The Effect of Feeding Laying Hens with Nonindustrial Hemp Seed on the Fatty Acid Profile, Cholesterol Level, and Tocopherol Composition of Egg Yolk
1Laboratory for Agricultural Production Improvement, Biotechnology and Environment, Faculty of Sciences, Mohammed I University, BP-717, 60000 Oujda, Morocco
2Laboratory of Sustainable Agriculture Management, Higher School of Technology Sidi Bennour, University Chouaib Doukkali, Street Jabran Khalil Jabran BP, 299-24000 El Jadida, Morocco
3Higher School of Education and Training, Mohammed I University, BP-410, 60000 Oujda, Morocco
4Royal Institute of Livestock Fouarat, Kenitra, Morocco
Abstract
The purpose of this study was to evaluate how cannabis-derived nonindustrial hemp seed (HS) inclusion in laying hen diets, as well as treatment duration, affected the fatty acid (FA) profile, cholesterol level, and tocopherol composition of egg yolks. Ninety-six (n = 96) Lohmann Brown classic laying hens were randomly assigned to one of the four groups: control (standard diet) and HS-containing diets (10% HS, 20% HS, and 30% HS). The study was conducted for a period of 4 months. The findings demonstrated that the FA profile and the tocopherol composition are strongly impacted by the addition of HS to the diet of laying hens (p < 0.05), but the cholesterol content remained unaffected. The increase in the dose of cannabis incorporated into the hen's diet (HS-30% group) led to a significant increase in the amounts of the polyunsaturated fatty acids n-3 and n-6 content of egg yolk. This enrichment was accompanied by a considerable decrease in the n-6/n-3 ratio (p < 0.001) from 8.19 to 4.88, on day 84 of the experiment. The total tocopherol content significantly increased (p < 0.05) from 281.44 (control) to 327.02 μg/g yolk (HS-30%) on day 84. Finally, in the context of warfare, these seeds might be used as a feed additive for laying hens to produce higher nutritive value eggs with affordable prices.
1. Introduction
The fatty acid (FA) content of animal-based foods, such as meat, dairy products, and egg yolks, is a perfect reflection of the lipid profile and the constitution of nutrition. Due to the biohydrogenation phenomena, this association is more frequently found in monogastric [1] than polygastric ruminants [2]. Because polyunsaturated fatty acids (PUFAs) are vital for humans, there is a growing awareness of their health advantages [3]. Furthermore, the n-6/n-3 polyunsaturated fatty acid (PUFA) ratio remains an important criterion for assessing fat quality and should be less than the value of 4 [4]. Moreover, nutritional survey shows that PUFA n-6 predominates over PUFA n-3 by a factor of 10 to 30, which indicates a PUFA n-3 deficiency. These latter nutrients are crucial for human health and important in preventing behavioral issues, diabetes, some cancers, inflammations, and cardiovascular disease (CVD) [5].
Furthermore, international health experts advise consuming enough PUFAs, namely, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), mostly found in fishery products. Nevertheless, this recommendation is not applicable in countries with low-income workforce (such as Morocco), where fish consumption is low due to its limited availability and high price. However, eggs are consumed more frequently in almost all countries around the world due to their availability to the consumers as well as their low cost. Therefore, enriching eggs with PUFA n-3 is a sustainable option for a healthy and well-balanced diet, as well as a way to reduce the intensive exploitation of fishery resources [6].
For a long time, egg consumption has been one of the simplest and cheapest ways to provide populations with the essential nutrients for a healthy and balanced diet (protein, fat, vitamins, and minerals). Eggs have been prevented from consumption by dieticians for a long time due to their high cholesterol and saturated fatty acid content [1]. Alexander et al. [7] reported in an interesting review that there are substantial correlations between cholesterol intake and heart disease. However, recent studies have revealed that there is minimal up to no connection between dietary cholesterol and cardiovascular risk indicators due to the interaction of several other factors of risk such as a sedentary lifestyle, smoking, obesity, and other physiological dysfunctions [7]. According to several studies, animal nutrition has a significant impact on both biochemical constitution and its nutritional value [8, 9].
Further studies found that the incorporation of n-3- and tocopherol-rich nutrients in laying hens' diet produced eggs with higher levels of these constituents and thus higher nutritional value [10]. Furthermore, incorporating flaxseed and fish oil in farm livestock feed items is a frequent method for enhancing meat with n-3 PUFA and eggs [11–13]. Nowadays, the development of innovative and alternative methods based on new natural plant resources would be of considerable interest.
Cannabis sativa seeds and derivatives have a long history of usage in ethnomedicine [14]. Recent research has shown interest in using cannabis seeds as a supplement of livestock feed, particularly as a source of PUFA n-3 [10, 15, 16]. These studies focused on supplementing the animal's diet with industrial hemp seed, seed meal, or flour, which revealed an increase in n-3 content while reducing the n-6/n-3 ratio. In addition, hemp seeds increased hen laying performance, such as the egg laying rate, as well as egg quality, namely, the average egg weight, eggshell strength, and the biochemical composition. Furthermore, Kasula et al. [17] showed that laying hens fed hemp seedcake have a high polyunsaturated fatty acid content as well as no tetrahydrocannabinol or cannabinoid residues in eggs, internal organs, or body tissue indicating that there are no transfer cannabinoid residues in hens' fats. According to a survey conducted in Morocco [18], 80% of the participants thought that the availability of eggs fortified with n-3 would be interesting, and 74% stated that they would be willing to pay more for them than ordinary eggs.
Therefore, the objective of this research was to investigate how the incorporation of the Moroccan nonindustrial cannabis seed affects the composition of tocopherols, cholesterol, and the fatty acid profile of yolk eggs. For this purpose, experiments were conducted using different ratios of nonindustrial cannabis seeds in a laying hen diet over different periods. The findings of this study will support the industry in reducing imports of more expensive oilseeds, such as soya, sunflower, and maize, and improving feed formulation to produce eggs that are n-enriched utilizing Moroccan nonindustrial cannabis seeds.
2. Material and Methods
2.1. Experimental Animals
The animal experiment was conducted in accordance with the European regulations CO 74/99 regarding stocking density, lighting, ventilation, and vaccination, at the Royal Institute of Livestock in Kenitra, Morocco. The trials on laying hens were carried out at the Royal Institute specialized in breeding of Kenitra, Morocco, in conformity with the European code number 74/99. The layers used in this study were randomly distributed in four groups of the Lohmann Brown classic (LBC) strain repeated six times (4 × 4 × 6 = 96), including a control group. During the rearing period, no new husbandry practices were introduced. The hens were raised in a semiautomatic coop in cages with the following characteristics: 61 cm long, 57 cm wide, and 50 cm high. The birds were housed with 4 hens per cage equipped with feeders and nipple drinkers, the building being equipped with automatic dejection mats. The environmental parameters were managed according to the guidelines of the exploited strain, with an average temperature between 18 and 20°C, humidity between 55 and 60%, and light duration of 16 hours/day, from 6:00 to 22:00. The laying hens come from a modern certified farm, at the age of 22 weeks.
2.2. Diets and Experimental Approach
To standardize the energy level of 3000 kcal/kg, ingredients were added to the hen's foods to generate diets with similar levels of nutrient composition. The formulation of the diet was developed in consultation with BENWAY, a company specializing in poultry feed. The feed has been formulated in collaboration with BENWAY, a company specialized in the feed manufacture for laying hens. Maize-/soya bean-based diets were utilized by the inclusion of different hemp seed levels of the local ecotype “Beldiya”: 0.0% (HS-0) control, 10% (HS-10), 20% (HS-20), and 30% (HS-30). The inclusion of hemp seeds of the local ecotype “Beldiya” on a feed composed of corn/soybean was on four levels: 0.0% (HS-0) control, 10% (HS-10), 20% (HS-20), and 30% (HS-30). Table 1 shows the HS compositions utilized in this investigation. The chemical composition of the cannabis-based diet used in this study was described in our previous article (Table 2) [19]. For two weeks, the hens were given time to adapt to their new environment system (cage and feeding method). In the first week, hens were given a commercial layer meal, and in the second week, the experimental diets were gradually introduced from 25%, 50%, and 75% to finally 100%. In the first week, the birds were fed a standard layer meal, and in the next week, the experimental regimes were gradually introduced at 25%, 50%, 75%, and finally 100%.
The birds were fed three times a day while given unlimited access to water. The study was conducted for a period of 4 months (from January 30th to the 31st of May 2021). Eggs were randomly sampled during the last three days of each period (28 days/period). 18 eggs per group (3 eggs × 6 replicates) were collected for a period of 3 consecutive days at 28, 56, and 84 days of treatment corresponding to 28, 32, and 36 weeks of age. The eggs were analyzed for lipids, fatty acids, cholesterol, and tocopherols.
2.3. Chemical Analysis
2.3.1. Diet and Egg Yolk Fatty Acid Extraction
Three eggs were randomly selected from each treatment with 6 replicates. Yolk and white were carefully separated to avoid mixing. The 3 yolks were pooled and homogenized in a 50 mL tube and stored at -20°C until analysis time. The four tested diets, on the other hand, were powdered and kept at -20°C for chemical composition analysis. Lipid extraction from all the samples was performed according to the method described by Bligh and Dyer [20] using a mixture of chloroform/methanol/water solvent (2/1/1; v/v/v). The solvents were evaporated using a Hahnvapor Rotary Evaporator HS-2005S-N (AM-486). The extracts were tested for fatty acids, cholesterol, and tocopherols.
2.3.2. Fatty Acid Profile
The fatty acids were converted into fatty acid methyl esters (FAMEs) before being analyzed using our previously published method [21]. The composition of FAME was evaluated using gas chromatography (GC Agilent 6890, Agilent Technologies) coupled with a flame ionization detector (FID). FAMEs (injection of 1 μL of the sample in splitless mode) were separated on a BPX70 capillary column with the following characteristics (60 m length, 0.32 mm internal diameter, and 0.25 μm film thickness; SGE Europe). Helium was used as carrier gas at a flow rate of 1 mL min−1. The oven's temperature was set at 50°C and afterwards increased to 170°C at a rate of 30°C per minute and then increased by 4°C per minute to 220°C. The temperature was then held for 10 minutes. After identifying the fatty acids and comparing them to a standard from Sigma-Aldrich containing 37 FAMEs (Supelco, Bellefonte, PA, USA), the results were presented as percentages. Other sums of FA are determined as odd-chain fatty acids (OFA) and have beneficial effects on human health [22–25]. The desirable fatty acids (DFA) are also calculated by the following formula: DFA = (C18 : 0 + UFA) according to Vlaicu et al. [13] and Belhaj et al. [26]. These latter are considered as hypocholesterolemic FAs by reducing the low-density lipoprotein impacts [22].
2.3.3. Tocopherol Content
Tocopherols were quantified according to the Ben Moumen et al. [27] protocol using an HPLC system (Shimadzu LC-6AD system) coupled with a DAD detector. The separation was carried out on an Uptisphere 120 A NH2 silica column (4.6 × 250 mm, the particle size of 5 μm) using a mobile phase composed of an n-hexane/isopropanol mixture (99/1; v/v) with a flow rate of 1 mL min−1. The identification was carried out using commercial standards for tocopherols (Sigma-Aldrich, St. Louis, USA) at 292, 296, and 298 nm. The tocopherol concentration was calculated from the external calibration curve with commercial tocopherols obtained from Sigma-Aldrich (St. Louis, MO, USA).
2.3.4. Cholesterol Content
The cholesterol content of egg yolk was measured using the method described by Vanderplanck et al. [28]. After saponification with KOH and extraction with diethyl ether, cholesterol was separated from the fat. The mixture was analyzed by gas chromatography using a Hewlett-Packard chromatograph (HP 6890 series GC) equipped with a capillary column (HP 5 MS (5 %-phenylmethylpolysiloxane, 30m × 0:25mm, 0.25 m film thickness), Agilent Technologies, Palo Alto, CA, USA) and a flame ionization detector. The injector was operated in splitless mode. The operating parameters were as follows: carrier gas: helium at 1 mL min−1; column temperature: 275°C; injector and detector temperatures: 250 and 300°C, respectively; and injection volume: 1 μL. The cholesterol concentration in egg yolk was calculated and expressed as mg per g of egg yolk.
2.4. Statistical Analysis
The statistical analyses were carried out using the Statistical Package for the Social Sciences (IBM SPSS 21). The normal distribution was checked using the Shapiro-Wilk test for quantitative variables. A two-way analysis of variance (ANOVA) was performed for fatty acid profile, tocopherol content, and yolk cholesterol content. The Tukey post hoc test was used for the comparison of means. The difference was considered significant at p < 0.05. A principal component analysis (PCA) was performed on the data set to differentiate the results according to the age of the hens and the feed distributed.
3. Results and Discussion
3.1. Fatty Acid and Cholesterol Composition
The results in Tables 3tab4tab5–6 show the effect of Moroccan nonindustrial hemp seed incorporation in the diet of laying hens on the fatty acid profile of the egg yolk during the experimental period. The results obtained show varying fluctuations concerning the dose and duration of the experiment; however, the main variations concern the increase in ALA content (Tables 3tab4tab5–6). We note a statistically significant difference (p < 0.05) in the amount of n-3 in the eggs depending on the diet used throughout the experiment and the amount of cannabis seeds supplied. After 84 days (12 weeks) of the feeding period, which corresponded the end of the experiment, the n-3 content increased as the incorporated dose of cannabis seeds increased (Tables 3 and 5). Thus, the percentage of n-3 escalated from 3.01% in the control (0% HS) to 3.41, 5.45, and 7.05% in the eggs from the hens fed, respectively, with 10%, 20%, and 30% of cannabis seeds (Table 5). The detailed analysis of fatty acids reveals that this increase in n-3 correlates specifically with an increase in ALA, with the level rising from 1.62% (control) to 5.47% (HS-30%). However, there are no significant differences in DHA and EPA levels. This last result contradicts most of the previously published results (Fabro et al. [29]; Mierliță [30]. The long-chain AGPI metabolism in animals allows the conversion of ALA to DHA and EPA, only if the ratio of n-6 to n-3 is not too high; otherwise, the path of converting LA to ARA will be promoted. In contrast and based on our results, the ARA rates no longer exhibit significant and noticeable variations. These results could be explained by a potential oxidative degradation of DHA, EPA, and ARA (long-chain PUFA) during the extraction of the lipid phase from egg yolks and throughout the other phases of the fatty acid analysis of. In fact, no antioxidants were added during the different phases of the lipid analysis, compared to other studies [13]. This deliberate decision to exclude the antioxidant agents was made to mimic the conditions of an egg consumer or an industry who would be using products made from raw eggs. Moreover, other experiments are being conducted in our laboratory to study the effect of the antioxidant's addition on the fatty acid composition (data not shown). Furthermore, other studies on chicken fed cannabis seeds or oil revealed a significant reduction in the expression of gene coding for hepatic desaturases responsible for long-chain PUFA desaturation [31]. The incorporation of cannabis seeds in poultry feed has also increased in LA, which has risen from 21.92% (control) to 31.78% (HS-30%) after 84 days (12 weeks) of the feeding period (Table 3). Several previous studies have found the same trend in the LA rate [32]. However, in other research, the LA rate did not change despite the increase in HS [15, 33]. Our results can be elaborated through the fact that the HS used are rich in ALA but also in LA (Table 1). Nevertheless, the observed increase in n-3 would possibly be responsible for a decrease in egg weight from hens (data not shown). Gonzalez-Esquerra and Leeson [34] as well as Castillo-Badillo et al. [35] observed a reduction in egg weight in treatments including tuna oil or menhaden oil in hen feed layers. This decline would be a result of low blood triglyceride levels, which restricts the amount of lipids available for the development of the egg yolk, as well as from modified estradiol circulation, which would also have an impact on the development of the egg [36]. However, there is currently no clear explanation of how n-3 FAs decrease egg weight. We also note an increase in the rate of another n-6 (GLA), whose level ranges from 0.15% (control) to 0.35% (HS-30%) (Table 3). This increase, which has been shown also in previous studies [29], is explained by the presence of GLA at quite high levels in the cannabis seeds incorporated into food. Indeed, one of the most important characteristics of cannabis seeds is their relatively high GLA content, compared to other oilseeds traditionally used in laying hen feeds, such as soybean or sunflower. Even though LA is the precursor for the synthesis of the long-chain n-6 fatty acids gamma-linolenic acid (GLA), dihomogamma-linolenic acid (DGLA), and arachidonic acid (ARA), none of these n-6 acids react in the same way. LA and ARA promote inflammation, while GLA, through its conversion to DGLA, can reduce inflammation [37]. ARA, which is derived from the elongation and desaturation of GLA, does not exhibit any change with a rate of around 2%, regardless of the diet of the animals, as we have already indicated. The same result had been obtained by other authors [38, 39], who supported their findings by pointing out that the significant increase in ALA levels promotes the n-3 pathway at the detriment of the n-6 pathway; both ways use the same enzymes, in particular the Δ5 and Δ6 desaturases. The balance of the metabolic pathway depends on the most bioavailable precursor. An excess of n-6 will promote the synthesis of ARA and DPA (C22:5n-6) to the detriment of EPA and DHA. On the other hand, a sufficient amount of n-3 will promote the synthesis of EPA and DHA and inhibit the synthesis of long-chain n-6. Moreover, at the end of the experimentation (day 84), the results show a very significant (p < 0.05) decrease in the n-6/n-3 ratio which goes from 8.19 (control) to 9.54, 6.15, and 4.89 for HS-10%, HS-20%, and HS-30%, respectively (Table 4). All previous studies have found the same phenomenon with usual marked decreases. For example, Mierliță [30] obtained a reduction of 11.07% (control) to only 2.98%, while Shahid et al. [39] obtained a decrease of 16.83% to 4.22%. These differences may be explained by differences in the experimental protocol and duration, or they might be linked to the variety or subspecies of the Cannabis sativa used. In almost all previous experiments, the seed cannabis used belongs to the industrial hemp variety; however, in our experiment, we used the Beldiya ecotype, which is native to the north of Morocco and is also part of the plants cataloged as nationally prohibited drugs since its THC content exceeds 0.4%.
Results, resumed in Tables 3 and 5, show that regardless of the proportion of cannabis seeds supplied to the poultry diet, the rate of SFA does not significantly change. The main SFA is the palmitic acid (C16:0), its rate slightly decreases (p < 0.05) from 24.38% (control) to 22.46% (HS-30%), and this reduction is partially offset by an increase in the stearic acid (C18:0) rate, which rises from 8.98% (control) to 11.17% (HS-30%). The rate of MUFA decreases significantly (p < 0.05) passing from 38.51 (control) to 24.42% (HS-30%). This decrease in MUFA specifically affects oleic acid (C18:1). Considering that, in fatty acid metabolism, C16:0 gives way to C18:0 via elongation and eventually to C18:1 by desaturation, the observed fluctuations are now well known. An interesting meta-analysis conducted by Fabro et al. [29], which encapsulates the majority of scientific studies investigating the effects of incorporating seeds, oil cakes, or cannabis oil on the fatty acid profile of egg yolk, allows us to validate our findings. In fact, throughout the majority of the publications reviewed in this meta-analysis, the SFA rate does not change while the MUFA rate decreases. These variations are explained by the fact that a high level of n-3 (provided by cannabis seeds) might inhibit the expression of the gene coding for stearoyl-CoA desaturase (Δ9 desaturases) which reduces the conversion of C18:0 to C18:1, leading to a decrease in C18:1 and an increase in C18:0.
The addition of Beldiya cannabis seed HS did not lead to significant differences in cholesterol content. Table 7 shows that this content ranges between 7.99 and 10.73 mg/g of egg yolk, which is slightly lower than the values reported by Mierliță [30], who did not observe any differences after the cannabis-incorporated diet. However, other studies, including those conducted by Skřivan et al. [32] and Shahid et al. [40] had obtained a reduction in cholesterol content that decreased from 19.25 (control) to 11.67 mg/g of egg yolk (HS-25%). According to these authors, this decrease is due to cannabis seeds containing a high concentration of phytosterol, specifically sitosterol, which limits cholesterol absorption through crystallization and coprecipitation, but most importantly through competition at the intestinal absorption level. Kovacs et al. [41] revealed that the cholesterol content of eggs varied according to the species, variety, or stress of the laying cycle rather than the diet.
3.2. Tocopherol Composition
The HPLC-DAD analysis of tocopherols from lipid egg yolk (Table 8) revealed that the main tocopherols detected correspond to the isomers α- and γ-isomers, while the β- and δ-isomers were not detected. These results are consistent with those of several other subsequent studies: Ko et al. [42], Skřivan et al. [32], and Moghadam et al. [43]. However, other authors including Cherian et al. [44] and McLaughlin and Weihrauch [45] were able to detect all 4 isomers. Regardless of the treatment (age and dose), α-tocopherol is the major isomer constituting between 56.03% and 96.6% of total tocopherols while the percentage of γ-tocopherol ranges from 3.34 to 43.97% of total tocopherols. Several publications refer to preferential intakes of the α-isomer compared to other isoforms in mammals [46, 47] and poultry [44]. This preference is explained by the presence of a specific protein that binds α-tocopherol in hepatocytes and its subsequent transfer by LDL to other tissues, as well as by the potential existence of selection processes that discriminate the uptake or accumulation of γ-tocopherol over α-tocopherol. Results (Tables 8 and 9) show that the total tocopherol content varies depending on the age and dose of cannabis seeds incorporated into the diet (Table 9). According to Wang et al. [48], the main factors influencing the tocopherol content of egg yolk are nutrition, age, and laying hen variety or genotype. A significant increase in total tocopherol content depending on the dose of cannabis seeds incorporated is observed only after 84 days of treatment varying from 281.45 (control) to 327.03 μg/g egg yolk (HS-30%) (Tables 8 and 9). Thus, 28 days of treatment appear, according to our results, insufficient for the fortification of eggs produced with tocopherols. An in-depth analysis of the composition of the two detected isomers (alpha and gamma) reveals dissimilar variations. So, the eggs produced are enriched with alpha-tocopherol during the treatment both in the control and for the different doses of cannabis seeds added varying from 50.52 (control) and 36.3 (HS-30%) after 28 days to 272.03 (control) and 306.3 μg/g egg yolk (HS-30%) after 84 days. However, the most important outcome, which can be derived from the data obtained, is in particular the stabilization of the γ-tocopherol content. Indeed, regardless of the duration of treatment, the amount of γ-tocopherols is of the order 16.54-32.9 μg/g yolk in eggs produced after the inclusion of various doses of cannabis seeds, whereas in the control eggs, there is a very significant decrease in the γ-tocopherol content since there is an apparent drop from 25.01 (28 days) to only 9.41 μg/g yolk after 84 days of treatment. Few studies have investigated the impact of age on the incorporation of tocopherols in egg yolk. Furthermore, Tang et al. [49] observed a significant age-related increase in α-tocopherol from 92.984 μg/g egg yolk (24 weeks) to 225.0784 μg/g egg yolk (36 weeks), as well as constant values of γ-tocopherols (11 to 9.84 μg/g egg yolk). On the other hand, Ko et al. [42] found that the level of α- and γ-tocopherols decreased with age in laying hens. According to Chen et al. [50], the concentration of tocopherols increases with age until the 14th day of the experiment, where it becomes stable. It is not entirely clear from the articles published the reason behind the differences in the tocopherol content in accordance to age. It may be due to variations in the extraction and measurement of tocopherol methods or even due to a metabolic state that is more active or less active depending on age and experimental conditions. It can also be noted that after a 28- or 84-day experiment, the increase in the amount of cannabis seeds in the diet does not lead to an increase in γ-tocopherol level (Table 8). Quite the contrary, a slight, nonsignificant decrease can be observed; it could be explained by poor absorption of γ-tocopherol from cannabis seeds, which at high concentrations contains a high amount of fiber and other antinutritional compounds which decreases and hinders the absorption of various nutrients including tocopherols.
Since animals are unable to synthesize tocopherols, provided exclusively by the diet [51], and since the vitamin premixes used did not contain vitamin E, the tocopherols came only from the dietary components of the different formulations. The increase and stability in γ-tocopherol content, compared to the control group, can therefore be explained by an enrichment of the diet with γ-tocopherols since the Beldiya cannabis seeds used contain approximately 21 times γ more than α-tocopherol [21]. Chen et al. [50] already showed that α-tocopherol has a positive effect on the stability of the yolk as an antioxidant at different degrees of concentration, 25, 45, and 50 μg/g of yolk, yet, it turns into a prooxidant at 75 μg/g and higher. Skřivan et al. [32] conducted a similar 12-week study with different hemp seed varieties, whereas in our study, we used indigenous North Moroccan Cannabis sativa L. seeds cultivated for their recreational use. Moreover, Skřivan et al. [32] have also shown a significant increase in the α-tocopherol content of egg yolk products when 60 g of cannabis seeds/kg was incorporated into the diet. On the other hand, the level of γ-tocopherol increased gradually in the control group, rising from 11 mg/kg MS to 29, 39, and 43 mg/kg MS for cannabis seed incorporation rates of 30, 60, and 90 g/kg, respectively. Similarly, studies conducted on diets supplemented with vitamin E [52, 53] or α-tocopherol [54] have shown an increase in the tocopherol content of egg yolks.
3.3. Chemometric Analysis of the Yolk Egg Composition
A principal component analysis (PCA) was performed to identify and determine the correlations between the hens and their yolk egg composition. The PCA allows further exploration of the above results. This analysis was conducted using 32 variables, including fatty acid profile, cholesterol content, and tocopherol composition. It provides an intelligible visualization of the relationship between hens and their egg's fatty acid profile, cholesterol, and tocopherol content. Figure 1 presents the projection of the different elements in the plane defined by the two first principal components (PC).
The PC1 and PC2 accounted for 44.60% and 11.96% of the variation, accordingly (Table 10). The PC1 was correlated positively with PUFA, PUFA n-3, PUFA n-6, ALA, and LA and negatively with MUFA and C16:0. The PC2 was characterized positively by C20:2n6, trans-C18:2n6, and SFA. In opposite direction, it was defined by C20:3n3 and UFA. The projection of the studied groups on the plane defined by the two first PC shows clear discrimination between hens based on egg composition (Figure 2). Regardless of the period, the hens of the control groups were located on the left side of Figures 1 and 2 differentiated from the others. However, the SH-20% and SH-30% groups were on the right side of Figure 1, with PUFA, PUFA n-3, PUFA n-6, ALA and LA. The discrimination between the animals under study shows a significant difference which is certainly linked to the impact of hemp seed incorporation in the diet of laying hens.
4. Conclusion
Based on the results of these experiments, we can conclude that the cannabis seed ecotype Beldiya, grown in the Moroccan Rif mountains, can be promoted and valued by using it as a supplement in the diet of poultry to replace, at least partially the oilseeds (soya and sunflower) imported by Morocco at a high expense. This application of cannabis in the hen's diet would provide Moroccan consumers with higher nutritional quality eggs enriched with ALA and tocopherols. Despite being advantageous nutritionally, enriching eggs with PUFAs can unfortunately promote lipid changes (increase unsaturation), leading to undesirable or even toxic products. For this reason, 20% of HS incorporation in laying hen feed is considered as a safe dose. Further studies are required to see whether adding natural antioxidants would be feasible in order to prevent these flaws in lipid oxidation and enhance the oxidative stability of the produced eggs.
Acknowledgments
This work was supported by the Ministry of National Education, Professional Training, High Education and Scientific Research in collaboration with “National Agency for Medicinal and Aromatic Plants” and “Royal Institute of Specialized Technicians in Livestock-Fouarat” through the VPMA2/ref 2020/1 project.
Data Availability
The original data from the paper are available from the corresponding author upon reasonable request.
Ethical Approval
We used data from other papers, and no ethical statement is required.
Conflicts of Interest
The contact author has declared that none of the authors has any competing interests.
| Elements | Percentage of fresh matter |
|---|---|
| Dry matter | 88.27 |
| Total phosphorus∗ | 11.60 |
| Total proteins | 22.00 |
| Lipids | 33.00 |
| Calcium | 1.725 |
| Humidity | 11.73 |
| Minerals | 49.70 |
| Crude cellulose | 14.68 |
| Fatty acids (g/100 g fatty acids) | |
| C16:0 | 7.680 |
| C18:1n9 | 18.050 |
| C18:2n6 | 51.010 |
| C18:3n3 | 16.460 |
| Saturated fatty acids | 11.910 |
| Unsaturated fatty acids | 88.090 |
| Monounsaturated fatty acids | 19.720 |
| Polyunsaturated fatty acids | 68.380 |
| Diet ingredient | Control | HS-10% | HS-20% | HS-30% |
|---|---|---|---|---|
| Hemp seed | 0 | 10 | 20 | 30 |
| Sunflower meal | 1.685 | 13.000 | 13.000 | 13.000 |
| Soybean meal | 25.585 | 10.459 | 9.563 | 11.094 |
| Calcium | 8.609 | 7.379 | 8.934 | 8.927 |
| DDGS | 0.896 | 7.000 | 7.000 | 3.572 |
| Corn | 58.829 | 46.214 | 38.001 | 21.294 |
| Dicalcium phosphate | 1.368 | 3.726 | 1.162 | 2.000 |
| Soybean oil | 2.028 | 1.000 | 0.000 | 0.000 |
| Premix 1 | 0.500 | 0.500 | 0.611 | 8.000 |
| Sodium sulfate | 0.000 | 0.237 | 0.300 | 0.200 |
| Salt | 0.183 | 0.112 | 0.200 | 0.200 |
| DL-methionine | 0.273 | 0.215 | 0.217 | 0.200 |
| L-Lysine HCl | 0.004 | 0.146 | 0.999 | 1.500 |
| Premix 2 | 0.040 | 0.012 | 0.013 | 0.013 |
| Total | 100 | 100 | 100 | 100 |
| Dry matter (%) | 88.312 | 88.536 | 86.682 | 81.242 |
| Calculated nutritional composition | ||||
| Metabolizable energy (kcal kg−1) | 2989.920 | 3000.000 | 3000.002 | 3000.003 |
| Humidity (%) | 11.195 | 10.972 | 10.352 | 9.537 |
| Crude protein (%) | 17.799 | 17.800 | 18.000 | 18.000 |
| Total lipids (%) | 5.057 | 7.143 | 8.480 | 11.102 |
| Ash (g/kg) | 126.960 | 140.863 | 189.627 | 244.972 |
| Calcium (g/kg) | 38.996 | 40.000 | 40.000 | 40.000 |
| Phosphorus available (g/kg) | 4.396 | 8.510 | 27.762 | 27.752 |
| Sodium (g/kg) | 0.083 | 0.160 | 0.160 | 0.160 |
| Linoleic acid (%) | 2.491 | 7.454 | 12.092 | 17.370 |
| Lysine (g/kg) | 9.352 | 8.642 | 8.705 | 8.735 |
| Methionine (g/kg) | 5.580 | 5.589 | 5.823 | 6.138 |
| Leucine (g/kg) | 14.921 | 14.528 | 14.140 | 13.522 |
| Methionine+cysteine (g/kg) | 8.779 | 8.638 | 8.576 | 8.616 |
| Threonine (g/kg) | 6.902 | 6.634 | 6.944 | 6.913 |
| Tryptophan (g/kg) | 1.760 | 1.826 | 1.944 | 1.917 |
| Day 28 | Day 56 | Day 84 | ||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Control | HS-10% | HS-20% | HS-30% | Control | HS-10% | HS-20% | HS-30% | Control | HS-10% | HS-20% | HS-30% | |
| C8:0 | 0.002ab ± 0.001 | 0.003b ± 0.002 | 0.0032b ± 0.006 | 0.003ab ± 0.001 | 0.002ab ± 0.000 | 0.003b ± 0.002 | 0.002ab ± 0.001 | 0.002ab ± 0.000 | 0.002ab ± 0.000 | 0.001a ± 0.000 | 0.001a ± 0.000 | 0.001a ± 0.001 |
| C11:0 | 0.003a ± 0.002 | 0.005a ± 0.002 | 0.005a ± 0.000 | 0.006a ± 0.004 | 0.015b ± 0.002 | 0.004a ± 0.001 | 0.006a ± 0.002 | 0.004a ± 0.002 | 0.020c ± 0.006 | 0.018a ± 0.001 | 0.014b ± 0.003 | 0.011c ± 0.006 |
| C12:0 | 0.004abc ± 0.000 | 0.004abc ± 0.00 | 0.003a ± 0.000 | 0.004d ± 0.001 | 0.004cd ± 0.000 | 0.003ab ± 0.000 | 0.005e ± 0.000 | 0.006f ± 0.000 | 0.004cd ± 0.000 | 0.003a ± 0.00 | 0.003a ± 0.001 | 0.003a ± 0.000 |
| C14:0 | 0.242abc ± 0.018 | 0.255abcde ± 0.015 | 0.269e ± 0.007 | 0.258cde ± 0.005 | 0.268de ± 0.005 | 0.239ab ± 0.012 | 0.249abc ± 0.006 | 0.236a ± 0.007 | 0.266de ± 0.020 | 0.256bcde ± 0.019 | 0.266de ± 0.007 | 0.249abc ± 0.008 |
| C14:1 | 0.335b ± 0.006 | 0.035b ± 0.001 | 0.022a ± 0.002 | 0.036b ± 0.012 | 0.547d ± 0.003 | 0.020a ± 0.004 | 0.074e ± 0.008 | 0.046c ± 0.006 | 0.535cd ± 0.016 | 0.033b ± 0.002 | 0.033b ± 0.006 | 0.022a ± 0.003 |
| C15:0 | 0.089bcd ± 0.005 | 0.077ab ± 0.001 | 0.088bc ± 0.004 | 0.103d ± 0.022 | 0.075ab ± 0.005 | 0.069a ± 0.005 | 0.084bc ± 0.005 | 0.128e ± 0.015 | 0.066a ± 0.003 | 0.075ab ± 0.006 | 0.083bc ± 0.012 | 0.092cd ± 0.005 |
| C15:1 | 0.006d ± 0.002 | 0.003abc ± 0.002 | 0.003bc ± 0.002 | 0.002abc ± 0.002 | 0.002abc ± 0.001 | 0.002abc ± 0.001 | 0.001ab ± 0.000 | 0.000a ± 0.000 | 0.004cd ± 0.000 | 0.003abc ± 0.002 | 0.002abc ± 0.002 | 0.003abc ± 0.002 |
| C16:0 | 23.258ef ± 0.523 | 23.639f ± 0.426 | 23.153de ± 0.072 | 22.728bcd ± 0.167 | 25.463i ± 0.313 | 23.384efg ± 0.196 | 22.680abc ± 0.153 | 22.209a ± 0.153 | 24.381h ± 0.430 | 23.808g ± 0.168 | 23.106cde ± 0.178 | 22.461ab ± 0.301 |
| C16:1n9 | 1.824c ± 0.123 | 1.037b ± 0.073 | 1.047b ± 0.056 | 0.900ab ± 0.025 | 2.815e ± 0158 | 0.997ab ± 0.057 | 1.015ab ± 0.070 | 0.883a ± 0.077 | 2.382d ± 0.173 | 0.998ab ± 0.077 | 1.033b ± 0.082 | 0.911ab ± 0.054 |
| C16:1n7 | 0.743b ± 0.031 | 0.585ab ± 0.254 | 0.590ab ± 0.190 | 0.620b ± 0.167 | 0.677b ± 0.040 | 0.731b ± 0.160 | 0.358a ± 0.141 | 0.561ab ± 0.202 | 0.616ab ± 0.019 | 0.548ab ± 0.227 | 0.573ab ± 0.183 | 0.586ab ± 0.164 |
| C17:0 | 0.008bc ± 0.001 | 0.009de ± 0.001 | 0.009e ± 0.001 | 0.009de ± 0.001 | 0.008cde ± 0.001 | 0.008cd ± 0.001 | 0.006a ± 0.001 | 0.009de ± 0.001 | 0.006ab ± 0.001 | 0.006ab ± 0.000 | 0.007ab ± 0.001 | 0.007abc ± 0.001 |
| C17:1 | 0.003abc ± 0.001 | 0.005cd ± 0.001 | 0.003abc ± 0.001 | 0.003abc ± 0.001 | 0.002ab ± 0.000 | 0.006de ± 0.001 | 0.002a ± 0.000 | 0.002ab ± 0.000 | 0.004cd ± 0.001 | 0.007e ± 0.001 | 0.004bcd ± 0.004 | 0.012f ± 0.000 |
| C18:0 | 9.603b ± 0.172 | 9.744bc ± 0.084 | 10.713e ± 0.224 | 12.689h ± 0.225 | 9.112a ± 0.163 | 9.797bc0.312 | 10.319d ± 0.181 | 11.922g ± 0.215 | 8.980a ± 0.117 | 9.813bc ± 0.089 | 9.991c ± 0.082 | 11.170f ± 0.127 |
| C18:1n9 | 30.853e ± 0.315 | 28.433d ± 0.373 | 25.197b ± 0.271 | 21.844a ± 0.224 | 37.187g ± 0.762 | 27.925cd ± 0.240 | 24.824b ± 0.266 | 21.802a ± 0.151 | 33.984f ± 0.662 | 27.421c ± 0.526 | 24.695b ± 0.243 | 21.850a ± 0.202 |
| C18:1n7 | 0.911a ± 0.051 | 0.844a ± 0.362 | 0.807a ± 0.136 | 0.821a ± 0.232 | 1.143b ± 0.018 | 0.840a ± 0.135 | 1.434b ± 0.224 | 1.039a ± 0.145 | 1.147b ± 0.174 | 1.027a ± 0.282 | 1.065a ± 0.190 | 1.043a ± 0.166 |
| C18:2n6t | 0.024b ± 0.002 | 0.025bc ± 0.001 | 0.022b ± 0.002 | 0.026bc ± 0.008 | 0.043d ± 0.002 | 0.022b ± 0.003 | 0.047d ± 0.005 | 0.032c ± 0.008 | 0.040d ± 0.005 | 0.024b ± 0.004 | 0.019b ± 0.003 | 0.012a ± 0.003 |
| C18:2n6 (LA) | 25.454c ± 0.464 | 29.108d ± 0.242 | 29.999e ± 0.173 | 30.554f ± 0.253 | 17.922a ± 0.599 | 29.859e ± 0.389 | 30.709f ± 0.234 | 31.454gh ± 0.233 | 21.923b ± 0.440 | 29.911e ± 0.226 | 31.020fg ± 0.237 | 31.785h ± 0.166 |
| C18:3n6 (GLA) | 0.181c ± 0.020 | 0.219c ± 0.007 | 0.288d ± 0.019 | 0.353e ± 0.025 | 0.160ab ± 0.003 | 0.225c ± 0.021 | 0.288c ± 0.021 | 0.350e ± 0.029 | 0.145a ± 0.005 | 0.232c ± 0.023 | 0.292d ± 0.017 | 0.348e ± 0.033 |
| C18:3n3 (ALA) | 1.898c ± 0.070 | 1.932c ± 0.051 | 3.693d ± 0.179 | 5.014f ± 0.271 | 0.549a ± 0.029 | 1.968c ± 0.091 | 3.815de ± 0.128 | 5.248g ± 0.049 | 1.623b ± 0.162 | 1.966c ± 0.061 | 3.938e ± 0.066 | 5.468h ± 0.052 |
| C20:0 | 0.025cd ± 0.002 | 0.030de ± 0.004 | 0.030de ± 0.002 | 0.027cd ± 0.008 | 0.036e ± 0.011 | 0.027cd ± 0.003 | 0.021bc ± 0.003 | 0.021c ± 0.003 | 0.021bc ± 0.005 | 0.0134ab ± 0.001 | 0.029cde ± 0.007 | 0.010a ± 0.001 |
| C20:1n9 | 0.012e ± 0.001 | 0.015f ± 0.002 | 0.005ab ± 0.001 | 0.010de0.003 | 0.008bcd0.001 | 0.005a ± 0.001 | 0.008cd ± 0.001 | 0.006abc ± 0.000 | 0.006abc ± 0.000 | 0.005ab ± 0.003 | 0.007abc ± 0.002 | 0.006abc ± 0.003 |
| C20:2n6 | 0.019abc ± 0.002 | 0.024bcd ± 0.005 | 0.030cde ± 0.002 | 0.034de ± 0.004 | 0.044e ± 0.004 | 0.024bcd ± 0.002 | 0.126f ± 0.023 | 0.101g ± 0.018 | 0.025bcd ± 0.002 | 0.015ab ± 0.001 | 0.012ab ± 0.001 | 0.009a ± 0.001 |
| C21:0 | 0.049bc ± 0.005 | 0.052bc ± 0.006 | 0.052bc ± 0.005 | 0.050bc ± 0.006 | 0.057c ± 0.003 | 0.056c ± 0.009 | 0.119f ± 0.012 | 0.074e ± 0.011 | 0.058c ± 0.008 | 0.051bc ± 0.001 | 0.042ab ± 0.006 | 0.033a ± 0.004 |
| C20:3n6 | 0.215ab ± 0.017 | 0.220ab ± 0.045 | 0.292c ± 0.019 | 0.239ab ± 0.050 | 0.207a ± 0.019 | 0.242ab ± 0.033 | 0.259bc ± 0.027 | 0.290c ± 0.021 | 0.215ab ± 0.040 | 0.224ab ± 0.040 | 0.208a ± 0.030 | 0.258bc ± 0.021 |
| C20:4n6 (ARA) | 2.695d ± 0.115 | 2.144bc ± 0.094 | 2.028ab ± 0.069 | 1.988a ± 0.035 | 2.623d ± 0.087 | 2.070ab ± 0.153 | 1.970a ± 0.063 | 1.960a ± 0.035 | 2.283c ± 0.091 | 2.048ab ± 0.059 | 1.980a ± 0.114 | 1.992a ± 0.100 |
| C20:3n3 | 0.006a ± 0.001 | 0.014b ± 0.002 | 0.0125ab ± 0.002 | 0.013ab ± 0.005 | 0.018b ± 0.008 | 0.012ab ± 0.001 | 0.014b0.003 | 0.017b ± 0.005 | 0.057c ± 0.003 | 0.087d ± 0.004 | 0.093d ± 0.004 | 0.126e ± 0.006 |
| C22:0 | 0.010b ± 0.001 | 0.013cd ± 0.001 | 0.015de ± 0.002 | 0.012c ± 0.002 | 0.007a ± 0.001 | 0.012c ± 0.002 | 0.017e ± 0.001 | 0.013cd ± 0.003 | 0.008ab ± 0.001 | 0.011c ± 0.001 | 0.008a ± 0.002 | 0.008ab ± 0.001 |
| C22:1n9 | 0.004abc ± 0.000 | 0.004abc ± 0.001 | 0.008d ± 0.002 | 0.004abc ± 0.001 | 0.004abc ± 0.001 | 0.006cd ± 0.002 | 0.004abc ± 0.001 | 0.003ab ± 0.001 | 0.003ab ± 0.001 | 0.003ab ± 0.001 | 0.005bc ± 0.003 | 0.002a ± 0.000 |
| 20:5n3 (EPA) | 0.016a ± 0.004 | 0.036b ± 0.005 | 0.067c ± 0.004 | 0.096e ± 0.005 | 0.028b ± 0.011 | 0.035b ± 0.004 | 0.063c ± 0.003 | 0.089de ± 0.006 | 0.017a ± 0.001 | 0.035b ± 0.006 | 0.061c ± 0.002 | 0.085d ± 0.004 |
| C22:2n6 | 0.021c ± 0.004 | 0.023c ± 0.003 | 0.022c ± 0.002 | 0.015b ± 0.007 | 0.022c ± 0.002 | 0.003a ± 0.001 | 0.003a ± 0.001 | 0.002a ± 0.000 | 0.003a ± 0.000 | 0.023c ± 0.002 | 0.025c ± 0.006 | 0.024c ± 0.003 |
| C24:0 | 0.021abc ± 0.000 | 0.024abc ± 0.003 | 0.028bc ± 0.005 | 0.025abc ± 0.005 | 0.024abc ± 0.002 | 0.020abc ± 0.001 | 0.047e ± 0.009 | 0.045cd ± 0.009 | 0.021abc ± 0.004 | 0.014a ± 0.001 | 0.016ab ± 0.003 | 0.031cd0.024 |
| C24:1n9 | ND | 0.003b ± 0.000 | 0.005c ± 0.001 | 0.005c ± 0.001 | ND | 0.003b ± 0.000 | 0.004bc ± 0.001 | 0.005c ± 0.002 | ND | ND | 0.003b ± 0.001 | ND |
| 22:6n3 (DHA) | 1.764c ± 0.238 | 1.435b ± 0.135 | 1.489b ± 0.103 | 1.506b ± 0.221 | 1.130a ± 0.032 | 1.383b ± 0.175 | 1.424b ± 0.156 | 1.439b ± 0.107 | 1.313ab ± 0.048 | 1.321ab ± 0.118 | 1.364b ± 0.175 | 1.370b ± 0.040 |
| df | Mean squares | F-value | p value | |
|---|---|---|---|---|
| C18:0 | ||||
| Dose | 3 | 36.495 | 1138.033 | <0.001 |
| Period | 2 | 4.425 | 137.981 | <0.001 |
| Dose × period | 6 | 0.975 | 30.411 | <0.001 |
| Error | 96 | 0.032 | ||
| C18:1n9 | ||||
| Dose | 3 | 728.574 | 4582.418 | <0.001 |
| Period | 2 | 17.344 | 109.088 | <0.001 |
| Dose × period | 6 | 25.279 | 158.992 | <0.001 |
| Error | 96 | 0.159 | ||
| C18:2n-6 (LA) | ||||
| Dose | 3 | 525.677 | 4792.460 | <0.001 |
| Period | 2 | 18.368 | 167.455 | <0.001 |
| Dose × period | 6 | 39.113 | 356.585 | <0.001 |
| Error | 96 | 0.110 | ||
| C18:3n6 (GLA) | ||||
| Dose | 3 | 0.178 | 410.749 | <0.001 |
| Period | 2 | 0.000 | 0.893 | <0.5 |
| Dose × period | 6 | 0.001 | 2.311 | <0.050 |
| Error | 96 | 0.000 | ||
| C18:3n3 (ALA) | ||||
| Dose | 3 | 85.106 | 5697.618 | <0.001 |
| Period | 2 | 1.174 | 78.626 | <0.001 |
| Dose × period | 6 | 1.334 | 89.333 | <0.001 |
| Error | 96 | 0.015 | ||
| C20:4n-6 (ARA) | ||||
| Dose | 3 | 1.843 | 222.301 | <0.001 |
| Period | 2 | 0.172 | 20.750 | <0.001 |
| Dose × period | 6 | 0.099 | 11.979 | <0.001 |
| Error | 96 | 0.008 | ||
| 20:5n-3 (EPA) | ||||
| Dose | 3 | 0.026 | 880.377 | <0.001 |
| Period | 2 | 0.000 | 7.656 | 0.001 |
| Dose × period | 6 | 0.000 | 6.395 | <0.001 |
| Error | 96 | 2.929−005 | ||
| 22:6n-3 (DHA) | ||||
| Dose | 3 | 0.018 | 0.866 | <0.500 |
| Period | 2 | 0.506 | 24.281 | <0.001 |
| Dose × period | 6 | 0.186 | 8.921 | <0.001 |
| Error | 96 | 0.021 | ||
| Day 28 | Day 56 | Day 84 | ||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Control | HS-10% | HS-20% | HS-30% | Control | HS-10% | HS-20% | HS-30% | Control | HS-10% | HS-20% | HS-30% | |
| SFA | 33.315a ± 0.547 | 33.855abc ± 0.444 | 34.370cd ± 0.253 | 35.914f ± 0.324 | 35.071e ± 0.384 | 33.622ab ± 0.398 | 33.557ab ± 0.257 | 34.669de ± 0.335 | 33.834abc ± 0.381 | 34.071bc ± 0.169 | 33.565ab ± 0.241 | 34.088bc ± 0.348 |
| UFA | 66.685f ± 0.547 | 66.141def ± 0.445 | 65.627cd ± 0.253 | 64.082a ± 0.324 | 64.929b ± 0.385 | 66.372ef ± 0.398 | 66.441ef ± 0.257 | 65.328bc ± 0.335 | 66.166def ± 0.381 | 65.922de ± 0.169 | 66.431ef ± 0.240 | 65.899de ± 0.348 |
| MUFA | 34.389e ± 0.277 | 30.959d ± 0.610 | 27.684b ± 0.254 | 24.243a ± 0.486 | 42.181g ± 0.791 | 30.529cd ± 0.279 | 27.722b ± 0.201 | 24.344a ± 0.390 | 38.519f ± 0.670 | 30.038c ± 0.272 | 27.416b ± 0.370 | 24.424a ± 0.376 |
| PUFA | 32.296c ± 0.612 | 35.182d ± 0.253 | 37.943f ± 0.188 | 39.840h ± 0.563 | 22.748a ± 0.704 | 35.843de ± 0.404 | 38.719g ± 0.315 | 40.984i ± 0.331 | 27.647b ± 0.539 | 35.884e ± 0.218 | 39.015h ± 0.302 | 41.477i ± 0.225 |
| PUFA n-6 | 28.610c ± 0.502 | 31.765d ± 0.235 | 32.682e ± 0.217 | 33.209de ± 0.223 | 21.022a ± 0.655 | 32.446e ± 0.487 | 33.402g ± 0.208 | 34.189h ± 0.253 | 24.636b ± 0.403 | 32.472e ± 0.261 | 33.559f ± 0.301 | 34.428h ± 0.176 |
| PUFA n-3 | 3.685c ± 0.282 | 3.417c ± 0.102 | 5.262d ± 0.224 | 6.630e ± 0.374 | 1.726a ± 0.058 | 3.400c ± 0.225 | 5.316d ± 0.245 | 6.794ef ± 0.120 | 3.011b ± 0.162 | 3.409c ± 0.138 | 5.456d ± 0.135 | 7.049f ± 0.725 |
| n-6/n-3 | 7.801c ± 0.554 | 9.302d ± 0.295 | 6.222b ± 0.292 | 5.022a ± 0.262 | 12.185e ± 0.228 | 9.590d ± 0.691 | 6.295b ± 0.289 | 5.033a ± 0.077 | 8.198c ± 0.347 | 9.540d ± 0.427 | 6.155b ± 0.173 | 4.884a ± 0.042 |
| DFA | 76.288de ± 0.531 | 75.885cd ± 0.422 | 76.340def ± 0.075 | 76.772fg ± 0.163 | 74.041a ± 0.308 | 76.169cde ± 0.204 | 76.760fg ± 0.291 | 77.250h ± 0.159 | 75.146b ± 0.423 | 75.735c ± 0.169 | 76.422ef ± 0.176 | 77.070gh ± 0.287 |
| OFA | 0.158abc ± 0.006 | 0.150a ± 0.006 | 0.161abc ± 0.004 | 0.173c ± 0.021 | 0.159abc ± 0.006 | 0.144a ± 0.113 | 0.219d ± 0.014 | 0.218d ± 0.013 | 0.159abc ± 0.013 | 0.161abc ± 0.009 | 0.151ab ± 0.011 | 0.168bc ± 0.008 |
| SFA | ||||
| Dose | 3 | 6.719 | 53.592 | <0.001 |
| Period | 2 | 2.153 | 17.174 | <0.001 |
| Dose × period | 6 | 5.142 | 41.017 | <0.001 |
| Error | 96 | 0.125 | ||
| UFA | ||||
| Dose | 3 | 6.744 | 53.798 | <0.001 |
| Period | 2 | 2.120 | 16.916 | <0.001 |
| Dose × period | 6 | 5.134 | 40.956 | <0.001 |
| Error | 96 | 0.125 | ||
| MUFA | ||||
| Dose | 3 | 970.495 | 4703.078 | <0.001 |
| Period | 2 | 31.944 | 154.802 | <0.001 |
| Dose × period | 6 | 35.677 | 172.893 | <0.001 |
| Error | 96 | 0.206 | ||
| PUFA | ||||
| Dose | 3 | 900.335 | 5049.344 | <0.001 |
| Period | 2 | 31.078 | 174.296 | <0.001 |
| Dose × period | 6 | 61.527 | 345.062 | <0.001 |
| Error | 96 | 0.178 | ||
| PUFA n-6 | ||||
| Dose | 3 | 486.461 | 3816.600 | <0.001 |
| Period | 2 | 16.787 | 131.707 | <0.001 |
| Dose × period | 6 | 40.015 | 313.948 | <0.001 |
| Error | 96 | 0.127 | ||
| PUFA n-3 | ||||
| Dose | 3 | 91.235 | 2289.228 | <0.001 |
| Period | 2 | 2.237 | 56.123 | <0.001 |
| Dose × period | 6 | 2.392 | 60.012 | <0.001 |
| Error | 96 | 0.040 | ||
| PUFA n-6/PUFA n-3 | ||||
| Dose | 3 | 139.298 | 1112.580 | <0.001 |
| Period | 2 | 15.565 | 124.319 | <0.001 |
| Dose × period | 6 | 12.553 | 100.264 | <0.001 |
| Error | 96 | 0.125 | ||
| DFA | ||||
| Dose | 3 | 17.423 | 197.216 | <0.001 |
| Period | 2 | 0.746 | 8.444 | <0.001 |
| Dose × period | 6 | 4.008 | 45.366 | <0.001 |
| Error | 96 | 0.088 | ||
| OFA | ||||
| Dose | 3 | 0.007 | 52.041 | <0.001 |
| Period | 2 | 0.007 | 55.367 | <0.001 |
| Dose × period | 6 | 0.004 | 31.124 | <0.001 |
| Error | 96 | 0.000 | ||
| Day 28 | Day 56 | Day 84 | ||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Control | HS-10% | HS-20% | HS-30% | Control | HS-10% | HS-20% | HS-30% | Control | HS-10% | HS-20% | HS-30% | |
| Cholesterol (mg/g) | 9.27 ± 0.720 | 8.368 ± 0.570 | 9.648 ± 1.002 | 9.698 ± 1.449 | 8.284 ± 3.651 | 9.970 ± 0.671 | 7.991 ± 0.836 | 10.739 ± 0.844 | 8.284 ± 3.651 | 9.970 ± 0.671 | 7.991 ± 0.836 | 10.739 ± 0.844 |
| Day 28 | Day 84 | |||||||
|---|---|---|---|---|---|---|---|---|
| Control | HS-10% | HS-20% | HS-30% | Control | HS-10% | HS-20% | HS-30% | |
| α-Tocopherol (μg/g) | 50.520ab ± 3.295 | 40.686ab ± 0.905 | 52.863b ± 3.709 | 36.306a ± 1.964 | 272.037e ± 6.842 | 128.816d ± 2.288 | 86.829c ± 1.054 | 306.303f ± 12.412 |
| γ-Tocopherol (μg/g) | 25.012bc ± 1.409 | 32.919c ± 11.286 | 16.546ab ± 0.156 | 21.546abc ± 0.118 | 9.411a ± 1.106 | 32.332c ± 2.379 | 30.579c ± 5.224 | 20.726abc ± 0.765 |
| α-Toco/γ-Toco | 2.019a ± 2.019 | 1.322a ± 0.389 | 3.194a ± 0.193 | 1.685a ± 0.083 | 29.109c ± 2.580 | 3.997a ± 0.200 | 2.888a ± 0.428 | 14.780b ± 0.299 |
| Total tocopherol (μg/g) | 75.535a ± 4.677 | 73.606a ± 10.183 | 69.408a ± 3.864 | 57.853a ± 2.079 | 281.447d ± 7.946 | 161.148c ± 4.228 | 117.409b ± 5.991 | 327.028e ± 13.085 |
| df | Mean squares | F-value | p value | |
|---|---|---|---|---|
| α-Toco | ||||
| Dose | 3 | 16071.283 | 543.464 | <0.001 |
| Period | 1 | 140625.354 | 4755.364 | <0.001 |
| Dose × period | 3 | 18460.871 | 624.270 | <0.001 |
| Error | 16 | 29.572 | ||
| γ-Toco | ||||
| Dose | 3 | 275.048 | 275.048 | <0.001 |
| Period | 1 | 5.927 | 0.289 | >0.500 |
| Dose × period | 3 | 219.788 | 10.711 | <0.001 |
| Error | 16 | 20.520 | ||
| α-Toco/γ-Toco | ||||
| Dose | 3 | 217.636 | 241.916 | <0.001 |
| Period | 1 | 679.060 | 754.816 | <0.001 |
| Dose × period | 3 | 229.937 | 255.589 | <0.001 |
| Error | 16 | .900 | ||
| Total Toco | ||||
| Dose | 3 | 13592.350 | 251.142 | <0.001 |
| Period | 1 | 139826.796 | 2583.541 | <0.001 |
| Dose × period | 3 | 15803.113 | 291.990 | <0.001 |
| Error | 16 | 54.122 | ||
| Cholesterol | ||||
| Dose | 3 | 6.733 | 2.354 | >0.050 |
| Period | 2 | 1.370−007 | 0.000 | 1.000 |
| Dose × period | 6 | 2.456 | 0.859 | >0.500 |
| Error | 24 | 2.861 | ||
| Variables | Principal component | ||
|---|---|---|---|
| 1 | 2 | 3 | |
| C18:1n9 | -0.986 | 0.101 | -0.044 |
| MUFA | -0.979 | 0.132 | -0.028 |
| PUFA | 0.964 | -0.212 | -0.087 |
| PUFA n-3 | 0.949 | 0.017 | 0.093 |
| C18:3n-3 (ALA) | 0.945 | 0.046 | 0.136 |
| C18:3n-6 (GLA) | 0.930 | 0.046 | 0.188 |
| C16:00 | -0.907 | 0.120 | 0.301 |
| PUFA n-6 | 0.904 | -0.291 | -0.155 |
| DFA | 0.902 | -0.151 | -0.296 |
| C18:2n6 (LA) | 0.901 | -0.296 | -0.149 |
| EPA | 0.885 | 0.202 | 0.310 |
| n-6/n-3 | -0.879 | -0.003 | 0.032 |
| C16:1n9 | -0.865 | 0.280 | 0.108 |
| C18:0 | 0.863 | 0.267 | 0.331 |
| C20:4n-6 (ARA) | -0.761 | 0.059 | -0.020 |
| C15:0 | 0.667 | 0.370 | 0.118 |
| C24:1n9 | 0.628 | 0.352 | -0.079 |
| C20:3n6 | 0.521 | 0.230 | -0.050 |
| C20:2n6 | 0.291 | 0.753 | -0.440 |
| C18:2n6t | -0.390 | 0.723 | -0.278 |
| OFA | 0.492 | 0.622 | -0.281 |
| C22:2n6 | -0.106 | -0.535 | 0.438 |
| γ-Tocopherol | -0.025 | -0.418 | 0.174 |
| UFA | -0.166 | -0.538 | -0.791 |
| SFA | 0.164 | 0.541 | 0.789 |
| C20:3n-3 | 0.139 | -0.602 | 0.301 |
| α-Tocopherol | -0.044 | -0.288 | 0.174 |
| C20:1n9 | -0.089 | 0.059 | -0.125 |
| C22:1n9 | 0.057 | -0.005 | -0.060 |
| DHA | 0.280 | -0.140 | -0.409 |
| C16:1n7 | -0.237 | -0.183 | 0.118 |
| Cholesterol | 0.198 | -0.112 | 0.137 |