A Comprehensive Exploration of the Fatty Acids Profile, Cholesterol, and Tocopherols Levels in Liver from Laying Hens Fed Diets Containing Nonindustrial Hemp Seed

This study investigates the impact of dietary nonindustrial Moroccan hemp seed (HS) on the fatty acid profile, cholesterol, and tocopherol levels, in the liver of 120 Lohmann brown laying hens aged 22 weeks during 12 weeks of treatment. The hens are randomly allocated into four treatment groups, each subdivided into six replicates with five birds in each replicate. The dietary treatments consist of 0% HS (control), 10% HS, 20% HS, and 30% HS. Results indicate a substantial increase (p < 0.01) in polyunsaturated fatty acids, including omega 3 (n-3) and omega 6 (n-6) types, with the inclusion of HS in the diet. The n-6/n-3 ratio is significantly reduced (p < 0.01), and there is a significant reduction (p < 0.01) in saturated fatty acids only for the 30% HS treatment, indicating a more favorable fatty acid composition. Cholesterol levels remain largely unaffected by HS inclusion, except for the 10% HS group, which shows a significant decrease (p < 0.05). Moreover, hepatic tocopherol levels are significantly elevated (p < 0.01) in subjects receiving the HS diet, with the 30% HS group exhibiting the highest tocopherol content. In summary, incorporating HS into the diet up to 30% appears to offer promising benefits for hepatic lipid composition, particularly in terms of n-3 polyunsaturated fatty acids, the n-6/n-3 ratio, and tocopherol levels, while having minimal impact on cholesterol levels.


Introduction
Eggs are an important human foodstuf, consumed in millions of tonnes every year.According to statistics, world production is estimated at 86.4 million tonnes in 2021 and is set to rise further due to the strong demand for animal proteins [1].Hens' eggs constitute a great source of proteins and fats as well as an excellent and balanced source of essential amino acids, certain minerals (calcium, zinc, and iron), and vitamins both liposoluble and hydrosoluble [2][3][4][5].Daily consumption of eggs provides almost all of the recommended amount of these nutrients [6].
Poultry farming in Morocco has undergone rapid growth in recent years, with a 44% surge in investment recorded between 2010 and 2019.Nevertheless, the sector's heavy reliance on imported poultry feed inputs remains a signifcant challenge.Hence, a key research focus has been to explore the feasibility of utilizing local ingredients that can lower feed costs while maintaining high nutritional standards for the hens.One promising alternative is the incorporation of fsh-based products such as fsh oils and meals produced within Morocco, as well as seaweed-based ingredients.Nevertheless, the high cost and the sustainability of the world's fsh stocks remain a serious concern.In addition, some research has indicated their adverse impact on both hens' productive performance and the sensory attributes of the resultant eggs [7].A further alternative would be to use oilseeds produced in Morocco, such as local nonindustrial cannabis seeds (named beldiya).Beldiya is considered a hashish terroir.Te hashish production in Morocco relies on a specifc cultivar named NLD, an abbreviation for narrow-leaf drug cannabis, often colloquially termed "sativa."Tis particular cultivar, Cannabis indica ssp.indica var.mediterranean, may have emerged from the interbreeding of Asian NLD with narrow-leaf Cannabis varieties (NLH) from southern Europe.Tis hybridization could account for the cultivar's moderate delta-9-tetrahydrocannabinol (THC) content, ranging from 2% to 5%, and its comparatively elevated cannabidiol (CBD) levels [8].
In previous studies conducted in our laboratory [9,10], the variability of oil, tocopherol, phenol, and fatty acid content across various nonindustrially produced Moroccan hemp seed (HS) varieties was investigated.Te fatty acid composition predominantly consisted of unsaturated fatty acids (87.3-88.96%),with linoleic acid (LA) being the dominant form, accounting for approximately 56% of total fatty acids, while α-linolenic acid (ALA) levels ranged from 14 to 17%.Te LA (omega-6) to ALA (omega-3) ratio was found to be approximately 3.4.Furthermore, our fndings [10,11] demonstrate that HS collected from diferent regions of the Rif region exhibited signifcant concentrations of phenolic compounds and gamma tocopherols, along with potent antioxidant activity that could provide protection to PUFA, particularly very long-chain fatty acids (VLCs), against oxidation.
Until now, most investigations on the impact of incorporating hemp seed (HS) or hemp oil in poultry diets have primarily focused on productive performance and egg/ meat nutritional quality [12][13][14].
Numerous research studies have been conducted to assess the infuence of industrial hemp seeds on the modifcation of lipid profles in the hepatic tissue of chickens [15][16][17].Yang et al. [18] revealed that dietary alterations can induce variations in lipogenesis and lipid storage in the livers of chickens.By combining elevated quantities of hemp seeds with faxseeds, the hepatic vitamin E content was augmented [17].Elkin et al. [19] established that the addition of DHArich microalgae to the diets of White Leghorn hens mitigated substantial liver enlargement, induced changes in the ovarian follicle hierarchy, reduced the circulation of triglycerides, as well as suppressed hepatic expression of critical genes linked to the biosynthesis of triglycerides.
In a previous study [20], it was documented that the utilization of nonindustrial Moroccan hemp seeds in the diets of Lohmann Brown laying hens resulted in a marked enhancement in nutritional egg quality, which was attributed to elevated levels of n-3 fatty acids and tocopherols.Te objective of the current investigation was to explore the impact of integrating nonindustrial Moroccan hemp seeds from the Beldiya ecotype into the diets of laying hens on the hepatic fatty acid profle, tocopherols, and cholesterol content, while concurrently correlating these alterations with observed nutritional composition changes in eggs.

Materials and Methods
Tis experiment has been performed in the poultry building at the Royal Institute of Livestock, Kenitra, kingdom of Morocco.

Birds and Husbandry.
In this investigation, a group of 120 Lohmann Brown laying hens, each aged 22 weeks, was procured from a licensed commercial poultry facility.Tese hens underwent an initial weight assessment and were subsequently assigned randomly to one of four distinct treatment groups, which encompassed a control group receiving no hemp seed supplementation.Each treatment group was subdivided into six replicates, each consisting of fve birds, and a completely randomized design was employed.Te dietary interventions involved the substitution of soybean-corn components in the control basal diet with varying levels of hemp seed, specifcally 0% (control), 10% (HS 10), 20% (HS 20), and 30% (HS 30).Te laying hens were accommodated in a designated animal housing facility equipped with automated drop belts and battery cages, with the following dimensions: 0.61 m long, 0.57 m wide, and 0.5 m high, adhering to rigorous sanitary and veterinary standards.Individual nipple drinkers were provided for each bird, and the metallic feeders were partitioned to prevent interreplicate feed consumption.Feeding occurred thrice daily, and ad libitum access to water was ensured.Environmental conditions were meticulously regulated, maintaining a controlled temperature range of 18-22 °C, humidity levels at 55-60%, and a photoperiod of 16 hours of light followed by an 8-hour dark cycle.Te experiment lasted 12 weeks; throughout the experimental duration, no instances of mortality were observed, and vaccination procedures were executed under the oversight of a licensed veterinarian.

Experimental Diets.
Te composition of the diets and ingredients used were described in our previous article (Table 1) [20].As for the content of the hemp seeds used in this study, they are shown in (Table 2).Te diets administered were carefully formulated to be isocaloric and isonitrogenous, meticulously tailored to meet the specifc nutritional needs of laying hens [21], receiving a daily ration of 100 grams of feed [18].Te diets were administered on a daily basis and stored at a temperature of 4 °C throughout the duration of the trial.Te dietary formulations encompassed incremental quantities of HS (0%, 10%, 20%, and 30%), designated as control, HS-10, HS-20, and HS-30, 2 Scientifca respectively, incorporated into a primarily soybean-cornbased mixture.Tese diets were produced by a specialized poultry feed company called BENWAY.

Diet Analyses.
In the research, an examination was conducted on the nutritional elements within the diet, encompassing moisture, ash, crude protein, crude fber, calcium, total phosphorus, and ether extract contents, as per the methodology outlined by [22].Te apparent metabolizable energy (AME) of the key dietary components was assessed following the European reference approach described by [23].Tis method involves calculating the difference between energy intake and energy expenditure.

Sampling.
At the end of the experiment, when layers had reached 36 weeks of age, the hens were transported to a slaughterhouse.Six hens from each treatment group (one hen from each replicate) were randomly selected and weighed.Prior to slaughter, the layers were subjected to a 12hour feed withdrawal, then slaughtered according to the halal cutting method before being plucked and eviscerated.
Tis method of slaughter involves frst holding the hens' legs with the right foot and the wings with the left.Te protocol begins with the recitation of Allah's name and the Tasmiyah (Bismillah Allahu Akbar).Ten, using a very sharp knife, the neck of the chicken was cut down to the bone.Te weight of the carcass was recorded prior to tissue sampling.Te liver was expeditiously removed from the carcass, weighed, and rapidly cooled using liquid nitrogen and then preserved at a temperature of −20 °C for future analyses, including fatty acid assessment.All relevant international and national regulations and ethical protocols governing the humane treatment and utilization of animals were meticulously adhered to throughout the study [24].

Liver Lipid Content and Cholesterol. Te Folch technique
was employed to extract total lipids from liver and diet samples.Tis process involved homogenization in a solution composed of chloroform and methanol (2 : 1, v/v), as initially outlined by the 2 : 1 (v/v) solution [25].Following centrifugation, the organic phase was collected, and the resulting all-lipid extracts were retained for subsequent analyses.To determine the levels of cholesterol in the liver, the Liebermann-Burchard (LB) technique, as detailed by Sabir et al. [26], was employed.Specifcally, 0.1 g of hepatic fat was diluted in 10 ml of chloroform, and 3 ml aliquots were extracted, followed by the addition of 2 ml of LB reagent (comprising 0.5 ml sulfuric acid dissolved in 10 ml of acetic anhydride).Te fnal volume was adjusted to 7 ml with  Scientifca chloroform.In parallel, a standard cholesterol solution (1 mg/ml) was prepared, and 5 aliquots ranging from 0.5 ml to 2.5 ml were treated with 2 ml of LB reagent, with the fnal volume adjusted to 7 ml using chloroform.Te solution was then incubated in darkness for 15 minutes, and the absorbance was measured at 640 nm using a UV spectrophotometer (Rayleigh UV1800, UV-Visible).

Fatty Acid Analyses.
To analyze fats, the AOCS "Ce 1k-09" method was employed to transform fatty acids from diet and liver samples into fatty acid methyl esters (FAMEs) [27].Subsequently, these fats were subjected to analysis via gas chromatography, coupled with a fame ionization detector (GC Agilent 6890, Agilent Technologies).Te injection of FAMEs into the system was performed in a splitless mode, with 1 μL of the sample injected.Separation of FAMEs took place in a BPX70 capillary column (60 m long, 0.32 mm internal diameter, and 0.25 μm flm thickness; SGE Europe).Helium gas served as the carrier gas at a fow rate of 1 mL per minute.Initially, the oven temperature was set at 50 °C, subsequently increased to 170 °C at a rate of 30 °C per minute, and then raised by 4 °C per minute until reaching 220 °C.Tis temperature was maintained for 10 minutes.Identifcation of the fatty acids was accomplished by comparing them to a standard containing 37 FAMEs (Sigma-Aldrich, Supelco, Bellefonte PA, USA), and the results were reported as percentages.(1)

Tocopherols Analyses.
To examine tocopherol compounds in liver fat, the methodology by Ben Moumen et al. [31].We employed high-performance liquid chromatography with diode array detection (HPLC-DAD) for this purpose.Initially, the samples were dissolved in hexane and then separated using an Uptisphere 120 A NH2 silica column (250 mm × 4.6 mm, 5 μm).Te mobile phase used was a hexane/isopropanol mixture (99 : 1, v/v) maintained isocratically.Te fow rate was set at 1 mL/min, and a UV detector was used to identify the separated compounds at wavelengths of 292, 296, and 298 nm.For quantifcation, we relied on external calibration, utilizing a commercially available tocopherol standard from Sigma-Aldrich (St. Louis, MO, USA).

Results and Discussion
Te use of hemp as a poultry feed ingredient is not common practice due to strict legislation on hemp cultivation, particularly in and outside European countries; however, in Morocco, in March 2021, the government passed Law No. 13-21 on the legal uses of cannabis, which authorizes the cultivation of cannabis for medical and industrial purposes, while prohibiting its use for recreational purposes.
Several studies have indicated that incorporating alphalinolenic acid (ALA)-rich diets can strongly infuence the fatty acid composition in the egg yolk and hen's liver, particularly in regards to ALA, EPA, and DHA [16,32].Research in the realm of incorporating hemp seed (HS) into the dietary regimen of laying hens predominantly centers on the utilization of whole hemp seeds, hemp oil, or other derivatives and products derived from the hemp plant [32][33][34][35].Both birds and humans depend strongly on their liver for lipid metabolism [36].Hepatic tissue occupies a central position in the orchestration of serum cholesterol and triglyceride levels through the modulation of their biosynthetic pathways and metabolic processes, as well as the intricate mechanisms governing the assembly, reutilization, and efux of lipoproteins [37].Tis present study demonstrates that the inclusion of HS at rates of 10%, 20%, and 30% in the diets of hens signifcantly raises levels of PUFAs n-3 in their liver when compared to the control group.
SFAs have been linked to an increased risk of developing heart disease.As a result, it is recommended to reduce their consumption.A diet enriched with HS impacted the concentration of SFAs (Table 3), composed mainly of palmitic acid (C16:0) and stearic acid (C18:0).When HS intake was raised from 0 to 30%, the level of SFAs in the liver fat reduced signifcantly (p < 0.01) from 38.6% to 36.9%.In agreement with these fndings, Huang et al. [38] reported a noteworthy decrease in SFAs within the livers of hens that consumed 10% faxseed.On the contrary, Juodka et al. [15] reported that there was no diference in the level of SFAs in the liver lipids of cannabis-cake fed ducks.
Te total MUFAs in the liver are infuenced by endogenous synthesis and/or intestinal absorption from the diet [39].In the present study, the total MUFAs content was found to be signifcantly decreased from 29.6 to 22, as a result of a notable reduction of 0.2% in palmitoleic acid (C16:1) and oleic acid (C18:1) in the livers treated with (HS) diet, in comparison to the control group.Similar fndings were also reported by Neijat et al. [16,34], who demonstrated a signifcant reduction in hepatic MUFA levels at varying inclusion levels of HS (10%, 20%, and 30%).In addition, we observed a decrease in MUFA content in the egg yolk lipids when fed with HS [20].Tese variations in MUFA levels can be attributed to a high level of omega-3 fatty acids, provided by cannabis seeds, which would reduce the expression of stearoyl-CoA desaturase SCD-1 in the liver, thereby decreasing oleic acid [40].
PUFA-n-3 fatty acids play crucial roles in numerous physiological activities, particularly in regulating the physical properties of cellular membranes, eicosanoid signaling, and gene expression of enzymes responsible for triglyceride storage [41].However, it is noteworthy that the dietary requirements for animals with respect to these fatty acids are still not clearly defned [42].It has been observed that the synthesis of omega-3 long-chain fatty acids is primarily regulated by substrate levels rather than gene expression [43].
Like many monogastric animals, laying hens exhibit a restricted endogenous enzymatic capacity to enzymatically alter the molecular structure of dietary fatty acids [44].Nonetheless, they are capable of converting alpha-linolenic acid (ALA) to eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids, as well as linoleic acid (LA) to arachidonic acid (ARA), by using specifc elongase and desaturase enzymes [45].Te well-known competition between n-6 and PUFAs n-3 for their substrates, LA and ALA, respectively, by the same LC-PUFA-converting enzymes (desaturases and elongases) has been established [46].Our results on PUFAs demonstrated a signifcant increase in the amount of PUFAs with the inclusion of HS, which is consistent with the recognized process of laying hens, converting ALA into long-chain metabolites [47].A signifcant increase (p < 0.01) in DHA and ALA, and consequently PUFAs n-3, was observed in the livers of hens fed an HS-enriched diet, compared to the control group, which increased from 2.68%, 4.72%, 5.96% to 6.14%, respectively, for the control group, 10%, 20%, and 30% HS, respectively.However, for EPA, we found a nonsignifcant increase when compared with control (p > 0.05).Some authors suggest that this may be due to the limited conversion of ALA to EPA in both animals and humans [48][49][50], or the subsequent conversion of EPA to eicosanoids to fulfll other functional requirements in vivo [51].Te HS supplementation also increased PUFAs n-6 levels in liver lipids from 29.04% for the control diet to 31.59%, 33.67%, and 34.7% for diets containing 10%, 20%, and 30% (HS), respectively.Te enriched diets induced a signifcant rise in LA and ARA compared to the control diet, which is expected for two main reasons.First, the HS are abundant in LA, which is recognized as a substrate for PUFAs n-6 [45].Second, there is co-occurrence of n-3 and PUFAs n-6 on the same PUFA conversion LC enzyme type.[46].Te competitive nature of n-3 and n-6 fatty acids for desaturation and elongation enzymes has been widely investigated in diferent experiments [52][53][54].Most of these studies suggest that 18 : 3 n-3 is a preferential substrate for A6 desaturase to 18 : 2 n-6, given that an n-6/n-3 ratio of up to 3 is maintained [53,55].Consequently, the high concentration of ALA in Cannabis seeds can efectively promote the conversion of ALA to EPA and DHA over that of LA to ARA [56].Te marked increase in PUFA n3 relative to PUFA n-6 results in a substantial decrease in the n-6/n-3 ratio, which decreases as the level of HS inclusion increases until it reaches a plateau at 20% HS.Tese fndings align with the majority of previously documented research outcomes [16,34].PUFAs and their ratios are important for regulating hypocholesterolemic indices.Te PUFAs n-3 seem to play an essential role in the regulation of the thrombogenic index (TI), while the PUFAs n-6 are thought to have a major infuence on the atherogenic index (AI).Te consumption of healthy animal foods, such as meat products, should be characterized by low AI and TI values and high HDL-C indices [50].According to other reports, the recommended levels for AI and TI should be below 1.0 [51].Te results of our study indicate that the hepatic lipid profle has a signifcant impact on lipid health indices.Te AI was comparable in all treatments (0.6), except for HS-30% which showed a signifcant decrease compared to the control, while the TI was substantially lower in HS-10, HS-20, and HS-30 (0.85, 0.83, and 0.77, respectively) versus the control (1.02).We also found that the value of hepatic fatty acid saturation index decreased in hens subjected to the HS 10%, 20%, and 30% diets (22.65, 18.97, and 19.26, respectively) compared to the control diet (23.94).Te value of the HDL-C/HDL-C + LDL-C (hH) indices obtained from the hens fed the HS diet was signifcantly higher than the value obtained from the control, which is benefcial for human health.Our fnding is consistent with the results obtained by other authors based on the fatty acid profle of laying hens [23,30,57,58].According to the Food and Agriculture Organization (FAO), peroxidability index (PI), oxidative susceptibility (OS), desirable fatty acid (DFA), and nutritive value index (NVI) should be as high as possible and should be considered to have neutral or cholesterol-lowering efects [58,59].Our results show that the inclusion of HS in the diet of laying hens allows the increase of these diferent in dices compared to the control, and therefore the Cannabis seeds ecotype beldiya would allow to further improve the nutritional quality of laying hens (Table 4).
Te liver is the primary site of cholesterol biosynthesis in laying hens, and the resulting cholesterol is incorporated into vitellogenin and/or very low-density lipoprotein particles that are secreted into the bloodstream and subsequently taken up by the oocytes [60].In a previous study [61], we demonstrated that local ecotype hemp seed oil (HS) had an interesting efect on mice plasma lipid parameters, reducing the atherogenic index and LDL/HDL ratio but the hen's egg cholesterol content remained unafected [20].In the present study, we found that diets containing HS led to a reduction in cholesterol levels, which was signifcant at a level of p < 0.05 for the HS-10% diet, with levels of 94 and 82 mg/100 g for the control and HS-10% diet, respectively (Table 5).Although the observed decrease in cholesterol levels was small, it is likely due to a combination of factors related to the chemical composition of cannabis seeds (HS are particularly rich in phytosterols, protein, and insoluble fber).Phytosterols mainly β-sitosterol have potential benefts in reducing hypercholesterolemia by blocking cholesterol absorption [62,63].Tey can also reduce cholesterol biosynthesis in the liver by modulating the activity of HMG-CoA reductase, thereby limiting the level of cholesterol formation [64].Additionally, HS is rich in insoluble fber and proteins.Plant proteins have been shown to have a cholesterol-lowering efect, which is largely due to the indigestible fraction that decreases intestinal cholesterol absorption, increases fecal sterol excretion, and improves the catabolism of lipoproteins containing the bulk of cholesterol by increasing the number or activity of LDL receptors [65].Increasing fber concentrations in diets reduces hepatic fat concentrations in laying hens by increasing fatty acid oxidation and decreasing fatty acid synthesis in the liver [66].Te viscosity produced by fber is considered to be one of the key factors in reducing intestinal cholesterol absorption by binding directly to cholesterol, reducing its difusion to the apical surface of the intestinal mucosa cells and being involved in the cholesterol emulsifcation in the intestinal lumen [67].Concerning the liver weight, fat, and relative fat liver content, our fndings show that the HS diet has no efect (p > 0.05) on these parameters.
Hemp seeds are acknowledged for their high levels of antioxidant constituents, including tocopherols, specifcally c-tocopherol [68,69].In the current study, there was a signifcant increase (p < 0.01) in total tocopherol levels, as well as α-and c-tocopherol levels, only in the livers of chickens fed a diet containing a high amount of cannabis seeds (30-HS), than in the control group, while for the other 6 Scientifca treatments (10, 20-HS), there was an increase, compared to the control group, although not signifcant (p > 0.01).Skřivan et al. [17,70] demonstrated that upon the inclusion of HS in the dietary regimen of hens, a substantial transference of α-and c-tocopherol occurred in the egg yolk, liver, and muscle tissues and therefore enhanced their antioxidant potential.Korošec et al. [71] had reported that both isomers (α-and c-tocopherol) played an important role in   Scientifca the expression of genes involved in infammatory processes, immune response, and the metabolism of lipids and cholesterol.
To unravel the intricacies of the complex mixture comprising fatty acid classes, health lipid indices, tocopherols, and cholesterol, a principal component analysis (PCA) was conducted.Te PCA model (depicted in Figures 1 and 2) revealed two signifcant components that accounted for 69.4% and 14.8% of the total variance of the original parameters (PC1 and PC2).Specifcally, PC1 showed a positive correlation with tocopherols, NVI, and unsaturated fatty acids and a negative correlation with saturated fatty acids and SI, while PC2 revealed a positive correlation with DHA, polyunsaturated fatty acid and omega 3 fatty acid, and a negative correlation with cholesterol and AI.
Based on our data, we have observed a positive correlation between the concentration of ALA, EPA, DHA, and PUFA n-3 and some health indices.Furthermore, a robust correlation was discovered between tocopherols and long-chain fatty acid n-3.From the PCA conducted, we have identifed four distinct groups, of which the control and 30-HS groups were the most prominent.Te control group exhibited high values for SFAs, n-6/n-3 ratio, MUFAs, saturation index (SI), and thrombogenicity index (TI), while the 30-HS group showed high values for UFAs PUFA n-3, PUFA n-6, and tocopherols.Te occurrence of these two distinct clusters therefore confrms the benefcial health efect of HS incorporation on the hepatic lipid profle.

Conclusion
Te primary aim of our study was to assess the benefcial infuence of incorporating nonindustrial Moroccan ecotype hemp seed (HS Beldiya) into the diet of laying hens on hepatic lipid metabolism.Te results obtained in this study corroborate and extend our prior research on the productive performance and egg characteristics of laying hens.Te results demonstrated that HS supplementation up to a level of 30% leads to advantageous changes in liver lipid composition, including a noteworthy increase in PUFAs n-3 levels, a decrease in cholesterol levels, and an increase in tocopherol levels.Te most suitable concentration for the supplement appeared to be at a very low level, not exceeding 10% HS.Since the liver is the primary location of lipid metabolism, these modifcations are expected to lead to favorable changes in plasma lipid parameters and may also improve the quality of meat produced.Consequently, future work may be conducted to investigate this hypothesis by analyzing the lipids of diferent muscles in laying hens fed nonindustrial Moroccan HS.

Figure 1 :
Figure 1: Correlation load of fatty acids, health indices, cholesterol, and tocopherol of the liver samples analyzed in the plane defned by 2 principal components.

Table 2 :
Proximate and major fatty acid composition of hemp seeds (beldiya) utilized as a dietary constituent for laying hens.

Table 1 :
Formulation, proximate chemical composition, and nutrient profle of diets containing hempseed (HS) compared with the control.
* DDGS: distiller's dried soluble grains; * * * AME available metabolizable energy.* Scientifcaconcentrations among the laying hens.For comparing means, Tukey's test was applied, and statistical signifcance was established at a threshold of p < 0.01.Te outcomes are presented in the form of means ± standard error of the mean (SEM).Furthermore, to elucidate the interrelationship among the examined parameters, a principal component analysis (PCA) was conducted.
Analysis.Te collected data were subjected to rigorous statistical analysis employing the one-way analysis of variance (ANOVA) test, conducted using IBM SPSS software (version 20; SPSS Inc., Chicago, IL, USA).Tis analytical approach was employed to assess the treatmentinduced variations in the hepatic fatty acid profle, lipid health indices, cholesterol levels, and tocopherol 4

Table 3 :
Efect of dietary hemp seed on fatty acids profle (% total FA) in liver.

Table 5 :
Efect of dietary hemp seed on weight, fat content, relative fat content, cholesterol, and tocopherol concentration of the liver of studied laying hens.
abcMean marked with a diferent superscript letter within each row are signifcantly diferent.

Table 4 :
Efect of dietary hemp seed on health-related lipid indices of the liver of studied laying hens.SI, saturation indices; DFA, desirable fatty acids; AI, atherogenicity indices; TI, thrombogenicity indices; OS, oxidative susceptibility; hH, hypo/hypercholesterolemic; HSFA, hypercholesterolemic saturated fatty acids; NVI, nutritive value indices; SEM, standard error of the mean, p signifcance.abcd Mean marked with a diferent superscript letter within each row are signifcantly diferent.