With the development of economy and living standards, there is an increasing consumption for livestock and poultry products (meat, eggs, milk, etc.), which promotes the vigorous development of animal husbandry and demand for feedstuffs. In the past a few years, the lack of feedstuffs has become an important factor restricting the development of the livestock industry due to the shortage of traditional feed resources and the rising prices (Li et al., 2019). To reduce the dependency on the imported feeds and reduce the costs of production, seeking new feed sources with high quality and yield has become a hot spot in the field of animal nutrition (Reda et al., 2022).

Ramie (Boehmeria nivea) is a perennial herbaceous plant that belongs to the Urticaceae (Boehmeria) family; it is originated in China, commonly known as “Chinese grass” (Ni et al., 2018). As a traditional textile raw material, only 5% of ramie are utilized, resulting in a great waste of resources (Liu et al., 2013). In recent years, the development and utilization of high-quality unconventional feed resources have attracted much attention. Ramie is a source rich in nutrients, such as proteins, fatty acids (especially oleic acid, palmitic acids and linolerc acid), minerals, and vitamins. It is also well balanced in amino acids with the total amount up to 18.36% (Wang et al., 2019; Wang et al., 2021a). Ramie contains many biologically active compounds in its roots and leaves, such as flavonoids (Rutin, Rhoifolin, beta-ionone) and polyphenols compounds (chlorogenic acid, ferulic acid, caffeic acid), which have been shown to possess antibacterial and anti-inflammation activities in vitro (Xiao et al., 2014; Wang et al., 2019).

Previous studies have proved that other unconventional feedstuffs, such as almond hulls, alfalfa and nettle plants, are able to improve egg quality and reduced feed costs (Wang et al., 2021b; Grela et al., 2020; Zhang et al., 2020). It also have been proved that supplementary nettle (Urtica dioica), which belong to the same family as ramie, can significantly reduce the lipid metabolism and reduce the total cholesterol, triglyceride contents in blood plasma of mice (Avci et al., 2006). To date, most studies regarding to nettle as an alternative ingredient in laying hens’ feeds have confirmed that it can increase yolk color, decrease total cholesterol contents, improve egg yolk fatty acid composition (Loetscher et al., 2013a; Zhang et al., 2020). Ramie is a plant of Urticaceae, and its high nutritional content makes it a potential new material feed. Therefore, the aim of this study was to assess the potential of ramie as a dietary ingredient for the laying hens, by determining the effects of dietary ramie supplementation on the production performance of hens, and the quality, nutrients composition, and oxidative status of the eggs.

All experiment procedures in the present study were approved by the Animal Care Committee of the Institute of Bast Fiber Crops, Chinese Academic of Agricultural Sciences.

Ramie leaves, which are commonly used for medicinal and edible purposes, are effective in reducing serum cholesterol and improving meat quality of farmed animals (Avci et al., 2006; Tang et al., 2021). However, there is only limited research on the effects of dietary ramie supplementation on laying hens. Thus, the aim of this study is to investigate whether the production performance, egg quality traits, yolk antioxidation indices and egg nutrients composition of laying hens are affected by the dietary supplementation of ramie. Ramie had no adverse effect on the overall production performance of hens in the present study. Many researches showed that the egg production increased in response to the nettle supplementation, such as Urtica dioica, Urtica cannabina and Urtica fissa (Loetscher et al., 2013a; Zhang et al., 2020; Liu et al., 2010). Ramie as a perennial dicotyledon of the Urticaceae family (Wei et al., 2014), might possess the same effect as nettle. Previous studies reported that egg production remained unchanged (Wang and Jie, 2012) or even tended to increase (Luo et al., 1989) with the supplement of ramie. In line with these studies, the supplementation of 3 and 6% ramie powder tended to increase the egg production in our study. Ramie is rich in cellulose, flavonoids compounds, polyphenol compounds, vitamin C, and minerals. It might partially because of the antioxidant properties of ramie contribute to the improved production performance. Recent studies showed that adding flavonoids to the diet can improve the performance of laying hens by improving the body’s antioxidant capacity, reducing the occurrence of oxidative stress and promoting the absorption of nutrients in the intestine of laying hens (Brisibe et al., 2008; Galal et al., 2008; Seven, 2008; Liu L. L. et al., 2014; Zhu et al., 2021).

Yolk color is an important quality trait of eggs which is extremely critical to consumers. In our study, the yolk color was enhanced significantly by adding a certain amount (6–9%) of ramie. Similar results were also observed that yolk color gradually deepened as the addition levels of urtica dioica or urtica cannabina in diets (Like Boehmeria nivea, they are Urtica plants) (Loetscher et al., 2013a; zhang et al., 2020). U. dioica was found to be equally as effective as synthetic pigmentation. It was 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 (Kregiel et al., 2018). Lutein was considered an important natural pigment for ameliorating egg yolk and broiler skin color, which could be deposited in egg yolks (Hencken, 1992; Loetscher et al., 2013a; Loetscher et al., 2013b). Besides, nettle was reported to be rich in yellow-colored xanthophylls, with lutein (184 μg/g) being the predominant compound, followed by β-carotene (6.7 μg/g) (Marchetti et al., 2018). And xanthophylls were found to be absorbed in the digestive tract and deposited in subcutaneous fat and yolk leading to higher yolk color scores (Wen et al., 2019). Therefore, the higher yolk color observed in ramie (6–9%) treated groups in the present study might be due to the high levels of effective polar xanthophylls, such as lutein. Additionally, supplemental antioxidants to layer feed could also improve the yolk color (Yuan et al., 2016). Neohesperidin dihydrochalcone is a hydrogenated flavonoid derivative and has antioxidant, antimicrobial and anti-inflammatory properties in animals (Yeomans et al., 2007; Marti et al., 2008). It could improve the egg yolk color because it reduced the effects of lipid peroxides and free radicals on organisms (Zhu et al., 2021). It could also be possible that ramie (rich in flavonoid constituent) prevented the lutein from being oxidized and therefore increased the pigment deposition (Wang et al., 2019).

Egg yolk index can be used to measure the egg yolk nutrient concentration and egg freshness, and the higher egg yolk index means better egg processing grade and edible value. Previous studies showed that adding 2% nettle powder to the diet of laying hens could significantly increase the yolk index (Mansoub, 2011). Our study showed that egg yolk index was significantly increased in the 9% ramie group. The increase in egg yolk index might be related to the stability of yellow pigment in the lipid molecules located in egg yolk membrane, as ramie prevent the occurrence of the oxidative stress.

Nettle could also reduce lipid peroxidation (serum MDA content decreased) and liver enzyme activity in CCl4-treated rats, and improved the activity of antioxidant defense system (Kanter et al., 2003). Our study showed that ramie supplmentation significantly reduced MDA content in egg yolk. In addition, supplementation of 3% ramie powder significantly increased the T-AOC capacity of egg yolk. These indicated that by adding ramie powder, the antioxidant capacity of egg yolk was improved.

Total cholesterol content in eggs can be reduced by adding natural plants, cellulose and trace elements (Zhang et al., 2010). The addition of 15% Urtica cannabina in the diet of laying hens could significantly reduce the cholesterol content in yolk, without affecting the CP and EE content in eggs (Zhang et al., 2020). The addition of 6% Urtica dioica could significantly reduce the cholesterol content in quail egg yolk (Moula et al., 2019). Our study showed that ramie supplementation had no significant effect on the nutritional composition of whole eggs, but the total cholesterol concentration in yolk with 3 and 6% ramie treated groups was significantly decreased. This might be due to the phytosterol components in ramie which could reduce the absorption of cholesterol in intestinal tract, thereby lowering cholesterol levels in the blood, and subsequently, in animal products (Avci et al., 2006).

N-3PUFA, including ALA, EPA and DHA, has been recognized for its beneficial effects on the growth, health and immune function of humans and animals (Lee et al., 2009; Chen et al., 2014; Zhang et al., 2020). Numerous fatty acid desaturases played key roles in synthesizing PUFA. Several desaturases are absent in animals and humans, such as delta-12 and delta-15 desaturases (Lee et al., 2016). Thus, ALA must be obtained from the diet and be converted into EPA and DHA by delta-6 desaturase catalyzed dehydrogenation and the addition of two carbons by an elongase (Fraeye et al., 2012). Dietary supplementation with fresh nettle reduced the ratio of n-6/n-3 and increased the total n-3PUFA in yolk (Zhang et al., 2020). Similarly, it was reported that dietary supplementation with fresh nettle increased the contents of linoleic acid and linolenic acid, improved the proportion of PUFA and n-3PUFA and reduced the ratio of n-6/n-3 in the breast meat of broiler (Stojcic et al., 2016). Moreover, it was showed that the fat metabolism of pigs was modulated by 500 mg/kg nettle extract in diet, that the MUFA was decreased and PUFA was increased in muscle fat (Szewczyk et al., 2006). The composition of fatty acids stored in monogastric animals indicated the possibility that the lipid and fatty acid composition of poultry eggs could be altered by the diet (Kouassi et al., 2020). In the present study, 6% ramie significantly increased the content of icosaic acid and ALA in egg yolk, and significantly increased the content of n-3PUFA. In addition, the ramie supplementation was associated with a significant elevation in the LA, PUFA and EFA proportion compared to the control diet. This might be related to the rich sources of essential fatty acids in ramie leaves. ALA is the main fatty acid accounting for 40.7% of the fatty acids in mature leaves (Guil-Guerrero et al., 2003). Hence, eggs might receive the high content of n-3PUFA when the laying hens were fed by diets rich in n-3PUFA (Wen et al., 2019; Grˇcevi´c et al., 2019). Another possible explanation for the higher total n-3PUFA content in the egg yolks of the groups treated with ramie could be because of the protective function of the antioxidative compounds such as lutein, tocopherol, flavonoids and phenolic compounds. 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 (Loetscher et al., 2013b; Zhang et al., 2020). Lutein and flavonoids compounds are potent antioxidants due to their free radical quenching activities (Irgin et al., 2016; Kamoshita et al., 2016). In previous study, the addition of marigold powder (rich in lutein) to the laying hens’ feed significantly increased egg lutein content, which helped to preserved a higher content of DHA in the yolks. Adding Urtica cannabina to the feed of laying hens (or due to the presence of antioxidant compounds) can maintain high DHA content in egg yolk (Zhang et al., 2020). Based on these results, we assume that the richness of antioxidants in ramie had an impact on the conservation of PUFA in the lipids of the egg yolks by protecting them from oxidation and damage, thereby maintaining a higher level of PUFA in the ramie group. Collectively, these findings indicated that, as an important n-3 PUFA source, ramie contributed to the excessive production of n-3 PUFA in the eggs.

As one of the main animal products, eggs are welcome in the market as excellent protein source (Perić et al., 2011). The nutritional value of protein mainly depended on the variety and content of the amino acids. Sufficient content of essential amino acid and balance amino acid composition played an extremely important role in the nutritional value and edible flavor of food (Wu et al., 2013). Many studies showed that the nutrition and flavor of eggs were affected by factors such as dietary nutrients and feeding methods (Perić et al., 2011; Bashir et al., 2015; Dong et al., 2018; Bagheri et al., 2019). The tender stems and leaves of ramie contained high level of crude protein. After Boer goats feeding with silage of ramie tender stems and leaves, the content of umami amino acids and proline in the mutton were increased, which was beneficial in the umami taste, the protein bioavailability, and the quality of mutton (Gao et al., 2016). The addition of silage ramie as a source of roughage to Simmental beef cattle diets not only maintained the quality and nutritional value, but also improved the composition of flavor amino acids (umami amino acids, bitter amino acids, and sour amino acids) in beef (Yang et al., 2017). The results of the present experiment show that supplementation of ramie had no effect on the content of umami amino acids in eggs. It was different from previous researches, and the possible explanations might be the different processing methods of ramie before feeding (without silage), and the difference in experiment animals, who possessed different digestion and absorption capabilities. It was reported that 9% canola meal supplementation in diet increased the total amino acid content in eggs of Roman laying hens (Wang et al., 2013). As in the present study, supplementation of 6% ramie significantly increased phenylalanine content in the whole eggs, and different levels of ramie significantly increased EAA/TAA and EAA/NEAA in whole eggs. The trend of increased content of essential amino acids in the whole eggs in ramie treated groups might be because the amino acid content in ramie leaves is quite rich (EAA/TAA value was greater than 40%, and EAA/NEAA value was greater than 69%) (Sun et al., 2013). After the essential amino acids in ramie leaves being digested and absorbed by laying hens, they were deposited in eggs. It was also possible that ramie contains a variety of active substances, which improves intestinal health and nutrient absorption, thereby increased the content of essential amino acids in eggs (Graf, 1992; Nardini et al., 1995; Van Acker et al., 2000; Miyake et al., 2003; AOAC International, 2005).

In summary, with a dietary supplement of ramie, no negative effects on egg production and egg quality in laying hens were found, but increase of the antioxidant capacity of the egg yolk and the nutritional content of the whole egg were achieved. The yellow color of egg yolk deepened as the increase of ramie content, and 9% ramie supplements contributed the darkest egg yolk. Compared with other groups of laying hens, supplementation of 6% ramie in the diet significantly reduced the total cholesterol content in the egg yolk; and increased the content of n-3 polyunsaturated fatty acids and essential amino acids in the whole eggs. However, how the biologically active ingredients in ramie affect the content of unsaturated fatty acids in eggs needs further research. These findings support the potential application of ramie as a dietary supplementation for laying hens (Table 7).