Impact of Cannabis Seed Incorporation in Layer Diet on Productive Performance and Egg Quality Traits

The production of nonindustrial cannabis is highly developed in the Moroccan Rif region; however, local farmers consider hemp seeds which are rich in omega 3 and tocopherols, only as by-products of cannabis cultivation with low market value. The local ecotype is considered to be a plant with a cannabinoid content of more than 0.4%. So, the objective of this research is to investigate how the incorporation of this local hemp seed affects productive performance and egg quality traits. The experiment is conducted to evaluate the effects of hemp seed (HS) incorporation on hen laying performance and physical egg quality at three levels: 10% (HS-10% group), 20% (HS-20% group), and 30% (HS-30% group). Ninety-six Lohmann Brown classic laying hens were randomly assigned to a control group and three feed treatments. The sampling was taken after the 28-week rearing period (peak egg laying). Throughout the experiment, low-rate HS inclusion (HS-10%) showed no significant differences in egg-laying performance (p > 0.05). However, the high incorporation rates of HS (20% and 30%) negatively affected the egg-laying performance (84–94% and 80–86%, respectively). The albumen quality was also improved by the HS inclusion, where the highest values of the Haugh unit were recorded, ranging between 68.69 and 73.91 for the HS-30% groups. The results also show that HS inclusion and duration influence significantly the yolk color (p < 0.001). The yellow intensity decreases with HS incorporation and aging, from a dark yellow (b∗ = 38.63 for the control group) to a very pale yellow (b∗ = 26.29 for HS 30% group). Based on these findings, we can conclude that the incorporation of nonindustrial Moroccan cannabis seeds (ecotype Beldiya) at low rate in the diet of laying hens does not alter the laying performance or the quality of the egg; therefore, they could be used in poultry feeding as an alternative constituent to partially replace high-cost imported ingredients, such as corn and soybeans.


Introduction
Egg production is one of the most important poultry industries, attracting substantial investment worldwide. Consequently, there is an urgent need to improve layer productivity and egg quality by using natural feed substances [1,2]. Consumers have become more aware and demand healthy foods that are exempt from synthetic substances [3].
Using phytogenic extracts, as a source of bioactive compounds, is becoming an appealing strategy to improve the health-related properties of animal-source foods (ASF) [4]. To accomplish this requirement, food scientists and animal breeders are developing diferent strategies to improve the nutritional profle of ASF. Te latter are important sources of essential amino acids, vitamins, and oligoelements, but their lipid composition is often criticized. Enriching animal products with n-3 PUFAs remains a sustainable solution for a healthy diet [5]. Furthermore, the use of phytogenic feed additives is becoming an attractive and sustainable strategy to improve animal products' lipid quality and functional properties.
Drought has always been present in the history of Morocco, but its importance as a structural element of the country's climate has been increasing in recent decades. Te cultivation of oilseeds is often very dependent on irrigation; consequently, the production of these seeds is very limited. Morocco is indeed 80% dependent on imports. It has a signifcant defcit in oilseeds, which are an essential ingredient in poultry feed. Oilseed crops partially contribute to the country's food security by producing edible oils and providing cakes which are protein-rich by-products and essential for the animal feed industry (e.g., the poultry sector). In the current context of drought, war, and the Covid pandemic, the price of oilseeds has soared, leading to an increase in the price of poultry feed and, therefore, chicken and eggs.
Cannabis (Cannabis sativa L.) is one of the oldest and most versatile herbal plants cultivated by man. Tis plant has traditional and cultural roots in many countries around the world. Recent publications suggest that it originated in Central Asia [6,7], while others suggest its origins are in South Asia [8]. Cannabis seeds that have been unused in recent years by cannabis producers in Morocco could constitute an alternative source, even partial, to soybeans, sunfower, and maize seeds imported at increasingly high prices. Te hemp seed and its cake represent an interesting source of amino acids, phenols, tocopherols, minerals, and polyunsaturated fatty acids [9]. It was demonstrated in our previous study that the local hemp seed contains 94.08% of dry matter, including 20-23% crude protein, 30-34% lipids (80% of which are polyunsaturated fatty acids), 26-37% insoluble fber, and 3-5% ash [10]. Tese characteristics imply its potential use as a phytogenic ingredient or an additive in animal feed with health-promoting properties. So, hemp seeds could be a valuable alternative to improve and reduce the cost of imported feeding stuf for animals, mainly poultry. In fact, nonindustrial hemp seeds, considered a by-product of cannabis culture, have a low commercial value. However, they have excellent nutritional value in terms of lipid profles. According to the United Nations Ofce on Drugs and Crime, there were 47.196 hectares of hemp cultivation in Moroccan Rif [11]. It was prohibited because of the psychoactive and addictive properties of its tetrahydrocannabinol (Δ9-THC) component. However, in December 2020, in order to take advantage of the growing legal market and increase farmers' income, the Moroccan government initiated the legalization and regulation of cannabis production with low THC for innovative industrial applications. Actually, there is a growing interest in the plantation of cannabis for medicinal and cosmetic uses, and even in the use of whole hemp seeds in food as well as in the production of four and hemp seed oil. Much research has been performed on adding hemp to animal feed. Due to its positive efects, much attention has been oriented recently to the use of Cannabis sativa in farm animal diets, especially poultry [12]. However, the efect of the Moroccan ecotype of Cannabis sativa seeds was never studied.
Te ultimate goal of the research is to promote the Moroccan hemp seeds produced in the Rif by including them in poultry diets. With this in mind, this study aims to evaluate the efect of diets based on diferent incorporation rates of these seeds on egg production and quality traits.

Birds and Housing.
Te animal experiment was conducted at the Royal Institute of livestock, Kenitra, Morocco, in compliance with the European Code CO 74/99 concerning stocking density, lighting, vaccination, and other procedures. No breeding practices other than those normally employed were introduced during the trial.
Ninety-six (n � 96) Lohmann Brown Classic (LBC) laying hens were randomly assigned to three feed treatments and a control group; each treatment was repeated 6 times (4 Hens/replicates). Te hens used in this experiment were selected from a legitimate industrial farm at 22 weeks of age with an initial weight varying between 1.70 and 1.80 kg. Te birds were carried out in a semi-automatic facility with automatic dung mats; whereas, the hens were housed, 4 per cage, equipped with trough feeders and nipple drinkers with the following dimensions: 61 cm long, 57 cm wide, and 50 cm high. Te hens were confned in optimal conditions with temperature and humidity controlled at 18-20°C and 55-60%, respectively. Te lighting regimen was 16 h/day from 06:00 am to 10:00 pm. Te laying hens were kept in welfare conditions.

Diets and Experimental
Approach. Diets were formulated using similar nutrients and ingredients to standardize the energy level of the studied diets (3000 kcal/kg). Te formulation was developed in consultation with BENWAY, a company specializing in poultry feed. Maize/soya beanbased diets were utilized by the inclusion of diferent levels of hemp seed of the local ecotype "Beldiya: 0.0% (HS-0), 10% (HS-10), 20% (HS-20), and 30% (HS-30). At 22 weeks of age, all hens were allowed to adapt to the new environment system (cage and feed) for 2 weeks. During the frst week, hens were fed a commercial layer diet, and during the second week, the studied diets were progressively incorporated (25%, 50%, 75%, then 100%). Feed was distributed 3 times per day, and the water was made ad libitum to the hens. Eighteen eggs per group were sampled randomly during the last three days of each period 24-28, 28-32, and 32-36 for egg quality traits and yolk color measurement. Te chemical compositions of the HS and experimental diets used in this study are given in Tables 1 and 2, respectively.

Hen Performance.
In total, 216 eggs were collected for the 4 study groups, which corresponds to 18 eggs per experimental group for 3 days, resulting in 54 eggs for each treatment. Egg production (EP) and egg weight (EW) were measured and recorded daily. Total feed consumption was determined as the diference between feed ofered and residual feed remaining in the feeders (g feed ofered/g feed remaining). Hen body weight (BW) was measured weekly, on the same day as the weight of the feed. Feed conversion ratio (FCR) per dozen eggs was calculated by recording the total feed consumed in a week divided by the total number of eggs produced in that week and multiplied by 10 [13]. Mortality was recorded daily.

Egg Quality Measurements.
For external and internal egg quality parameters, nine eggs were hand collected from each cage and enumerated. Each egg was weighed using an electronic balance (Radwag Wagi Elektroniczne). Eggs were broken on a fat glass surface, and eggshell weight (g) was measured. Te albumen and yolk were separated, the yolk was weighed, and the thick albumen and egg yolk height was measured within an electronic tripod micrometer (Ingco-HDCD01200). Te albumen and yolk weights were divided by the whole egg weight and then multiplied by 100 to determine the weight percentage. Egg shape index (%), egg yolk index (%), and egg yolk albumen index (%) were recorded according to Romanof and Romanof [14]. Haugh units were calculated using the following formula [15]: where H represents the white height and W represents the egg weight.

Color Yolk Analysis.
Te yolk color was measured using Chroma meter (Konica Minolta, CR400) according to the CIELAB system: L * lightness, a * redness (red-green), and b * yellowness (blue-yellow). Te hue angle H * and chromaticity C * were calculated by the following formula [16]:

Results and Discussion
3.1. Zootechnical Performances. Te efect of hemp seed incorporation on production efciency and egg quality has been the subject of several works, some of which revealed a benefcial efect while others showed the opposite [17]. However, most research focused on industrial cannabis, whereas our experiment examined the efects of seeds devoid of THC that originated from ecotype Beldiya, which is categorized as a nonindustrial variety. Te efects of the Moroccan ecotype Beldiya incorporation on the zootechnical performance, given in Tables 3-6, show that the hemp seed diet and duration considerably impact all the examined zootechnical parameters (p < 0.05). Te degree of the seeds' inclusion has a positive efect at specifc levels which should not be exceeded. Te highest levels of egg production (100%) were observed in the HS-10 treatment during the frst week, while the lowest levels were observed in the HS-30 treatment during the same week (80.56%). Tis study was carried out at the peak of egg laying (28-33 weeks of age) of the LBC strain according to the standard of this strain [18]. However, the pre-registered laying peaks in our experimentation were diferent from those of the treatments, indicating the efect of the incorporation rate of HS on hen laying peaks. Te peak of oviposition was recorded early in the W-28 for the HS-10% group with 100% of egg production, and then it stabilizes at 97.22%. On the other hand, the control group as well as the HS-20% group reached their intermediate peaks in the W-32 with 100% and 94.44%, respectively. In contrast, the peak egg production was recorded late for HS-30% in W-36 with 86.11%, below the peak of the Lohmann Brown Classic strain determined at 94%. Tis result could be explained by the diferent adaptation periods required for each studied group. Skřivan et al. [19] showed that the use of low doses of hemp seed in poultry feed can improve performance and product quality, but more than 30 g/kg can lead to poor performance due to antinutritional agents, including polyphenols and phytate. Indeed, if greater amounts of cannabis seeds are added, the adaption period is expected to be prolonged. Te recorded efects of the HS diet are comparable to those reported in Neijat et al. [20], where a study was conducted on the efect of industrial HS on the zootechnical traits of laying hens. Te fndings demonstrate that regardless of the dosage of cannabis seeds used, EW rises with age. Tus, the EW increases on average from 55 to 60 g (control), 53 to 56 g (HS 10%), 50 to 59 g (HS 20%), and 52 to 58 g (HS 30%). However, adding cannabis seeds to the feed did not result in an increase in egg weight compared to the control. Tus, after 32 weeks, the weight of the eggs decreases from control to 30% HS by an average of 10 g. Tese results are in accordance with [20], whose fndings demonstrated that hens receiving 30% HS had signifcantly lower EW than the control or lower HS doses (10 or 20%). [21] worked on 19-week-old hens with two weeks of adaptation, showing that egg weights increased between the frst and second weeks; the study also reported a steady EW increase until the ffth week before eventually remaining constant. However, March and MacMillan [22] demonstrated that a linolenic acid increase in the diet was an important factor in improving egg size and weight. Tis fnding can be explained by the need for long-chain fatty acids to synthesize lipoproteins, which can be transported to the ovary to be absorbed by developing eggs (March and MacMILLAN, 1990). Te observed increase in egg production and egg mass of laying hens in the present investigation for HS-10% was probably due to the presence of essential nutrients in hemp seeds such as amino acids, longchain fatty acids, oligo, and microelement components, and fbers in this phytogenic resource that has resulted in better laying performance at a low rate. It is also possible that hemp seeds might have stimulated the hepatic secretion of egg yolk precursors by protecting hepatocytes from oxidative damage with subsequent enhancement of yolk formation and ovulation [4]. In this sense, Gharaghani et al. [23] demonstrated that using fennel (Foeniculum vulgare) in poultry feeding, particularly in laying hens, decease the heat stress. Based on this proposition, this coproduct of cannabis culture can replace the chemical hepatoprotector used in laying hens.
Tese results are in agreement with those reported by Lee et al. [24], which showed that hempseeds meal rich with PUFA can reduce oxidative toxicity due to their antioxidant properties. Te hens' weight declines after 32 weeks but grows during the 36 th week. Depending on experiment duration, these variations would be most explained partly by the more or less rapid adaptation to diet-supplemented cannabis seeds and by the laying rate. In the control group, the variation in hen's weight could be explained by the increase in the laying rate, which accounts for a large part of the energy consumed. Te weight increased after the decline in the laying rate. Jing et al. [21] and Silversides and Lefrancois [25] observed a reduction in hen body weight over their study period regardless of the inclusion levels. Regarding the FI and FC, the results also show a signifcant diference (p < 0.05) between the studied groups. Te hen's feed intake showed a marked increase between W-28 and W-32 and a stability between W-32 and W-36 for all studied groups except for the HS-30% group. Tis increase positively afects the FC, logically leading to an increase in egg weight throughout the experiment. Tese results could be linked to the adaptation of the hens to the feed ration and to their increase in body     weight. Also, they can be related to the increase in egg weight. We also noted that FI was greater than 140 g/egg/day regardless of the duration of the experiment or the seed dose included in the diet, indicating that the addition of cannabis seeds did not afect the diet palatability.

Physical Egg Quality.
Te results of the hemp seed inclusion in the laying diet are illustrated in Tables 4 and 6. Te YW was negatively correlated with the incorporation rate of HS (p < 0.05), but positively correlated with the aging period (Table 4). Te higher value of YW was recorded in the egg of the control group at W28 (25.61%), while the lowest value was found for HS-30 at W28 (21.38%). Te AW was not afected by the duration for the control and HS-10% groups, but a decrease was recorded between W-32 and W-36 for HS-20% and between W-28, W-32, and W-36 for HS-30%. However, the lowest results were recorded for the control and HS-10% group at W-28 and W-36 (64.28% and 64.38%, respectively). Te highest result was registered at W-28 and W-32 for HS-30% and HS-20% groups. Cufadar et al. [26] reported that adding hemp seed meal to the diet of quails increases the albumen index. Tis result could be linked to the same bioactive components of HS, such as antioxidants, crude protein, and long-chain fatty acids that protect the magnum and uterus and promote albumin secretion in layers [27]. Haugh unit (HU) was calculated using the albumen height and egg weight. It is a parameter that makes it possible to evaluate the albumen quality [4]. Te current study results show that the inclusion of HS in a hen's diet signifcantly improves albumen quality (HU). Te HU in the HS-30% group was signifcantly higher than other groups, as depicted by the following results: 73.91 for HS-30, 73.91 for HS-10, 72.54 for HS-20, and 68.03 for control; all results are registered at W36. Furthermore, the HU was afected by age (p < 0.05). Tis improvement could be related to the HS richness of the high-quality protein, especially the edestin and albumin fractions. Tis result is in agreement with that reported by [28], which explains this improvement of HU by the HS inclusion rate. Moreover, several studies demonstrate that using phytogenic additives with antibacterial and antioxidant properties in laying hen diets could improve albumen quality [29,30]. Te Yolk index (YI) showed no diference between HS-10 and control (p > 0.05). Te average of the recorded index for the other groups was around 0.43. Te best results were recorded at W32 for HS-20 and HS-30 by an overall average of 0.48 and 0.47, respectively. Our data show that all the eggs tested are in the extra-fresh class (YI > 0.38). In addition, the incorporation rate of HS is positively correlated with the YI increasing and will consequently provide an improvement in egg quality and contribute to improving the conceivability of eggs. Our results are similar to those of Hosseini-Vashan and Ghiasi [31] who reported that an increase in the inclusion of industrial hemp seeds or hemp seed oil leads to a signifcant improvement in the yolk index. Concerning the shell thickness (ST), HS 10% is similar to control for all age groups. HS 20% and HS 30% have a higher value than control for only 36 weeks of age with an average of 0.44 mm. Tis result could be explained by the adaptation phenomenon and the efect of the accumulation of bioactive molecules.

Yolk Color.
Although there are diferences of opinion around the world regarding consumer preference for egg yolk color, all studies agree that this parameter is among the most important criteria in the purchase of eggs [32]. As shown in Table 5, the yolk color varies according to the duration of the experiment and the dose of cannabis seeds included in the diet. Tus, the comparison of eggs from control hens and those from hens on diets with cannabis seed supplementation (10, 20, and 30%) shows signifcant diferences (Tables 5 and 6) in the color parameters: L * (lightness), a * (redness), and b * (yellowness). Te most visible efect is the decrease of b * , which considerably afects the intensity of the yellow color, changing from a dark yellow to a very pale yellow. Indeed, after 36 weeks of experimentation, b * goes from a value of 38.63 (control) to 26.29 (H-S 30%). Tis decrease can be explained by the fact that the control lot hens were fed a diet containing corn seeds rich in carotenoids, and that they were gradually replaced by cannabis seeds whose carotenoid content was much lower than that of corn. Maoka [33] linked the dark yellow color to the content of carotenes and xanthophylls, while the orangered color would be linked to chlorophylls. Tis decrease in yolk intensity may be a handicap for marketing these eggs on the Moroccan market, as consumers prefer eggs with an intense yellow yolk color, which they generally link to a freerange diet. It will be necessary to think about improving the quality of the eggs produced by including doses of cannabis not exceeding 10% or by another formulation where the cannabis would be a substitution of soya or sunfower cakes and not of corn.

Principal Component Analysis.
Principal component analysis (PCA) was carried out to easily visualize the relationships among the studied groups of hens and evaluated parameters. Te analysis was conducted using 18 variables, including zootechnical performance, egg quality traits, and yolk color measurement. Te results show that the three frst principal components (PC) account for more than 85% of the total information (Table 7). Tese three PC explained 57.20%, 22.06%, and 6.63%, respectively. Te PC1 was mainly characterized by shell weight, egg weight, egg percentage, and yellowness index on the right side. Te left side was characterized by albumen weight and feed conversion (FC), Figure 1. Te PC2 was defned positively by feed intake (FI), Haugh units (HU), and the yolk index, and in the opposite direction, it was defned by lightness and body weight (BW). Te projection of groups on the factorial map reveals a discrimination between the studied hen groups and the  measured parameters (Figure 2). Tis discrimination allows a simple and summarized interpretation already mentioned above by ANOVA analysis. In fact, the control and the HS-10% were located on the right side of Figure 2 and were clearly diferentiated from all studied hens, where the EP, EW, and yellowness index lie. Te HS-30% groups were diferentiated from the other hen types and are located on the left side of Figure 2. Concerning PC 2, the relationship between the studied hen groups is unclear. Te two most important variables, which are regrouped into the PC 2, were HU and FI. Te PCA exposed the efect of Moroccan autochthonous nonindustrial hemp seed incorporation in the diet of laying hens on zootechnical performance and physical egg quality. Numerous researchers reported similar discrimination between animals reared under phytogenicbased feeding [34,35].

Conclusion
Tis study investigates the efect of the Moroccan ecotype nonindustrial hemp seed incorporation in the laying hens' diet on zootechnical performance and physical egg quality. Te results show that the incorporation of hemp seed at low doses (<20%) has no adverse efect on the laying performance. Furthermore, incorporating HS in the diet leads to improved albumen quality while the yolk index was not afected. However, this incorporation causes a diminution of the egg yolk's color intensity. Based on these conclusions, Moroccan ecotype nonindustrial hemp seeds can be introduced at low rates as a substitute for conventional ingredients of laying hen feed, such as corn, soybeans, or other oil seeds. Further studies are being conducted in our laboratory to evaluate the nutritional value of the eggs produced as a result of this incorporation.

Data Availability
Te 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
Te authors declare that there are no conficts of interest.

Authors' Contributions
YT and KB were responsible for designing the research methodology, carrying out experimental tasks, and writing the frst draft of manuscript. FM was responsible for statistical analysis and data work interpretation. YR and NE were responsible for designing the research methodology. AB and RM were responsible for data analysis and data work interpretation. EA was responsible for designing the research methodology, data analysis. CHS and AE were responsible study conception, data analyses, and fnal approval of the version.