. Competing interests The authors declare that they have no competing interests. Funding This work was supported by the Wrocław University of Environmental and Life Sciences (Poland) as part of research project no N070/0008/23. The article is part of a Ph.D. dissertation titled “Cannabidiol- a Feed Additive that Improves Birds’ Welfare and the Efficiency of Poultry Production”, prepared during the Doctoral School at the Wrocław University of Environmental and Life Sciences. The APC is financed by Wrocław University of Environmental and Life Sciences. Authors' contributions SF wrote the original manuscript and performed research; SF and SO designed the research; SF, AŚ, ŁB and AL conducted laboratory assessments and acquired data; AŚ and AL visualized the data; AŚ analyzed data; AŚ, AL and SO reviewed and edited the manuscript and validated data; SO and ŁB supervised the project; SO provided funding; MA and KO assisted in sample collection. All authors read and approved the final manuscript. Acknowledgements We gratefully acknowledge Dr. Anna Burek from the Department of Environmental Hygiene and Animal Welfare for her valuable support with the sample analysis of serum parameters. References Milinsk MC, Murakami AE, Gomes STM, Matsushita M, de Souza NE (2003) Fatty acid profile of egg yolk lipids from hens fed diets rich in n-3 fatty acids. Food Chem 83:287-292 Djoussé L, Gaziano JM (2008) Egg consumption in relation to cardiovascular disease and mortality: the Physicians’ Health Study. Am J Clin Nutr 87:964-969 Djoussé L, Gaziano JM, Buring JE, Lee I-M (2009) Egg consumption and risk of type 2 diabetes in men and women. Diabetes Care 32:295-300 Hopkins PN (1992) Effects of dietary cholesterol on serum cholesterol: a meta-analysis and review. 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Halle I, Schöne F (2013) Influence of rapeseed cake, linseed cake and hemp seed cake on laying performance of hens and fatty acid composition of egg yolk. J Verbr Lebensm 8:185-193 Raza T, Chand N, Khan RU, Shahid MS, Abudabos AM (2016) Improving the fatty acid profile in egg yolk through the use of hempseed ( Cannabis sativa ), ginger ( Zingiber officinale ), and turmeric ( Curcuma longa ) in the diet of Hy-Line White Leghorns. Arch Anim Breed 59:183-190 Kasula R, Solis F, Shaffer B, Connett F, Barrett C, Cocker R, Willinghan E (2021) Hemp seed cake increases fatty acids but does not transfer cannabinoids in eggs and tissues of laying hens. Int J Livest Prod 12:98-111 Neijat M, Suh M, Neufeld J, et al (2016) Hempseed Products Fed to Hens Effectively Increased n-3 Polyunsaturated Fatty Acids in Total Lipids, Triacylglycerol and Phospholipid of Egg Yolk. Lipids 51:601-614 Sopian Y, Sivapirunthep P, Jayanegara A, Chaosap C (2025) Dietary Hemp (Cannabis sativa L.) Products Enhance Egg Yolk Omega-3 Fatty Acids and Color Without Compromising Laying-Hen Performance: A Meta-Analysis. Animals 15:2062 Chen S, Kim JK (2024) The role of cannabidiol in liver disease: a systematic review. Int J Mol Sci 25:2370 Sato K, Fukao K, Seki Y, Akiba Y (2004) Expression of the chicken peroxisome proliferator-activated receptor-γ gene is influenced by aging, nutrition, and agonist administration. Poult Sci 83:1342-1347 Chang RC, Thangavelu CS, Joloya EM, Kuo A, Li Z, Blumberg B (2022) Cannabidiol promotes adipogenesis of human and mouse mesenchymal stem cells via PPARγ by inducing lipogenesis but not lipolysis. Biochem Pharmacol 197:114910 Monroy-Ramirez HC, Galicia-Moreno M, Sandoval-Rodriguez A, Meza-Rios A, Santos A, Armendariz-Borunda J (2021) PPARs as metabolic sensors and therapeutic targets in liver diseases. Int J Mol Sci 22:8298 O’Neill CM, Minihane AM (2017) The impact of fatty acid desaturase genotype on fatty acid status and cardiovascular health in adults. Proc Nutr Soc 76:64-75 Jeong HY, Moon YS, Cho KK (2024) ω-6 and ω-3 polyunsaturated fatty acids: inflammation, obesity and foods of animal resources. Food Sci Anim Resour 44:988-1010 Additional Declarations No competing interests reported. Supplementary Files SupplementaryFile.xlsx Cite Share Download PDF Status: Published Journal Publication published 12 Nov, 2025 Read the published version in BMC Veterinary Research → Version 1 posted Editorial decision: Revision requested 15 Sep, 2025 Reviews received at journal 11 Sep, 2025 Reviews received at journal 09 Sep, 2025 Reviewers agreed at journal 01 Sep, 2025 Reviewers agreed at journal 01 Sep, 2025 Reviewers invited by journal 26 Aug, 2025 Editor invited by journal 26 Aug, 2025 Editor assigned by journal 26 Aug, 2025 Submission checks completed at journal 25 Aug, 2025 First submitted to journal 25 Aug, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7387031","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":508751896,"identity":"c3d13d62-f609-49b9-a68d-57c27353c774","order_by":0,"name":"Sepideh Fallahi","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABIElEQVRIie3RMUsDMRQH8HcU7JLaNVDb+wonByJ08Ktc6HCLde5Q5BUhXSyuCoJfoeDimBDoFHUt9JZbnE44cHKw+FqFtpDTVTB/eCGE9yMvBMDH549GMVBQx9pIBcg734e1VeGPhFH/isRbJKgksEVA4G8kHE+0KgYZsLpG9f5wnE4Xl0clDLoCWyMniexTom/tCzAmUE8s708zG1+DTQUeaDfhp5FpSAMnNJJqSCLz/j0E0gjkwknCu4LI0gBr5qg/JE+jNVlWE5gzIkiEGug6nnwRrCaRPaO3zAxjPEfTlvzwJnt+g2SWxrLiLeH40ZTF0HRYs6fzV3ke7i9kD8pht33VulDOwehTNus6nCqh2uNusNO8IbsbHx8fn3+eT46TcSwkvfBSAAAAAElFTkSuQmCC","orcid":"","institution":"Wrocław University of Environmental and Life Sciences","correspondingAuthor":true,"prefix":"","firstName":"Sepideh","middleName":"","lastName":"Fallahi","suffix":""},{"id":508751897,"identity":"d41bac9f-c68c-47d1-9aed-fa5e5a5cee05","order_by":1,"name":"Agnieszka Śmieszek","email":"","orcid":"","institution":"Wrocław University of Environmental and Life Sciences","correspondingAuthor":false,"prefix":"","firstName":"Agnieszka","middleName":"","lastName":"Śmieszek","suffix":""},{"id":508751898,"identity":"067b2007-02c0-41e5-9970-de242fbfae1c","order_by":2,"name":"Łukasz Bobak","email":"","orcid":"","institution":"Wrocław University of Environmental and Life Sciences","correspondingAuthor":false,"prefix":"","firstName":"Łukasz","middleName":"","lastName":"Bobak","suffix":""},{"id":508751899,"identity":"85c80956-b255-4f8c-b670-43bcd811c3d4","order_by":3,"name":"Anna Lipińska","email":"","orcid":"","institution":"Wrocław University of Environmental and Life Sciences","correspondingAuthor":false,"prefix":"","firstName":"Anna","middleName":"","lastName":"Lipińska","suffix":""},{"id":508751900,"identity":"8ec49990-7228-461e-a0dd-41c6866078a7","order_by":4,"name":"Muhammad Umair Asghar","email":"","orcid":"","institution":"Wrocław University of Environmental and Life Sciences","correspondingAuthor":false,"prefix":"","firstName":"Muhammad","middleName":"Umair","lastName":"Asghar","suffix":""},{"id":508751901,"identity":"d4d42080-e2e9-40e1-9564-9d37d3983333","order_by":5,"name":"Katarzyna Olejnik","email":"","orcid":"","institution":"Wrocław University of Environmental and Life Sciences","correspondingAuthor":false,"prefix":"","firstName":"Katarzyna","middleName":"","lastName":"Olejnik","suffix":""},{"id":508751902,"identity":"60e2ac5b-abaf-48fa-a024-9266930e58e3","order_by":6,"name":"Sebastian Opaliński","email":"","orcid":"","institution":"Wrocław University of Environmental and Life Sciences","correspondingAuthor":false,"prefix":"","firstName":"Sebastian","middleName":"","lastName":"Opaliński","suffix":""}],"badges":[],"createdAt":"2025-08-16 11:08:12","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7387031/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7387031/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12917-025-05122-y","type":"published","date":"2025-11-12T15:57:36+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":90480348,"identity":"21879693-9160-492a-acdd-7dc0c2bac252","added_by":"auto","created_at":"2025-09-03 07:55:57","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":349922,"visible":true,"origin":"","legend":"\u003cp\u003eHistology of laying hens’ liver after 15 weeks of CBD administration.\u003c/p\u003e\n\u003cp\u003eC (Control): water without additive; B (Blank): water + emulsifier; I: water + CBD 20 mg/kg BW + emulsifier; II: water + CBD 40 mg/kg BW + emulsifier; III: water + CBD 80 mg/kg BW + emulsifier\u003c/p\u003e\n\u003cp\u003eScale bar: 5µm\u003c/p\u003e","description":"","filename":"Onlinefloatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-7387031/v1/83e483e9c2029ce8e3e4c109.png"},{"id":90480351,"identity":"f26f5e96-7290-4eb2-bd2f-811e89df0609","added_by":"auto","created_at":"2025-09-03 07:55:57","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":33008,"visible":true,"origin":"","legend":"\u003cp\u003eRelative mRNA expression levels of\u003cem\u003e PPAR-γ\u003c/em\u003e, \u003cem\u003eFADS1\u003c/em\u003e, and \u003cem\u003eFADS2\u003c/em\u003e in liver tissue across groups.\u003c/p\u003e\n\u003cp\u003eC (Control): water without additive; B (Blank): water + emulsifier; G (I): water + CBD 20 mg/kg BW + emulsifier; G (II): water + CBD 40 mg/kg BW + emulsifier; G (III): water + CBD 80 mg/kg BW + emulsifier\u003c/p\u003e\n\u003cp\u003eBars represent mean values ± standard deviation (SD). Statistically significant differences between groups are indicated in the graph: * p \u0026lt; 0.05, **p \u0026lt; 0.01 and *** p \u0026lt; 0.001. Non-significant differences are described as \u003cem\u003e“ns”\u003c/em\u003e.\u003c/p\u003e","description":"","filename":"Onlinefloatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-7387031/v1/84a41cd56f1cc3456784c333.png"},{"id":90479491,"identity":"aa8c210e-2088-41fd-a61f-840401f9b767","added_by":"auto","created_at":"2025-09-03 07:47:57","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":17785,"visible":true,"origin":"","legend":"\u003cp\u003eHeatmap displays of the expression patterns of the targeted lipid metabolic-related genes in the groups.\u003c/p\u003e\n\u003cp\u003eC (Control): water without additive; B (Blank): water + emulsifier; G (I): water + CBD 20 mg/kg BW + emulsifier; G (II): water + CBD 40 mg/kg BW + emulsifier; G (III): water + CBD 80 mg/kg BW + emulsifier.\u003c/p\u003e\n\u003cp\u003eDarker and lighter colors represent increased and decreased expression, respectively (color code). The stable expression of \u003cem\u003eFADS1\u003c/em\u003e and \u003cem\u003eFADS2\u003c/em\u003e indicates maintained function in the metabolic pathways of fatty acid synthesis, whereas a significant decrease in \u003cem\u003ePPAR-γ\u003c/em\u003e mRNA level is observed in group III.\u003c/p\u003e","description":"","filename":"Onlinefloatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-7387031/v1/05e8c9e140ccfefc8c1efe61.png"},{"id":96105172,"identity":"3cf061f4-a2d3-4cd4-89a6-320b3126cc92","added_by":"auto","created_at":"2025-11-17 16:09:37","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1648446,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7387031/v1/26718cbb-5c00-488a-ba36-31c9f1c88f85.pdf"},{"id":90479488,"identity":"69b0c8f5-977a-4eea-b616-1c101dc20cb1","added_by":"auto","created_at":"2025-09-03 07:47:57","extension":"xlsx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":26921,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFile.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7387031/v1/b0ac14c8d62cd2ae3f5c8689.xlsx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Dose-dependent effects of Cannabidiol in drinking water on yolk fatty acid profile and hepatic expression of PUFA-related genes in laying hens: insights into the CBD systemic and molecular mechanisms of action","fulltext":[{"header":"Introduction","content":"\u003cp\u003ePoultry health is a significant concern in the poultry industry due to concerns about animal welfare, longevity, and the quality of derived products. This is particularly relevant in terms of laying hens which are maintained for a long period due to their role in producing eggs. Eggs are a highly valued food in the diets of many people, and their quality leads to both direct and indirect effects on human health. Therefore, customers have become more conscious and selective about the products they purchase.\u003c/p\u003e\u003cp\u003eEggs, as a good source of essential fatty acids, are important for the proper functioning of the human body. The yolk is composed of approximately 30% lipids, making it a primary source of dietary fats [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Eggs were long considered as a health risk due to their high yolk cholesterol content, although more recent studies have questioned this association with cardiovascular disease, obesity, type 2 diabetes, and higher mortality rates in diabetic patients [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Beyond cholesterol levels, the fatty acid composition of eggs plays a crucial role in determining their overall impact on human health. Raising awareness of the health benefits of specific fatty acids in the diet has led to intensive research into options for changing the fat composition of animal products. The amount of saturated fatty acids (SFAs), monounsaturated fatty acids (MUFAs), and polyunsaturated fatty acids (PUFAs) is an important factor in lipid metabolism and cardiovascular risk [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Higher PUFA and MUFA, and lower SFA, in diets have been reported to mitigate the adverse effects of dietary cholesterol [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Furthermore, some evidence suggests that high egg consumption (i.e., one egg/day) is associated with less severe coronary atherosclerosis [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThe egg lipid profile is dynamic and varies according to the bird\u0026rsquo;s genetics, age, feeding, and the nature and amount of fat in its diet [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. The PUFAs are widely reported for their beneficial effects on the heart, brain, and general health; specifically, omega-3 (n-3), omega-6 (n-6), and omega-9 (n-9) [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. The poultry industry has responded to increased consumer interest in healthier and functional foods by enhancing the fatty acid profile of eggs, particularly by increasing the content of health-promoting polyunsaturated fatty acids [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. For this reason, eggs are now being considered not only as a source of high-quality protein but also as a functional food that possesses additional health benefits.\u003c/p\u003e\u003cp\u003eIn recent years, considerable attention has been directed towards plant additives in poultry feeding to improve poultry health, production, and welfare. These phytogenic compounds could be a natural alternative to synthetic additives and antibiotics.\u003c/p\u003e\u003cp\u003eCannabidiol (CBD), a non-psychoactive compound that is isolated from the hemp plant (\u003cem\u003eCannabis sativa\u003c/em\u003e), has attracted attention for its potential therapeutic effects. Research in mammals indicates that CBD exhibits anti-inflammatory, antioxidant, anxiolytic, and immunomodulatory effects [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. This has led to investigations on the use of CBD in poultry to determine its effects on stress relief, immunity, and performance. In addition, the endocannabinoid system (ECS) is a signaling system responsible for regulating numerous physiological functions, including pain, inflammation, appetite, immune response, as well as oxidative stress reduction [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. This system has been identified in both invertebrates [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e] and vertebrates, including poultry (chicken) [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], thus highlighting its evolutionary conservation and fundamental role in maintaining physiological balance across vertebrates. The ECS consists of endocannabinoids, cannabinoid receptors (CB1 and CB2), and metabolic enzymes [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Endocannabinoids are bioactive lipids produced from long-chain polyunsaturated fatty acids and consist of ethers, amides, and esters [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. CB1 receptors are localized in the central nervous system; however, they are also found in the peripheral nervous system, including the liver and adipose tissue. In contrast, CB2 receptors are found predominantly in the peripheral nervous system, e.g., in the liver, kidney, and adipose tissue. The enzymes involved in the ECS regulate the balance of endocannabinoid levels \u003cem\u003evia\u003c/em\u003e biosynthetic and catabolic pathways [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThe liver has a significant role in both human and animal health. In laying hens, the liver is essential for metabolic regulation as well as the synthesis of egg-yolk precursors, such as vitellogenin and yolk lipoproteins. Any changes in liver function can cause lipid metabolism disorders, damage nutrient transport, affect reproduction performance, and ultimately influence egg production and quality. Moreover, many metabolic and inflammatory responses are controlled at the cell level by gene expression and the corresponding proteins. Regulation of genes involved in inflammatory and lipid metabolism pathways is key to understanding how the body responds to dietary interventions and environmental challenges. Among these, the \u003cem\u003eFADS1\u003c/em\u003e and \u003cem\u003eFADS2\u003c/em\u003e genes are of particular interest. These genes encode delta-5 desaturase and delta-6 desaturase enzymes, respectively, which play a central role in the conversion of linoleic acid (LA) and alpha-linolenic acid (ALA) into longer-chain PUFAs, such as arachidonic acid (AA) and eicosapentaenoic acid (EPA). These products, in turn, are key players in immune and inflammatory reactions [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. In addition, peroxisome proliferator-activated receptor gamma (\u003cem\u003ePPAR-γ\u003c/em\u003e) is a nuclear receptor involved in adipogenesis, glucose metabolism, and anti-inflammatory responses. Activation of \u003cem\u003ePPAR-γ\u003c/em\u003e has been linked to reduced inflammation and enhanced lipid metabolism, and its expression in the liver and adipose tissues plays a crucial role in maintaining metabolic homeostasis [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Therefore, the expression of these genes provides valuable insights into the mechanisms of liver function in response to various physiological and dietary factors, including the modulation of inflammation and lipid metabolism in laying hens.\u003c/p\u003e\u003cp\u003eThe present study investigates, for the first time, the influence of the water-soluble emulsion of cannabidiol (CBD) on the egg yolk fatty acid profile, liver health, and the gene expression associated with lipid metabolism in laying hens. This represents a novel approach in poultry research, as the potential of CBD as a dietary modulator of lipid metabolism in avian species remains largely unexplored.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eExperiment Design\u003c/h2\u003e\u003cp\u003eThe research was conducted on 180 Lohmann Brown Classic laying hens (26-week-old, purchased from a commercial farm) at the Research and Education Station in Swojczyce, belonging to Wrocław University of Environmental and Life Sciences (Wrocław, Poland). The animals were obtained from a commercial farm. The experimental design used in this investigation was a Completely Randomized Design, consisting of 5 treatments with 12 replications for each treatment, using 3 hens per replication (36 hens per group). Each replication was a separate cage. Hens were allocated into standard furnished cages (0.125 m\u0026sup2; per bird) in a random order. The environmental conditions were maintained according to Lohmann\u0026rsquo;s recommendations i.e., average temperature of 18\u0026deg;C, humidity 65%, and a photoperiod of 14 hours of light and 10 hours of darkness (14L:10D) [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Water-soluble cannabidiol (CBD) emulsion was administered in the drinking water for 15 weeks.\u003c/p\u003e\u003cp\u003eThe birds were divided into five experimental groups: a control group receiving water without additives (C), a blank group receiving water with an emulsifier (B), and three CBD-treated groups, which received water supplemented with 20, 40, or 80 mg/kg BW of CBD along with an emulsifier (groups I, II, III, respectively). All experimental groups received the same basal diet \u003cem\u003ead libitum\u003c/em\u003e. Diets were formulated based on CVB [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e] nutrient standards. Additional information about diets is presented in the supplementary material (Tables S1 and S2).\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eCBD solution\u003c/h3\u003e\n\u003cp\u003eAn emulsion (CBD) was prepared with a cannabidiol (CBD) isolate (Purity- 99.14%; A-Sense Ltd., Puławy, Poland) and rapeseed lecithin as the emulsifier (Bunge Poland Ltd., Brzeg, Poland). The CBD content was verified by chromatographic analysis, using an Agilent 1290 Infinity II system equipped with a thermostated Ascentis Express C8 column (2.7 \u0026micro;m, 150 \u0026times; 3.0 mm; Merck 53853-U) maintained at 30\u0026deg;C. Isocratic elution was performed using a mobile phase composed of 27% solvent A (5 mM ammonium formate with 0.1% formic acid in water) and 73% solvent B (0.1% formic acid in acetonitrile). Calibration standards were prepared using a certified reference material (CRM, 1.0 mg/mL in methanol; MERCK C-045). A stock solution was made, composed of 2% rapeseed phospholipids and 100 mg/mL of CBD isolate. Pre-homogenization was done by means of the T 25 digital ULTRA-TURRAX homogenizer (IKA, Warsaw, Poland) with an S 25 EC-T-C-18 G ST probe running at 12,000 rpm for 10 min. The resulting suspension was subjected to high-pressure homogenization (M-110P Microfluidizer, processor) at 200 MPa for five passes through the system.\u003c/p\u003e\u003cp\u003eThe emulsion was diluted at the necessary concentrations according to the average body weight (BW) of each experimental group. To verify the actual CBD intake, working solutions were sampled and analysed at the start and after 13 weeks of administration. The actual CBD consumption per kilogram of body weight was calculated at the start of the treatment period and reassessed after 13 weeks. Additional information supporting this assessment is presented in the supplementary Excel spreadsheet (Tables S3, S4, and S5).\u003c/p\u003e\n\u003ch3\u003eEggs collection and Fatty Acid Methyl Ester (FAME) analysis\u003c/h3\u003e\n\u003cp\u003eEggs were collected at two time points during the experiment: days 0 and 90, with three eggs per cage, 180 eggs per collection in total. Egg yolks were separated from the albumen, weighed, and transferred to plates. Each plate contained a pooled sample of three yolks from a single replication (cage). The yolks were thoroughly stirred to achieve a homogenized consistency.\u003c/p\u003e\u003cp\u003eHomogenized yolk samples from each group were lyophilized using a Labconco freeze dryer under the following conditions: initial shelf temperature of -40\u0026deg;C with a holding time of 1 hour, followed by heating at a rate of 1\u0026deg;C per minute to 25\u0026deg;C, where samples were held for 24 hours. For FAME preparation, 100\u0026thinsp;\u0026plusmn;\u0026thinsp;1 mg of lyophilized yolk was weighed into screw-capped tubes. Each tube was supplemented with 4 mL of 0.5 M NaOH in methanol and 14% BF₃ in methanol. The tubes were tightly sealed and incubated in a water bath at 70\u0026deg;C for 30 minutes. After incubation, the tubes were rapidly cooled in ice water and 6 mL of hexane was added. The mixture was vortexed for 30 seconds, and the hexane layer was collected. The extracted hexane phase was dried using an anhydrous MgSO₄ filtration system (PP tube with PE frits, 20 \u0026micro;m porosity) and evaporated under reduced pressure. The residue was dissolved in 1.5 mL of hexane and subjected to chromatographic analysis. FAMEs were analyzed using an Agilent 6890N gas chromatograph (GC) equipped with a 5973 MSD detector and a split/splitless injector. Separation was performed using an HP-88 column ((88%-cyanopropyl) aryl-polysiloxane; length: 100 m, ID: 0.25 mm, film thickness: 0.25 \u0026micro;m). The oven temperature program was as follows: initial temperature of 60\u0026deg;C (2 min hold), increasing at 20\u0026deg;C/min to 180\u0026deg;C, followed by a ramp of 3\u0026deg;C/min to 220\u0026deg;C (15 min hold), and a final increase of 5\u0026deg;C/min to 250\u0026deg;C (8 min hold), with a total run time of 50.33 minutes. The injection volume was 2 \u0026micro;L, applied in a 10:1 split mode [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e].\u003c/p\u003e\n\u003ch3\u003eBlood and liver sampling\u003c/h3\u003e\n\u003cp\u003eAt the end of the experiment, six hens per group (30 hens in total) were euthanized for the collection of blood and liver samples. Euthanasia, following Annex IV to Directive 2010/63/EU of the European Parliament and of the Council of 22 September 2010 on the protection of animals used for scientific purposes [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e], was carried out by stunning the birds with carbon dioxide and completed by exsanguination. Blood from each hen was immediately transferred to the tubes (volume: 10ml) containing the anticoagulant substance. Liver samples were collected under strictly aseptic conditions. Tissue sections measuring approximately 1 \u0026times; 1 \u0026times; 0.5 cm were promptly immersed in 10% neutral buffered formalin (pH 7.2\u0026ndash;7.4) at a minimum tissue-to-fixative volume ratio of 1:10.\u003c/p\u003e\n\u003ch3\u003eLiver histology\u003c/h3\u003e\n\u003cp\u003eFixation was performed at ambient temperature for 24 hours. Following fixation, the samples were rinsed under running tap water for 12 hours to remove residual fixing solution. The tissues were subsequently dehydrated through a graded ethanol series (70%, 80%, 90%, 96%, and 100%), cleared in xylene, and infiltrated with paraffin wax (melting point 56\u0026ndash;58\u0026deg;C). Serial sections of 5 \u0026micro;m thickness were cut using a rotary microtome and mounted on poly-L-lysine-coated glass slides. The sections were stained with hematoxylin and eosin (H\u0026amp;E; Merck, Darmstadt, Germany) according to standardized histological protocols, which included rehydration through decreasing ethanol concentrations, hematoxylin staining, differentiation in acid alcohol, counterstaining with eosin, dehydration, and permanent mounting. Histomorphological evaluation was performed using a Stemi 508 stereo microscope (Carl Zeiss, Jena, Germany) equipped with an Axiocam 208 high-resolution color camera. Digital images were acquired and processed using ZEN\u0026trade; image analysis software (Carl Zeiss, Jena, Germany).\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eBlood serum assessment\u003c/h2\u003e\u003cp\u003eTubes containing blood samples were transferred to the laboratory. In order to separate the serum, samples were centrifuged at 6000 rpm for 10 minutes. The serum for each replication was transferred to the 1.5 ml tubes to analyze for AST, ALT, Total Cholesterol, HDL, LDL, and TG with ABX Pentra reagents (Horiba ABX). Serum biochemical assessment was conducted with a Pentra 400 spectrophotometric device (Horiba ABX- France).\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eAnalysis of gene expression in liver samples\u003c/h3\u003e\n\u003cp\u003eThe total RNA was isolated from liver tissue collected after the experiment. The 200 mg of the specimen was homogenized using 1 mL of TRI Reagent\u0026reg; (Sigma-Aldrich/Merck, Poznan, Poland). The procedure was performed according to the manufacturer\u0026rsquo;s protocol, with a few modifications, including a 1-hour centrifugation at 12,000 \u0026times; g during precipitation, and twice washing of RNA pellets with 80% ethanol to ensure the RNA purity. The extracted RNA was diluted in nuclease-free water (Sigma-Aldrich/Merck, Poznan, Poland), and its concentration and purity were assessed using a DS-11 Fx nanospectrophotometer (DeNovix, Wilmington, DE, USA). The samples with an A260/A280 ratio of 2.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2 were used for gene expression analysis.\u003c/p\u003e\u003cp\u003eTo ensure RNA integrity, gDNA digestion was performed using the DNase I, RNase-free enzyme (1 U/\u0026micro;L; Thermo Scientific, Warsaw, Poland). The obtained total RNA (1 ug) was reverse-transcribed using the Tetro cDNA Synthesis Kit (Bioline Reagents Limited, London, UK). Both reactions, gDNA digestion and reverse transcription) were performed in a T100 Thermal Cycler (Bio-Rad, Hercules, CA, USA) according to the manufacturer\u0026rsquo;s instructions.\u003c/p\u003e\u003cp\u003eQuantitative real-time PCR (RT-qPCR) was performed using the SensiFAST SYBR\u0026reg; \u0026amp; Fluorescein Kit (Bioline Reagents Ltd., London, UK) on a CFX Opus 384 Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA). Each 10 \u0026micro;L reaction contained 1 \u0026micro;L of cDNA, 5 \u0026micro;L of Master Mix, and gene-specific primers at a final concentration of 400 nM. Thermal cycling conditions included initial denaturation at 95\u0026deg;C for 2 minutes, followed by 40 cycles of 95\u0026deg;C for 15 seconds, annealing at primer-specific temperature, i.e., 62\u0026deg;C for 15 seconds, and 72\u0026deg;C for 15 seconds of elongation. The expression of target genes was normalized against the expression of two reference genes, i.e., coding GAPDH and β-actin. Relative gene expression was determined using the 2\u003csup\u003e\u0026ndash;ΔΔCq\u003c/sup\u003e method, with normalization based on the gene exhibiting the lowest expression level (RQMAX approach) [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e], and the data were presented in a logarithmic scale. The specificity of each product was determined based on its melting temperature. Primer sequences are described in detail in Supplementary Table S6.\u003c/p\u003e\n\u003ch3\u003eStatistical data analysis\u003c/h3\u003e\n\u003cp\u003eData analysis for egg yolk fatty acid composition and liver parameters was performed using R software, version 4.4.0 [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Descriptive statistics, including the mean and standard deviation, were calculated. Group comparisons for these parameters were conducted using one-way analysis of variance (ANOVA), followed by Fisher\u0026rsquo;s Least Significant Difference (LSD) post-hoc test. For molecular assessments, data were derived from both biological and technical replicates within each experimental group. Statistical comparisons were made using one-way ANOVA, followed by Tukey\u0026rsquo;s post-hoc test where applicable. These analyses were performed using GraphPad Prism (version 10.3.1; GraphPad Software, CA, USA), with statistical significance set at p\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cb\u003eCBD enhances yolk PUFA profile and HDL levels, indicating improved lipid transport, with no significant hepatic alterations\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe fatty acid profile of egg yolks across the experimental groups is presented in Tables\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and \u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. Except for the arachidonic acid (C20:4 n-6), all measured fatty acid levels showed significant differences between day 0 and day 90.\u003c/p\u003e\u003cp\u003e1) Saturated Fatty Acids (SFA)\u003c/p\u003e\u003cp\u003eAfter 90 days of cannabidiol (CBD) supplementation, Group I exhibited the highest concentrations of myristic acid (C14:0) and palmitic acid (C16:0). The highest level of stearic acid (C18:0) was detected in Group B. Total SFA levels differed significantly among the groups both at the beginning of the experiment and after three months. Notably, Group III had the highest SFA concentration at the start but the lowest by day 90 (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eYolk saturated fatty acids (% of total) on day 0 and 90 of CBD use\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"4\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eFatty Acid\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eGroup\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eDay 0 (% \u0026plusmn; SD)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eDay 90 (% \u0026plusmn; SD)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eC 14:0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eC\u003csup\u003e1\u003c/sup\u003e\u003c/p\u003e\u003cp\u003eB\u003c/p\u003e\u003cp\u003eI\u003c/p\u003e\u003cp\u003eII\u003c/p\u003e\u003cp\u003eIII\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.33\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003c/p\u003e\u003cp\u003e0.35\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09\u003c/p\u003e\u003cp\u003e0.37\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08\u003c/p\u003e\u003cp\u003e0.34\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003c/p\u003e\u003cp\u003e0.32\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.31\u003csup\u003eb\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e\u003cp\u003e0.29\u003csup\u003eb\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06\u003c/p\u003e\u003cp\u003e0.38\u003csup\u003ea\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08\u003c/p\u003e\u003cp\u003e0.39\u003csup\u003ea\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003c/p\u003e\u003cp\u003e0.31\u003csup\u003eb\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ep- value\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.51\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;0.001**\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eC 16:0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eC\u003c/p\u003e\u003cp\u003eB\u003c/p\u003e\u003cp\u003eI\u003c/p\u003e\u003cp\u003eII\u003c/p\u003e\u003cp\u003eIII\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e26.12\u0026thinsp;\u0026plusmn;\u0026thinsp;1.24\u003c/p\u003e\u003cp\u003e25.52\u0026thinsp;\u0026plusmn;\u0026thinsp;0.62\u003c/p\u003e\u003cp\u003e25.32\u0026thinsp;\u0026plusmn;\u0026thinsp;0.9\u003c/p\u003e\u003cp\u003e25.93\u0026thinsp;\u0026plusmn;\u0026thinsp;0.77\u003c/p\u003e\u003cp\u003e25.51\u0026thinsp;\u0026plusmn;\u0026thinsp;0.53\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e26.43\u003csup\u003ea\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;0.98\u003c/p\u003e\u003cp\u003e25.49\u003csup\u003eb\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;0.77\u003c/p\u003e\u003cp\u003e26.96\u003csup\u003ea\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;1.61\u003c/p\u003e\u003cp\u003e25.11\u003csup\u003eb\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;0.56\u003c/p\u003e\u003cp\u003e25.11\u003csup\u003eb\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;0.56\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ep- value\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.14\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;0.001**\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eC 18:0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eC\u003c/p\u003e\u003cp\u003eB\u003c/p\u003e\u003cp\u003eI\u003c/p\u003e\u003cp\u003eII\u003c/p\u003e\u003cp\u003eIII\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e21.7\u0026thinsp;\u0026plusmn;\u0026thinsp;3.6\u003c/p\u003e\u003cp\u003e23.95\u0026thinsp;\u0026plusmn;\u0026thinsp;1.68\u003c/p\u003e\u003cp\u003e22.14\u0026thinsp;\u0026plusmn;\u0026thinsp;3.71\u003c/p\u003e\u003cp\u003e22.84\u0026thinsp;\u0026plusmn;\u0026thinsp;2.41\u003c/p\u003e\u003cp\u003e24.32\u0026thinsp;\u0026plusmn;\u0026thinsp;1.55\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e20.19\u003csup\u003eb\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;0.85\u003c/p\u003e\u003cp\u003e24.94\u003csup\u003ea\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;0.61\u003c/p\u003e\u003cp\u003e21.1\u003csup\u003eb\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;1.83\u003c/p\u003e\u003cp\u003e21.24\u003csup\u003eb\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;1.54\u003c/p\u003e\u003cp\u003e21.03\u003csup\u003eb\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;1.63\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ep- value\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;0.001**\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSFA\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eC\u003c/p\u003e\u003cp\u003eB\u003c/p\u003e\u003cp\u003eI\u003c/p\u003e\u003cp\u003eII\u003c/p\u003e\u003cp\u003eIII\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e48.15\u003csup\u003ebc\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;2.54\u003c/p\u003e\u003cp\u003e49.82\u003csup\u003eab\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;1.33\u003c/p\u003e\u003cp\u003e47.83\u003csup\u003ec\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;3\u003c/p\u003e\u003cp\u003e49.1\u003csup\u003eab\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;1.82\u003c/p\u003e\u003cp\u003e50.15\u003csup\u003ea\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;1.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e46.93\u003csup\u003ec\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;1.05\u003c/p\u003e\u003cp\u003e50.72\u003csup\u003ea\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;0.94\u003c/p\u003e\u003cp\u003e48.44\u003csup\u003eb\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;0.95\u003c/p\u003e\u003cp\u003e46.74\u003csup\u003ec\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;1.28\u003c/p\u003e\u003cp\u003e46.44\u003csup\u003ec\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;1.46\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ep- value\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.04*\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;0.001**\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003csup\u003e1\u003c/sup\u003e C (Control): water without additive; B (Blank): water\u0026thinsp;+\u0026thinsp;emulsifier; I: water\u0026thinsp;+\u0026thinsp;CBD 20 mg/kg BW\u0026thinsp;+\u0026thinsp;emulsifier; II: water\u0026thinsp;+\u0026thinsp;CBD 40 mg/kg BW\u0026thinsp;+\u0026thinsp;emulsifier; III: water\u0026thinsp;+\u0026thinsp;CBD 80 mg/kg BW\u0026thinsp;+\u0026thinsp;emulsifier.\u003c/p\u003e\u003cp\u003e\u003csup\u003e2\u003c/sup\u003e Saturated Fatty Acids (SFA): Myristic (C14:0); Palmitic (C16:0); Stearic (C18:0);\u003c/p\u003e\u003cp\u003e\u003csup\u003ea,b,c\u003c/sup\u003e Values within a column with different superscripts differ significantly at the level P\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e\u003cp\u003e2) Monounsaturated Fatty Acids (MUFA)\u003c/p\u003e\u003cp\u003eGroup I and B demonstrated the highest levels of palmitoleic acid (C16:1 n-7). After 90 days, the overall MUFA content was the highest in Groups C and II (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e3) Polyunsaturated Fatty Acids (PUFA):\u003c/p\u003e\u003cp\u003eGroup III exhibited the highest concentrations of linoleic acid (C18:2 n-6), α-linolenic acid (C18:3 n-3), docosahexaenoic acid (C22:6 n-3), as well as total omega-3, omega-6, and overall PUFA levels (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). At the beginning of the experiment, the n3/n6 ratio for all groups ranged from 0.22 to 0.23. After 90 days, it ranged from 0.20 to 0.24, with Group B showing the lowest and Groups I and III the highest values, respectively.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eYolk MUFA and PUFA levels (% total) at day 0 and day 90 with CBD\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"4\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eFatty Acid\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eGroup\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eDay 0 (% \u0026plusmn; SD)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eDay 90 (% \u0026plusmn; SD)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eC 16:1 (n-7)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eC\u003csup\u003e1\u003c/sup\u003e\u003c/p\u003e\u003cp\u003eB\u003c/p\u003e\u003cp\u003eI\u003c/p\u003e\u003cp\u003eII\u003c/p\u003e\u003cp\u003eIII\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e2.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.37\u003c/p\u003e\u003cp\u003e2.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3\u003c/p\u003e\u003cp\u003e2.63\u0026thinsp;\u0026plusmn;\u0026thinsp;0.29\u003c/p\u003e\u003cp\u003e2.52\u0026thinsp;\u0026plusmn;\u0026thinsp;0.32\u003c/p\u003e\u003cp\u003e2.34\u0026thinsp;\u0026plusmn;\u0026thinsp;0.25\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e2.47\u003csup\u003eb\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09\u003c/p\u003e\u003cp\u003e3\u003csup\u003ea\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;0.45\u003c/p\u003e\u003cp\u003e2.86\u003csup\u003ea\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3\u003c/p\u003e\u003cp\u003e2.61\u003csup\u003eb\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;0.14\u003c/p\u003e\u003cp\u003e2.47\u003csup\u003eb\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ep- value\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.19\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;0.001**\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eC 18:1 (n-9)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eC\u003c/p\u003e\u003cp\u003eB\u003c/p\u003e\u003cp\u003eI\u003c/p\u003e\u003cp\u003eII\u003c/p\u003e\u003cp\u003eIII\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e24.1\u0026thinsp;\u0026plusmn;\u0026thinsp;3.14\u003c/p\u003e\u003cp\u003e23\u0026thinsp;\u0026plusmn;\u0026thinsp;2.16\u003c/p\u003e\u003cp\u003e24.49\u0026thinsp;\u0026plusmn;\u0026thinsp;3.55\u003c/p\u003e\u003cp\u003e23.63\u0026thinsp;\u0026plusmn;\u0026thinsp;2.56\u003c/p\u003e\u003cp\u003e22.28\u0026thinsp;\u0026plusmn;\u0026thinsp;1.27\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e25.57\u003csup\u003ea\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;0.96\u003c/p\u003e\u003cp\u003e20.1\u003csup\u003ec\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;1.12\u003c/p\u003e\u003cp\u003e24\u003csup\u003eb\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;0.68\u003c/p\u003e\u003cp\u003e25.81\u003csup\u003ea\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;1.33\u003c/p\u003e\u003cp\u003e23.99\u003csup\u003eb\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;1.18\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ep- value\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.28\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;0.001**\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMUFA\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eC\u003c/p\u003e\u003cp\u003eB\u003c/p\u003e\u003cp\u003eI\u003c/p\u003e\u003cp\u003eII\u003c/p\u003e\u003cp\u003eIII\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e26.6\u0026thinsp;\u0026plusmn;\u0026thinsp;3.42\u003c/p\u003e\u003cp\u003e25.4\u0026thinsp;\u0026plusmn;\u0026thinsp;2.31\u003c/p\u003e\u003cp\u003e27.12\u0026thinsp;\u0026plusmn;\u0026thinsp;3.48\u003c/p\u003e\u003cp\u003e26.15\u0026thinsp;\u0026plusmn;\u0026thinsp;2.74\u003c/p\u003e\u003cp\u003e24.62\u0026thinsp;\u0026plusmn;\u0026thinsp;1.32\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e28.04\u003csup\u003ea\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;1.03\u003c/p\u003e\u003cp\u003e23.1\u003csup\u003ec\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;1.07\u003c/p\u003e\u003cp\u003e26.85\u003csup\u003eb\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;0.97\u003c/p\u003e\u003cp\u003e28.42\u003csup\u003ea\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;1.33\u003c/p\u003e\u003cp\u003e26.47\u003csup\u003eb\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;1.22\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ep- value\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.21\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" 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colname=\"c4\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;0.001**\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePUFA\u003csup\u003e3\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eC\u003c/p\u003e\u003cp\u003eB\u003c/p\u003e\u003cp\u003eI\u003c/p\u003e\u003cp\u003eII\u003c/p\u003e\u003cp\u003eIII\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e25.25\u0026thinsp;\u0026plusmn;\u0026thinsp;1.04\u003c/p\u003e\u003cp\u003e24.77\u0026thinsp;\u0026plusmn;\u0026thinsp;1.35\u003c/p\u003e\u003cp\u003e25.05\u0026thinsp;\u0026plusmn;\u0026thinsp;0.94\u003c/p\u003e\u003cp\u003e24.75\u0026thinsp;\u0026plusmn;\u0026thinsp;1.29\u003c/p\u003e\u003cp\u003e25.22\u0026thinsp;\u0026plusmn;\u0026thinsp;1.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e25.03\u003csup\u003ec\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;1.02\u003c/p\u003e\u003cp\u003e26.19\u003csup\u003eb\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;1.08\u003c/p\u003e\u003cp\u003e24.7\u003csup\u003ec\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;1.58\u003c/p\u003e\u003cp\u003e24.84\u003csup\u003ec\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;0.74\u003c/p\u003e\u003cp\u003e27.09\u003csup\u003ea\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;0.8\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ep- value\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.74\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;0.001**\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003csup\u003e1\u003c/sup\u003e C (Control): water without additive; B (Blank): water\u0026thinsp;+\u0026thinsp;emulsifier; I: water\u0026thinsp;+\u0026thinsp;CBD 20 mg/kg BW\u0026thinsp;+\u0026thinsp;emulsifier; II: water\u0026thinsp;+\u0026thinsp;CBD 40 mg/kg BW\u0026thinsp;+\u0026thinsp;emulsifier; III: water\u0026thinsp;+\u0026thinsp;CBD 80 mg/kg BW\u0026thinsp;+\u0026thinsp;emulsifier.\u003c/p\u003e\u003cp\u003e\u003csup\u003e2\u003c/sup\u003e Mono Unsaturated Fatty Acids (MUFA): Palmitoleic (C16:1 (n-7); Oleic (18:1 (n-9);\u003c/p\u003e\u003cp\u003e\u003csup\u003e3\u003c/sup\u003e Poly Unsaturated Fatty Acids (PUFA): Linoleic (18:2 (n-6); Alpha-linolenic acid (ALA) (18:3 (n-3); Arachidonic (AA) (20:4 (n-6); Docosahexaenoic (DHA, Cervonic (22:6 (n-3).\u003c/p\u003e\u003cp\u003e\u003csup\u003ea,b,c\u003c/sup\u003e Values within a column with different superscripts differ significantly at the level P\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e\u003cp\u003eThe results of the serum biochemical parameters are presented in Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. Aspartate aminotransferase (AST) levels were significantly rose in Group C compared to the other groups. Besides, high-density lipoprotein (HDL) levels were higher in Groups I and III. No statistically significant differences were observed for the remaining serum parameters.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eAnalysis of biochemical blood serum after 15 weeks\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"5\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eParameter\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eGroup\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eMean\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eSD\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eP- value\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"4\" rowspan=\"5\"\u003e\u003cp\u003eAST (U/L)\u003csup\u003e1\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eC\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e98.68\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e19.18\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.033*\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eB\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e77.69\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e19.83\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eI\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e76.24\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e13.76\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eII\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e79.34\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e15.94\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eIII\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e81.40\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e21.47\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"4\" rowspan=\"5\"\u003e\u003cp\u003eALT (U/L)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eC\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e10.37\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e5.19\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.502\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eB\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e11.90\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e6.95\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eI\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e9.74\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e6.49\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eII\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e9.07\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e3.06\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eIII\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e8.15\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e3.75\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"4\" rowspan=\"5\"\u003e\u003cp\u003eCholesterol (mmol/L)\u003csup\u003e3\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eC\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1.75\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.87\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.336\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eB\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e2.32\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.97\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eI\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e2.05\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.70\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eII\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e2.17\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.80\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eIII\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e2.40\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.64\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"4\" rowspan=\"5\"\u003e\u003cp\u003eHDL (mmol/L)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eC\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.61\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.15\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.024*\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eB\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.69\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eI\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.76\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.18\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eII\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.73\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.24\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eIII\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.93\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.37\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"4\" rowspan=\"5\"\u003e\u003cp\u003eLDL (mmol/L)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eC\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.38\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.28\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.408\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eB\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.56\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.32\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eI\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.45\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.19\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eII\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.53\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.27\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eIII\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.58\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.27\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"4\" rowspan=\"5\"\u003e\u003cp\u003eTG (mmol/L)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eC\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e5.84\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e4.54\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.392\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eB\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e9.48\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e6.23\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eI\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e6.64\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e4.44\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eII\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e7.24\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e4.21\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eIII\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e6.58\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e3.38\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"5\"\u003e\u003csup\u003e1\u003c/sup\u003eUnits per liter\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd colspan=\"5\"\u003e\u003csup\u003e2\u003c/sup\u003eC (Control): water without additive; B (Blank): water\u0026thinsp;+\u0026thinsp;emulsifier; I: water\u0026thinsp;+\u0026thinsp;CBD 20 mg/kg BW\u0026thinsp;+\u0026thinsp;emulsifier; II: water\u0026thinsp;+\u0026thinsp;CBD 40 mg/kg BW\u0026thinsp;+\u0026thinsp;emulsifier; III: water\u0026thinsp;+\u0026thinsp;CBD 80 mg/kg BW\u0026thinsp;+\u0026thinsp;emulsifier\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd colspan=\"5\"\u003e\u003csup\u003e3\u003c/sup\u003e Millimoles per liter\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003csup\u003ea,b\u003c/sup\u003e Values within a column with different superscripts differ significantly at the level P\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e\u003cp\u003eThe liver histology was consistent across all experimental groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), showing a homogeneous tissue structure without signs of disruption, degeneration, or hepatocyte necrosis. There was no evidence of hepatocyte vacuolization (cytoplasmic vacuoles appearing as empty spaces), cellular swelling, or nuclear displacement. Furthermore, no vacuolar degeneration characterized by a diffuse, foamy, or feathery appearance of hepatocytes was observed. Taken together, the histological assessment of the liver revealed no significant alterations in any of the examined groups.\u003c/p\u003e\u003cp\u003e\u003cb\u003eHepatic levels of mRNA for\u003c/b\u003e \u003cb\u003ePPAR-γ\u003c/b\u003e \u003cb\u003edecrease in response to high-dose CBD, while\u003c/b\u003e \u003cb\u003eFADS1\u003c/b\u003e \u003cb\u003eand\u003c/b\u003e \u003cb\u003eFADS2\u003c/b\u003e \u003cb\u003eremain unchanged\u003c/b\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eRT-qPCR analysis revealed that expression of \u003cem\u003ePPAR-γ\u003c/em\u003e is modulated by the emulsion with the highest CBD concentration. The significant reduction in \u003cem\u003ePPAR-γ\u003c/em\u003e expression in group III suggests that the treatment may have an inhibitory effect on lipid metabolic pathways or adipogenic differentiation processes. This decrease could also indicate a cellular stress response or the activation of pro-inflammatory pathways that are known to downregulate \u003cem\u003ePPAR-γ\u003c/em\u003e transcription. On the other hand, the stable levels of \u003cem\u003eFADS1\u003c/em\u003e and \u003cem\u003eFADS2\u003c/em\u003e across all experimental groups suggest that the treatment did not adversely affect the genes coding major enzymes responsible for hepatic fatty acid desaturation. The heatmap illustration was also prepared to demonstrate that \u003cem\u003ePPAR-γ\u003c/em\u003e exhibited the lowest expression in the hen\u0026rsquo;s liver among the tested genes, simultaneously showing the most dynamic regulation. The expression signature also indicated that \u003cem\u003ePPAR-γ\u003c/em\u003e is responsive to CBD treatment and may represent a sensitive molecular target. The accumulation of \u003cem\u003eFADS1\u003c/em\u003e and \u003cem\u003eFADS2\u003c/em\u003e transcripts in the hen's liver was higher than \u003cem\u003ePPAR-γ\u003c/em\u003e. Moreover, the stability in mRNA expression in \u003cem\u003eFADS1\u003c/em\u003e and \u003cem\u003eFADS2\u003c/em\u003e implies that core lipid metabolic pathways are not significantly altered by CBD supplementation (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe use of bioactive substances in poultry diets to promote animal welfare and enhance the safety and quality of poultry-derived products has recently garnered significant interest. Most recently, cannabidiol (CBD), a non-psychoactive phytochemical derived from \u003cem\u003eCannabis sativa\u003c/em\u003e, has gained recognition as a promising phytogenic additive with multiple potential benefits for systemic metabolism. Its biological activity is primarily mediated through interaction with the endocannabinoid system, influencing key processes such as lipid metabolism, as well as inflammatory signaling, oxidative stress responses, and energy homeostasis. These biological properties make CBD a compelling candidate for applications aimed at supporting the metabolic health of animals. The role of CBD is particularly notable in the context of its use as a functional nutrition strategy for livestock and poultry. In the context of liver physiology, CBD has exhibited a range of beneficial effects across \u003cem\u003ein vitro\u003c/em\u003e, \u003cem\u003ein vivo\u003c/em\u003e, and clinical models of metabolic dysfunction. These include the attenuation of hepatic inflammation, reduction of oxidative stress, and modulation of lipid accumulation, indicating its multifaceted role as a regulator of hepatic metabolism, cellular homeostasis, and disease progression.\u003c/p\u003e\u003cp\u003eIn the current study, we aimed to investigate the effects of dietary CBD-emulsion on lipid metabolism and hepatic function in laying hens, with a particular focus on yolk PUFA deposition, liver histopathology, and gene expression.\u003c/p\u003e\u003cp\u003eSeveral studies have reported the beneficial effects of CBD in pets and rodents, including alleviation of stress and anxiety [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e] and relief of pain [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Despite growing interest in phytogenic additives, research on purified CBD in poultry nutrition remains scarce, with most studies to date concentrating on hemp seeds or processing byproducts rather than isolated CBD. Gakhar et al. [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e] reported that including 20% hemp seed in the diet of laying hens increased omega-3 fatty acids in egg yolk. Similarly, another study on broiler chicken showed that the addition of 7.5% of hemp seed meal decreased serum cholesterol concentration [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Halle and Sch\u0026ouml;ne [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e] reported that supplementation of diets containing 5%, 10% or 15% hemp seed cake in laying hens over six months decreased SFAs and MUFAs levels while linoleic and linolenic acid (PUFAs) levels increased. A 25% of hemp seed alone or combined with 2% of ginger or turmeric in a diet of laying hens increased omega-3 content in the egg yolk, improved the n-3/n-6 fatty acid, and decreased the proportion of SFAs [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Besides, an increased content of egg yolk PUFAs was reported by Kasula et al. [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e] when laying hens were fed with diets containing 10, 20, or 30% of hemp seed cake over 19 weeks.\u003c/p\u003e\u003cp\u003eIn the present study, the level of egg yolk PUFAs. e.g., omega-3 and omega-6 raised significantly in group III (CBD 80 mg/kg BW) after 90 days of CBD administration. This finding suggests that CBD may facilitate the mobilization and deposition of PUFAs into the yolk probably via regulation of the lipid transport mechanisms. Elevation of high-density lipoprotein (HDL) serum level supports the idea of increased lipid delivery to the ovary. According to Neijat et al. [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e], hempseed products enhanced egg yolk nutritional value and n-3 PUFA levels. Furthermore, a recent meta-analysis by Sopian et al. [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e] demonstrated that hemp-derived products can perform as functional feed ingredients for laying hens, enhancing yolk pigmentation and enriching long-chain omega-3 fatty acids, particularly DHA, without impairing performance parameters when inclusion levels are well managed.\u003c/p\u003e\u003cp\u003eUnderstanding the molecular mechanisms underlying CBD\u0026rsquo;s effects is crucial for explaining its influence on lipid metabolism and tissue-specific responses. CBD interacts with a variety of molecular targets, including cannabinoid receptors (CB1 and CB2), peroxisome proliferator-activated receptors (PPARs), G protein-coupled receptor 55 (GPR55), adenosine receptors, and transient receptor potential (TRP) channels, which collectively mediate all its biological effects [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. Among these, \u003cem\u003ePPAR-γ\u003c/em\u003e is of particular interest due to its key function in lipid metabolism and liver homeostasis. \u003cem\u003ePPAR-γ\u003c/em\u003e is a nuclear receptor that plays a central role in regulating gene expression and is also involved in adipocyte differentiation and adipogenesis. It functions as a transcriptional regulator that promotes the development and maturation of adipose tissue, making it a critical factor in maintaining energy balance and tissue remodeling [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. Chang et al. [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e] indicated that CBD can bind and activate \u003cem\u003ePPAR-γ\u003c/em\u003e; therefore, it could function as a \u003cem\u003ePPAR-γ\u003c/em\u003e agonist, which could improve adipogenesis. The interaction between CBD and \u003cem\u003ePPAR-γ\u003c/em\u003e is complex; whereas it may act as an agonist and stimulate adipogenesis, chronic high-dose exposure may cause a feedback inhibition or stress-related suppression of \u003cem\u003ePPAR-γ\u003c/em\u003e expression, contributing to hepatic lipid accumulation.\u003c/p\u003e\u003cp\u003eImportantly, \u003cem\u003ePPAR-γ\u003c/em\u003e may function as a metabolic sensor in the liver, responding to changes in cellular energy status and contributing to the regulation of pathways involved in lipid accumulation and early-stage metabolic disturbances that precede fibrogenesis [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eIndeed, the highest CBD dose (80 mg/kg BW) in the present study positively influenced the fatty acid composition of egg yolk with no signs of hepatic pathological changes. The liver is a primary site for energy storage and possesses remarkable ability to regenerate and adapt. Its adaptation to various conditions may involve, for instance, enhancing functional efficiency to maintain the body\u0026rsquo;s homeostasis. This adaptability is particularly crucial in responding to physiological challenges, such as exposure to an aqueous cannabidiol solution. However, at the molecular level, we observed a significant downregulation of \u003cem\u003ePPAR-γ\u003c/em\u003e expression. Such alterations may be attributed to the metabolic burden imposed by CBD biotransformation or to intensified lipid mobilization processes [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThe synthesis of longer-chain PUFAs from linoleic acid (LA) and alpha-linolenic acid (ALA) mainly depends on the action of Δ5 and Δ6 desaturase enzymes that are encoded by \u003cem\u003eFADS1\u003c/em\u003e and \u003cem\u003eFADS2\u003c/em\u003e genes, respectively [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. The Δ5 desaturase (encoded by \u003cem\u003eFADS1\u003c/em\u003e) is particularly important in the biosynthesis of longer-chain PUFAs, including arachidonic acid (ARA) and eicosapentaenoic acid (EPA) [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. In the current study, hepatic mRNA expression levels of both \u003cem\u003eFADS1\u003c/em\u003e and \u003cem\u003eFADS2\u003c/em\u003e were not significantly affected by the dietary levels of cannabidiol (CBD) administered to laying hens. The genes encode highly conserved enzymes, with an overall role in cellular homeostasis. Therefore, their stable expression across experimental conditions may reflect a tightly regulated mechanism necessary to maintain physiological balance, regardless of external dietary modulation. The signature of \u003cem\u003ePPAR-γ\u003c/em\u003e and \u003cem\u003eFASD1/2\u003c/em\u003e gene expression may also indicate that the observed increase in yolk PUFAs is more likely due to enhanced mobilization and transfer of existing fatty acids rather than increased hepatic synthesis.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis study provides novel insights into the impact of dietary CBD supplementation on lipid metabolism in laying hens, focusing on hepatic gene expression and fatty acid deposition in egg yolks. To our knowledge, this is the first report assessing the expression of \u003cem\u003ePPAR-γ\u003c/em\u003e, as well as \u003cem\u003eFADS1\u003c/em\u003e and \u003cem\u003eFADS2\u003c/em\u003e, in the context of CBD intake in poultry.\u003c/p\u003e\u003cp\u003eWe showed that the highest dose of CBD-emulsion in drinking water increased the egg yolk PUFAs content, possibly by boosting lipid mobilization and transport mechanisms. The liver did not show any pathological changes with the administration of different CBD doses. However, a decrease in \u003cem\u003ePPAR-γ\u003c/em\u003e expression at the mRNA level indicates a complex interaction between CBD, the ECS, and lipid metabolism pathways. Nevertheless, the stable expression of \u003cem\u003eFADS1\u003c/em\u003e and \u003cem\u003eFADS2\u003c/em\u003e genes suggests that desaturation activity is maintained, which may confirm continued PUFA synthesis.\u003c/p\u003e\u003cp\u003eMoreover, the elevated HDL levels may facilitate the transport of PUFAs from the liver to the developing yolk, providing a connection between liver lipid metabolism and yolk lipid deposition.\u003c/p\u003e\u003cp\u003eTogether, our findings contribute to the knowledge of the physiological and molecular effects of phytocannabinoids in animal production. We believe that such studies are essential for opening new avenues to optimize egg lipid profiles through non-traditional dietary strategies. Further research is required to clarify the precise role of the endocannabinoid system in poultry, to identify a safe CBD dose that enhances egg composition, and to expand future diagnostics to include assessments of enzyme activity in the liver.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll experimental procedures were approved by the Local Ethical Committee for Animal Experimentation in Wrocław (Resolution No. 017/2023/P1 of 19 April 2023). All animals were maintained under conditions specified by Directive 2010/63/EU on the protection of animals used for scientific purposes [26]. The animal-handling research team included the Ph.D. or M.S. in Animal Science, as well as experienced veterinary practitioners.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and analysed during the current study are available from the corresponding author on reasonable request.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the Wrocław University of Environmental and Life Sciences (Poland) as part of research project no N070/0008/23. The article is part of a Ph.D. dissertation titled \u0026ldquo;Cannabidiol- a Feed Additive that Improves Birds\u0026rsquo; Welfare and the Efficiency of Poultry Production\u0026rdquo;, prepared during the Doctoral School at the Wrocław University of Environmental and Life Sciences. The APC is financed by Wrocław University of Environmental and Life Sciences.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSF wrote the original manuscript and performed research; SF and SO designed the research; SF, AŚ, ŁB and AL conducted laboratory assessments and acquired data; AŚ and AL visualized the data; AŚ analyzed data; AŚ, AL and SO reviewed and edited the manuscript and validated data; SO and ŁB supervised the project; SO provided funding; MA and KO assisted in sample collection. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe gratefully acknowledge Dr. Anna Burek from the Department of Environmental Hygiene and Animal Welfare for her valuable support with the sample analysis of serum parameters.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eMilinsk MC, Murakami AE, Gomes STM, Matsushita M, de Souza NE (2003) Fatty acid profile of egg yolk lipids from hens fed diets rich in n-3 fatty acids. Food Chem 83:287-292\u003c/li\u003e\n\u003cli\u003eDjouss\u0026eacute; L, Gaziano JM (2008) Egg consumption in relation to cardiovascular disease and mortality: the Physicians\u0026rsquo; Health Study. Am J Clin Nutr 87:964-969\u003c/li\u003e\n\u003cli\u003eDjouss\u0026eacute; L, Gaziano JM, Buring JE, Lee I-M (2009) Egg consumption and risk of type 2 diabetes in men and women. 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Food Sci Anim Resour 44:988-1010\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-veterinary-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [BMC Veterinary Research](http://bmcvetres.biomedcentral.com/)","snPcode":"12917","submissionUrl":"https://submission.nature.com/new-submission/12917/3?","title":"BMC Veterinary Research","twitterHandle":"@BMC_series","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"CBD, Laying hens, Yolk deposition, Lipid metabolism, Gene expression","lastPublishedDoi":"10.21203/rs.3.rs-7387031/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7387031/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe present research evaluated the effect of water-soluble cannabidiol (CBD) emulsion on egg yolk fatty acid profile, liver health, and gene expression in laying hens. A total of 180 Lohmann Brown Classic hens were divided into experimental groups, including a control group, a blank group (emulsifier only), and three groups receiving CBD. Cannabidiol was administered in drinking water for 15 weeks at doses of 20, 40, and 80 mg/kg body weight in GI, GII, and GIII, respectively. The analysis indicated that the highest-dose CBD significantly increased yolk polyunsaturated fatty acids (PUFAs), particularly omega-3 and omega-6, as well as the serum HDL level. However, this was accompanied by downregulation of \u003cem\u003ePPAR-γ\u003c/em\u003e expression. The histopathology of the liver did not show any differences among the groups. The expression of fatty acid desaturation genes, such as \u003cem\u003eFADS1\u003c/em\u003e and \u003cem\u003eFADS2\u003c/em\u003e, remained stable, indicating preserved desaturation function and PUFAs biosynthesis. These results suggest that while the highest dose of CBD enhances lipid mobilization and yolk deposition, it simultaneously may affect \u003cem\u003ePPAR-γ\u003c/em\u003e-mediated lipid pathways (e.g., adipogenic programming or lipid storage/transport) despite intact desaturation pathways.\u003c/p\u003e\u003cp\u003eThis is the first study to investigate the molecular impact of purified CBD on lipid metabolism and liver function in laying hens, explaining its potential as a functional feed additive in poultry nutrition.\u003c/p\u003e","manuscriptTitle":"Dose-dependent effects of Cannabidiol in drinking water on yolk fatty acid profile and hepatic expression of PUFA-related genes in laying hens: insights into the CBD systemic and molecular mechanisms of action","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-03 07:47:53","doi":"10.21203/rs.3.rs-7387031/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-09-15T06:17:07+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-11T15:28:49+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-09T23:36:53+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"183032863733554776990719301150812948917","date":"2025-09-01T23:21:12+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"3110281067652789853295009607607179185","date":"2025-09-01T06:25:37+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-08-27T03:27:56+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-08-26T06:34:06+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-08-26T06:32:19+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-08-25T17:34:34+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Veterinary Research","date":"2025-08-25T17:31:08+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-veterinary-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [BMC Veterinary Research](http://bmcvetres.biomedcentral.com/)","snPcode":"12917","submissionUrl":"https://submission.nature.com/new-submission/12917/3?","title":"BMC Veterinary Research","twitterHandle":"@BMC_series","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"f6a69fa9-2ea1-4f7d-a48b-f3c4cb2cbc31","owner":[],"postedDate":"September 3rd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-11-17T16:04:51+00:00","versionOfRecord":{"articleIdentity":"rs-7387031","link":"https://doi.org/10.1186/s12917-025-05122-y","journal":{"identity":"bmc-veterinary-research","isVorOnly":false,"title":"BMC Veterinary Research"},"publishedOn":"2025-11-12 15:57:36","publishedOnDateReadable":"November 12th, 2025"},"versionCreatedAt":"2025-09-03 07:47:53","video":"","vorDoi":"10.1186/s12917-025-05122-y","vorDoiUrl":"https://doi.org/10.1186/s12917-025-05122-y","workflowStages":[]},"version":"v1","identity":"rs-7387031","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7387031","identity":"rs-7387031","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}