1. Introduction
With the rapid development of the global population and living standards, there is an increasing demand for livestock and poultry production, which had led to increased requirements for livestock and poultry feedstuffs. Over the last decade, the shortage of traditional poultry feedstuffs and the increased price of these has provided significant challenges and has inspired researchers to seek out unconventional or alternative feedstuffs. Alternative feed ingredients have garnered increased attention intending to develop a more sustainable, environmentally friendly, and economically viable poultry feed to reduce the current dependency on imported feeds and to reduce the costs associated with the production of high-quality poultry products [
1].
The nettle
Urtica cannabina (
U.
cannabina) belongs to the Urticaceae (nettle) family in the major group Angiosperms (flowering plants); it is distributed worldwide in temperate regions [
2]. Previously, nettle was considered a weed due to its soil coverage and rapid growth. However, the economic and ecological advantages of cultivating nettle are numerous. For example, nettle cultivation can improve soils over-fertilized with nitrogen and phosphate, reduce soil heavy metal content, and promote the biodiversity of local flora and fauna [
3]. In addition to these benefits, nettle is a rich source of nutrients, such as proteins and fatty acids (especially α-linolenic, cis-9,12-linoleic, and palmitic acids), it is well balanced in amino acids, and is an abundant source of minerals and vitamins [
4,
5]. It also contains many biologically active compounds in its roots and leaves, such as lignanoids, triterpenoids, steroids, polysaccharides, and isolectins, which have been shown to possess anti-inflammation activities in vitro [
6]. The young nettle leaf and stem are edible and thus, are commonly consumed as a food and are also used medicinally for various diseases [
7]. Previous research has demonstrated that mice fed a diet supplemented with nettle plants exhibited significant reductions in lipid metabolism, with reductions in triglycerides and total cholesterol contents in blood plasma [
8]. In monogastric animals, the composition of fatty acids stored in adipose tissue, muscle, or animal products such as eggs largely reflects the ingested lipids [
9]. The growing market demand for designed eggs with lower cholesterol and higher n-3 polyunsaturated fatty acids is gaining increased attention and a number of relevant studies have been conducted. In these studies, unconventional feedstuffs such as rubber seeds and sugar beet pulp, etc., have been found to produce specialty eggs and reduced feed costs [
10,
11]. Furthermore, organic egg production requires that poultry have access to forage materials, for instance, alfalfa, red clover, or nettle plants [
12,
13].
To date, most studies of nettle as an additive in poultry feeds have examined the growth and slaughter performance of broilers and their antioxidation and immune function [
14,
15,
16]. Limited data are available on the use of
U.
cannabina for laying hens. In particular, the studies have examined the effects of dietary nettle supplementation, as an alternative ingredient in poultry feeds, on vitamin and total cholesterol contents, and egg yolk fatty acid composition [
13]. Therefore, the aim of this study was to determine the effects of dietary
U.
cannabina supplementation on laying performance, egg quality, fatty acid composition, and cholesterol contents of yolks, and to assess the value of
U.
cannabina as a dietary ingredient for the production of high-quality eggs.
4. Discussion
In general, nettle plants are considered to be highly nutritional foods with multiple functional values [
22], including the potential to lower serum cholesterol in animals [
8]. However, there is only limited research on the effects of dietary
U. cannabina supplementation of laying hens. Thus, the purpose of the current research was to investigate whether the performance, egg quality traits, egg yolk lipid profile, and blood biochemical parameters of laying hens were affected by the dietary supplementation of
U. cannabina. The obtained results indicate that the addition of this unconventional feedstuff to the diets of laying hens can contribute to the production of high-quality eggs.
U. cannabina had no adverse effect on the overall production performance of hens in the present study. To date, the effect of nettle plants in poultry diets on the performance of laying hens has been a subject of debate. For instance, previous studies have reported that feed intake remained unchanged [
23,
24] or decreased upon dietary
Urtica dioica (
U. dioica) supplementation [
25]. Further, the feed conversion ratio has been reported to remain the same [
23] or even increased in response to the
U. dioica supplementation [
26,
27]. The primary terpenoids in
U. dioica (especially carvacrol and carvone) possess multiple biological functions, including growth promotion and antioxidant functions [
28]. It has been demonstrated that differential geographical distribution and environmental conditions influence these compounds and their derivatives, causing differences in their content in plants [
29]. This may be one reason for the discrepancies in the literature. Other factors such as the strain of birds studied (broiler, laying hen, or quail) and their physiological status may contribute to the varied findings.
In the current study, the inclusion of
U. cannabina in the diet of laying hens was associated with increased shell thickness and shell percentage. On the contrary, previous research reported that the inclusion of
U. dioica in the diets of 70-week-old Brown Nick laying hens and Japanese quails did not influence egg quality characteristics such as shell thickness, Haugh unit, and egg composition (albumen, yolk, and shell) [
23,
30]. Kregiel et al. [
3] reported that in addition to the large amounts of calcium in nettle leaves and stem, nettle leaves also have an abundance of zinc, magnesium, and iron. Enrichment of egg mineral elements was observed in the
U. cannabina group in the present study, which is consistent with the above results.
Moreover, previous research reported that the systemic administration of
U. dioica increased serum calcium concentration and bone volume and accelerated new bone formation due to its anti-inflammatory effect in rats [
31,
32]. The anti-inflammatory effect of
U. dioica was mainly attributed to its antioxidant constituents, especially the phenolic compounds, which inhibit nuclear factor (NF)-κB activation, thereby reducing pro-inflammatory gene products [
32]. Furthermore, as an important eggshell formation factor in the avian uterus, calbindin plays a primary role in Ca
2+ transportation [
33]. The expression of calbindin mRNA was found to be disturbed in the uteri of birds infected with the avian influenza virus via the influences of substances from cytotoxic cells and proinflammatory cytokines; this was found to produce a deterioration of eggshell quality [
34,
35]. Therefore, based on the presence of abundant calcium in the eggs from the
U. cannabina group in this study, it can be assumed that supplementation with
U. cannabina may increase the calcium transport by calbindin due to its anti-inflammatory effect, thus increasing shell calcification which consequently leads to improvements in shell quality features. However, the related gene expressions and concentrations of cytokines still require further research.
The yolk color also represents a vital indicator of egg quality; this quality is particularly important to consumers. In our study, the enhanced yolk color was found in the
U. cannabina group. A similar phenomenon was also observed in another nettle study. Loetscher et al. [
30] studied the effect of
U. dioica on egg quality traits and found that yolk yellowness (
b*) was increased in the
U. dioica group, depending on the dosage. Further,
U. dioica was found to be equally as effective as synthetic pigmentation. Nettle is rich in yellow-colored xanthophylls, with lutein being the predominant compound, followed by β-carotene and zeaxanthin [
3]. It has been shown that the inclusion of
U. dioica in food increases the enrichment and bioaccessibility of lutein and
β-carotene (provitamin A carotenoids) at the duodenum digestion stage [
22]. Furthermore, xanthophylls were found to be absorbed in the digestive tract and deposited in subcutaneous fat and yolk leading to higher yolk color scores [
11]. Therefore, the higher yolk color observed in the
U. cannabina group in this study might be due to its high levels of effective polar xanthophylls, such as lutein and zeaxanthin, which could be deposited in egg yolks [
36]. In addition, β-carotene is almost completely converted into vitamin A in poultry [
36]. This is consistent with the observation of increased vitamin A content in the
U. cannabina supplementation group in the present study.
Recently, designed eggs with improved lipid profiles, such as lower cholesterol and higher PUFA, have gained popularity among consumers. In the present study, the concentration of egg yolk cholesterol was decreased in the
U. cannabina supplementation group compared to the control group. These findings are consistent with the findings of Moula et al. [
23] in which dietary supplementation with the
U. dioica powder reduced the total cholesterol in the yolks of eggs from Japanese quails. Nettle plants have been shown to be rich in bioactive compounds such as phytosterol, pentacyclic triterpenoids, coumarins, and ceramides [
3]. The lower egg yolk cholesterol concentration in the
U. cannabina group can be attributed to its phytosterol components (such as astigmasterol and campesterol compounds) which can decrease the cholesterol concentration in micelles to reduce the absorption of cholesterol in the gut, thereby lowering cholesterol levels in the blood [
8], and subsequently, in animal products.
Nettle leaves are rich sources of essential fatty acids. ALA is the pre-dominant fatty acid, accounting for 40.7% of the fatty acids in the mature leaves [
5]. The composition of fatty acids stored in monogastric animals largely indicates that the lipid and fatty acid composition of poultry eggs can be altered by diet [
37]. Hence, eggs can be an important source of n-3 PUFA when laying hens are fed a diet containing a high level of n-3 PUFA [
11,
38]. Numerous fatty acid desaturases play key roles in synthesizing PUFA. Several desaturases are absent in animals and humans, such as delta-12 and delta-15 desaturases [
39]. Thus, ALA must be obtained from the diet; it can be converted into EPA and DHA by delta-6 desaturase catalyzed dehydrogenation and the addition of two carbons by an elongase [
40]. An n-3 PUFA-enriched diet was found to increase the expression level of delta-6 desaturase mRNA [
11]. In the present study, elevation of DHA and EPA contents was also considered to reflect primary dietary regulation of delta-6 desaturase gene. In addition, the
U. cannabina supplementation in the current study was associated with a reduction in the MUFA (predominantly C18:1n9c) proportion and a significant elevation in the ALA proportion compared to the control diet. This phenomenon is consistent with a previous study where hens fed with rubber seeds oil (an n-3 PUFA enriched ingredient) were found to lay eggs with a decreased content of C18:1n9 along with an increased ALA content; these findings were thought to be related to the conversion of C18:1n9 into ALA through desaturation [
11]. In our study, the content of n-3 PUFA was significantly higher in the
U. cannabina group than in the control group (
p = 0.009), which caused a great improvement in the ratio of n-6/n-3 in the
U. cannabina group (4.95 vs. 12.67), exceeding the recommended intake ratio of 4:1 (n-6 PUFA 10 g/d, n-3 PUFA 2.5 g/d) by the European Food Safety Authority (EFSA) [
41]. Stojčić et al. [
14] similarly reported that dietary supplementation with fresh nettle reduced the ratio of n-6/n-3 and increased the proportion of n-3 PUFA in the breast meat of broiler. It is important to maintain a low ratio of n-6/n-3 in the diet due to its role in lowering the risk of cardiovascular diseases [
42,
43]. A higher total n-3 PUFA content, especially DHA, in the group supplemented with
U. cannabina could be explained by the protective function of antioxidative compounds such as lutein, provitamin A, tocopherol, and phenolic compounds. Stinging nettle is an abundant source of lutein, which has been considered to be an important natural pigment for improving egg yolk and broiler skin color [
15,
30]. Lutein and phenolic compounds (particularly flavonoids) are potent antioxidants due to their free radical quenching activities [
32,
44]. Grčević et al. [
38] reported that the addition of marigold powder (rich in lutein) to the laying hens’ feed significantly increased egg lutein content and preserved the high content of DHA in the yolks. Loetscher et al. [
15] provided broilers with a diet supplemented with 2.5%
U. dioica and found that the tocopherols content of the breast meat was significantly increased. Based on these results, we assume that the richness of antioxidants in
U. cannabina had an impact on the conservation of DHA in the lipids of the egg yolks by protecting DHA from oxidation and damage, thereby maintaining a higher level of DHA in the
U. cannabina group. Further studies should determine the level of these antioxidants in both
U. cannabina and the eggs produced by hens fed the
U. cannabina supplemented diet. Nonetheless, these findings indicate that, as an important n-3 PUFA source,
U. cannabina contributed to the production of n-3 PUFA-enriched and stable eggs.
In the current study, the laying hens fed with
U. cannabina exhibited no health issues and normal serum biochemical parameters were observed. Hepatic serum levels of ALT and AST are frequently used to evaluate liver injuries. In this study, there were no significant changes in serum ALT and AST levels in the
U. cannabina group compared with the control group, which confirms that there was no diet-induced liver injury [
37]. A decrease in total cholesterol and triglycerides and an increase in HDL-cholesterol was observed in the serum of hens fed with
U. cannabina due to the rich sterols content of
U. cannabina. On the other hand, a higher HDL-cholesterol content in the
U. cannabina group facilitated the translocation of cholesterol from the peripheral tissues to the liver for catabolism [
8], which might be another reason for the reduced serum level of cholesterol.