Orychophragmus violaceus and/or chicory forage affects performance, egg quality, sensory evaluation and antioxidative properties in native laying hens

Abstract

Orychophragmus violaceus (OV) and chicory (Cichorium intybus L., CC) can be used as fresh or dry forage for animals. To determine whether OV and/or CC have beneficial effects on performance and egg quality, a total of 1212 28-wk-old Beijing You Chicken (BYC) laying hens with similar performance were randomly allocated to 4 groups with 3 replicate pens per group, and 101 birds per pen. The birds were fed a basal diet (control), the basal diet + OV (3.507 kg/d/pen), the basal diet + CC (2.525 kg/d/pen), and the basal diet + OV + CC (OVC, 1.7535 kg/d/pen OV + 1.2625 kg/d/pen CC) for 3 wks after one wk of adaptation. The results showed that egg-laying rate was not affected by OV, CC and OVC (p > 0.05), but weekly average egg mass was significantly increased by OV and CC (p < 0.05). The feed egg ratio in the CC group (2.82) was significantly lower than that in the other three groups (p < 0.05). The eggshell thickness (EST), albumen height (AH) and Haugh unit (HU) were decreased by OV and CC (p < 0.05); while yolk color (YC) was increased in the CC and OVC groups (p < 0.05). Egg grade was decreased by OV (p < 0.05). Sensory evaluation showed that there was a trend for increased YC in OV, CC and OVC (p = 0.089). Serum total protein was significantly lower in OV group than those in the control and CC group (p < 0.05); serum albumin content was significantly decreased in OV, CC and OVC groups (p = 0.006). Serum glutathione peroxidase activity in CC and OVC groups was significantly higher than that in the control group (p < 0.05). In conclusion, the present study suggests that CC had a better effect on the performance of the native laying hens than OV. The OV and CC affected egg quality, while YC was increased in CC and OVC groups. The OVC improved YC and serum antioxidative properties of native laying hens without affecting the performance.

Keywords:

• Orychophragmus violaceus
• chicory
• laying hens
• egg quality
• antioxidative properties

Introduction

Free range has attracted a lot of attention in recent years as an animal-friendly production system.¹ In free-range conditions hens can have access to sunlight, plenty of space to move around, and the freedom to express their natural behaviors. Free-range eggs are favored by consumers for their dark yolk color and distinctive flavor,² and often sold at a higher price than those produced by caged hens.³

China is the world’s largest producer and consumer of eggs. The output of egg poultry feed accounts for more than 13% of China’s total output of industrial feed each year. Egg poultry diets are mainly based on corn-soybean meal, but protein feed resources are seriously insufficient and soybean meal is mainly dependent on imports, so many farmers, especially free-range farmers are more inclined to use forages in their effort to improve production performance while reducing the amount of soybean meal used.⁴

On-range feeding of free-range laying hens is popular, especially common in farms using mobile housing sheds.⁵ Feed supplements offered to free-range hens included shell grit, limestone, hay, silage, and others such as vegetables, pasture, insects, and harvested grass.^5,6 Forage and forage meal can be valuable alternative sources of protein for animals, provided they are easily available and not expensive.^7,8 Many forages such as clover, chicory, alfalfa can be used in poultry diets.^9,10

Orychophragmus violaceus (OV) is a biennial herb of Brassicaceae, mainly distributed in China. It can be found in plains, mountains, near buildings and other environments, and is commonly used for feed, health care, gardening and other purposes.^11,12 It has a strong ability to reproduce itself. Once sown, it will reproduce every year. It contains 9 kinds of amino acids, including threonine, glycine, alanine, isoleucine, phenylalanine, lysine, histidine, valine and leucine, and is also rich in iron, calcium, carotene, vitamin C, etc. The biomass of stems and leaves is large, which can be used as fresh or dry forage.¹¹

Chicory (Cichorium intybus L., CC) is a perennial herb from the Cichorium genus, Asteraceae family, which is cultivated worldwide and has both medicinal and edible functions and is mainly used in animal feed, and in several cases in the food industry.¹³ It contains a certain amount of proteins, vitamins, minerals and different types of bioactive compounds,¹⁴ such as alkaloid, inulin, sesquiterpene lactones, coumarins, flavonoids, saponins, tannins, etc.¹⁵ The CC leaves are fresh and tender, with good palatability and high nutritional value, and are very popular among various domestic animals and poultry.^4,16 Feeding CC has been shown to stimulate the intestinal absorption of minerals in laying hens.¹⁷ Dietary addition of water-soluble extract of CC in laying hens had no adverse effect on production performance and egg quality of laying hens, but it had been shown to reduce the number of harmful microbial flora in the cecum and egg cholesterol content.¹⁸

As mentioned above, the two forages are nutritional and available and can be valuable feed additives for animals, but there is a void of information about the effects of OV and CC on animal production, and it is also unclear whether there is a possible synergistic effect between OV and CC. The present study aimed to investigate the effects of OV and/or CC forage on performance, egg quality, sensory evaluation, and serum biochemical indexes in a native chicken- Beijing You Chicken (BYC), which is commonly used for meat and eggs and mostly reared in free-range system in China,^19,20 in order to provide some reference for the rearing and management of free-range chickens, also utilizing the forages and developing low soybean meal diets in the future.

Materials and methods

Experimental design and management

The experiment was conducted at a BYC demonstration farm, Shunyi district, Beijing. The farm had 40 identical chicken pens and outdoor areas evenly distributed in the 32.95 acres of aspen trees. Each pen was 4 m wide and 5 m long, usually for 100–120 BYC laying hens, stocking density was 5–6 hen/m². The feed trough, water nipple line, nest boxes, multi-layer perches and rice hulls as litter were provided in each pen. The outdoor area was 4 m wide and 7.5 m long, enclosed with wire mesh, the ground was covered with sand for the hens to bathe in, and a sunshade net was set up in summer. There was also a feed trough in the outdoor area, outside the wire mesh. The indoor feeding was used in the winter and outdoor feeding was used in other seasons. The hens were free to stay in indoor pen and outdoor area between 8:00 and 18:00 via a pophole. The range area around the pens was planted with chicory. While the chicory was growing, the hens were kept in the pen and outdoor area, when the chicory was ready, the hens were released to the range area freely. For the past two or three years, the OV had grown naturally around the pen and roadside, almost growing bigger than the CC. This experiment was carried out between early April and mid-May when the OV and CC were flourishing on the farm (see Fig. 1).

Figure 1. The Orychophragmus violaceus and chicory in the experiment.

A total of 1212 28-wk-old BYC laying hens with similar performance were randomly allocated to 4 groups with 3 replicate pens per group, and 101 birds per pen. The treatment groups included the basal diet (control; Table 1), the basal diet + OV (3.507 kg/d/pen), the basal diet + CC 2.525 kg/d/pen), and the basal diet + OV + CC (OVC, 1.7535 kg/d/pen OV + 1.2625 kg/d/pen CC).

Table 1. Composition and nutrient levels of the basal diet (air dry-basis), %.

Ingredients	Amount	Calculated nutrient levels	Amount
Corn	65	ME (MJ/kg)	11.14
Wheat bran	3	Crude protein^b	15.22
Soybean meal	22	Calcium^b	2.88
Limestone	6	Total phosphorus^b	0.64
Premix^a	4	Nonphytate phosphorus^b	0.46
Total	100	Lysine	0.71
		Methionine	0.37
		Cystine	0.27

^a Provided the following per kg of the diet: Vitamin A 7200 IU, Vitamin D 3360 IU, Vitamin E 20 IU, Vitamin K 2.9 mg, thiamin 2.4 mg, riboflavin 6.2 mg, calcium pantothenate 13 mg, niacin 36 mg, pyridoxine 4.3 mg, biotin 0.24 mg, folic acid 1.5 mg, VB 12 0.032 mg, choline 800 mg; Mn 100 mg, I 1.8 mg, Fe 100 mg, Cu 8 mg, Zn 90 mg, Se 0.30 mg. ^bAnalyzed values, each value based on triplicate determinations.

The forage grass was provided outdoors every day. At 8:30 a.m., the fresh OV and CC was harvested manually, about 5 cm above the ground. Basic nutrients were measured before the experiment. There were shoots and leaves for CC (containing crude protein 0.93 g, Ca 19.5 mg, P 26.4 mg per 100 g fresh weight); shoots, leaves and flowers for OV (containing crude protein 4.23 g, Ca 166.25 mg, P 35.03 mg per 100 g fresh weight). Hens had access to the grass from 9:00 am to 4:00 pm daily. The grass was weighed before 9:00 am and then after 4:00 pm the unconsumed grass was collected and the amount the hens consumed each day was determined.

The provided forage amount and estimated feed intake was designed to be 1:4 according to Zhu et al.,²¹ who stated that the best weight gain was achieved when the feed to CC ratio was 4:1. The estimated feed intake was 100 g per hen per day during laying period,²² 10.1 kg for 101 hens per pen, then the estimated forage amount was 2.525 kg/d/pen. According to the pretest observation, the edible part of OV accounted for 72% of the whole plant (the stem was a little tough and often left), so the supplement of OV was 3.507 kg/d/pen; chicory was edible as a whole plant, so the supplement of CC was 2.525 kg/d/pen; half of 3.507 kg and 2.525 kg in OVC group, i.e., 1.7535 kg/d/pen OV + 1.2625 kg/d/pen CC.

The experiment lasted 3 wks after one wk of adaptation. The health status and feeding behavior of the laying hens were visually observed and recorded daily.

To keep the same ranging time for the birds, the light regime was 16 L:8D (6:00 ∼ 22:00), the birds were fed a corn-soybean-based diet at 8:00, ranged freely from 8:00 to 17:00. The nutrient levels of diets are based on ‘Technical Code of Practice for Feeding and Management of Beijing-You Chicken’,²² the composition and nutrient levels of the basal diet is shown in Table 1, and the nutritive values were calculated according to the Feed Database in China.²³

The study was performed in accordance with local ethical guidelines and met the requirement of the institutional animal care and use committee, the ethical agreement number is IHVM11-2203-1.

Measurement and methods

The number of eggs and egg weight of each replicate group were recorded daily, the feed intake was recorded weekly, and weekly average feed intake (AFI), weekly average egg mass (EM), feed egg ratio (FER), egg-laying rate and mortality rate were calculated for 29–32 wks.

Ten fresh eggs were randomly selected from each replicate pen, providing a total of 30 eggs for each group. Egg weight (EW), eggshell strength (ESS), eggshell thickness (EST), eggshell color (ESC), Haugh unit (HU), albumen height (AH), yolk color (YC), egg grade (EG), egg shape index (ESI), relative yolk weight (RYW), relative eggshell weight (RESW), and relative albumen weight (RAW) of these eggs were measured and calculated within 24 h. The method and apparatus were the same as those previously described by our group.¹⁹

At the end of the experiment, 15 fresh eggs were collected from each group for sensory evaluation. The eggs were numbered with a pencil on the eggshells. Only the person who marked the eggs knew which group they came from. Added cold water and eggs to a covered saucepan. When the water was boiling, boiled the eggs for 3 min, turned off the heat and simmered for 3 min. Then removed the eggs and soaked them under cold water for 5–10 s before placing them on a plate. Before each set of eggs was distributed to the participants, the eggs were cut into quarters and each participant was given a quarter egg at a time. The tasting and scoring method were explained to the participants before the sensory evaluation. The egg aroma, YC, yolk fineness, albumen tenderness, egg flavor was evaluated according to Table 2. The participants were asked to gargle with water after each evaluation. The scoring system consisted of 5-point scale: 1 is the worst, 5 is the best. The two people involved in the egg grouping and cooking were not involved in the evaluation. A total of 16 experienced participants, who were researchers, postgraduate students and farm technicians aged 23–55, took part in the sensory evaluation. A total of 15 evaluation forms were issued and 15 valid evaluation forms were recovered.

Table 2. Indicators of sensory evaluation of eggs.

Score/Item	Egg aroma	YC	Yolk fineness	Albumen tenderness	Egg flavor
1	Have a very fishy smell	Beige white	Rough	Very hard	Very bad
2	Have a fishy smell	Light Yellow	Not fine	Hard	Bad
3	Normal	Normal	Normal	Normal	Normal
4	Have subtle aroma	Orange yellow	Fine	Tender	Good
5	Have aroma	Dark yellow	Very fine	Very tender	Very good

Note: Smell the egg first, observe the albumen and yolk, taste, evaluate and score.

At the end of 32 wks, blood samples were collected in the morning by venepuncture. 5 ml of blood per hen, four hens per replicate. The blood was transferred into coagulation-promoting tubes by venipuncture and centrifuged at 3000 g for 10 min at 4 °C. The serum was frozen at −20 °C for later analysis of some biochemical parameters, including serum total protein, albumin, glucose, high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C) content, total cholesterol (TC) content, total antioxidant capacity (T-AOC), peroxidase (POD), glutathione peroxidase (GSH-Px), total superoxide dismutase (T-SOD) activities. All the kits were purchased from Nanjing Jiancheng Institute of Biological Engineering and determined by a spectrophotometer (Evolution 60, Thermal Fisher Scientific, Shanghai) and ELISA (Multiskan FC, Thermal Fisher Scientific, Shanghai).

Statistical analyses

The data were analyzed statistically using the SPSS 25.0 Software for Windows (SPSS Inc. Chicago, IL). One-way ANOVA was used to analyze the effects of OV and/or CC on performance, egg quality, sensory evaluation, and biochemical parameters. Duncan’s Test was used for multiple comparisons. The percentage was arcsine transformed before the normality test. p < 0.05 was regarded as statistically significant. There was a trend when P was from 0.05 to 0.10. The data are presented as mean and standard error of mean.

Results

Performance

Table 3 shows that the AFI, egg-laying rate and mortality rate of BYC laying hens were not affected by OV, CC and OVC (p > 0.05), but weekly average EM was significantly increased by OV or CC forage (p < 0.05). There was no significant difference between OVC group and control group. The FER in CC group (2.82) was significantly lower than that in other three groups (p < 0.05).

Table 3. Effects of Orychophragmus violaceus and/or chicory forage on performance of BYC laying hens during 29–32 wks.

Item	Control	OV	CC	OVC	SEM	p-Value
AFI (kg)	7.46	8.49	7.73	7.97	0.09	0.162
EM (kg)	2.48^b	2.74^a	2.74^a	2.53^b	0.04	0.012
FER (kg:kg)	3.01^a	3.10^a	2.82^b	3.15^a	0.03	<0.001
Egg-laying rate (%)	73.00	77.00	75.00	76.00	0.60	0.114
Mortality (%)	0	0	0	0.07	0.02	0.400

Note: OV: Orychophragmus violaceus; CC: chicory; OVC: Orychophragmus violaceus +chicory; AFI: average feed intake; EM: egg mass; FER: feed egg ratio. Values in the same line, with no letter or the same letter superscripts are not significantly different (p > 0.05), while those with different letter superscripts are significantly different (p < 0.05).

Egg quality

Table 4 shows that OV, CC and OVC had no effects on ESS, RESW and RAW (p > 0.05), but significantly affected the EST, ESC, ESI, AH, YC, HU and EG (p < 0.05). In detail, the EST was decreased by OV or CC (p < 0.05), but not affected by OVC; The ESC and ESI were increased by CC (p < 0.05); The AH and HU in the control and OVC groups were higher than those in the CC group, and the lowest values were observed in OV group (p < 0.05); The YC was not affected by OV, but significantly increased by CC and OVC (become more reddish in color, p < 0.05), reached the highest in the CC group (the reddest) (see Fig. 2). The EG was decreased by OV (p < 0.05), but not affected by CC or OVC. There was a trend for decreased EW following OV, CC and OVC supplementation (p = 0.071), and a trend for increased RYW following OV, CC and OVC supplementation (p = 0.099).

Figure 2. Effects of Orychophragmus violaceus and chicory forage on yolk color.

Table 4. Effects of Orychophragmus violaceus and/or chicory forage on egg quality of BYC laying hens at 32 wks of age.

Item	Control	OV	CC	OVC	SEM	p-Value
ESS/(kg/cm^2)	3.98	4.09	4.01	4.08	0.069	0.934
EST/(mm)	0.33^a	0.31^b	0.31^b	0.32^ab	0.003	0.027
ESC/(%)	54.78^b	53.16^b	59.65^a	51.85^b	0.610	<0.001
ESI	1.31^b	1.33^b	1.38^a	1.32^b	0.007	0.001
EW/(g)	45.67	44.81	42.96	43.84	0.377	0.071
AH/(mm)	8.25^b	4.12^d	5.38^c	9.07^a	0.239	<0.001
YC	7.74^c	8.16^bc	9.49^a	8.65^b	0.125	<0.001
HU	93.56^b	67.84^d	77.84^c	98.88^a	1.476	<0.001
EG	3.88^a	3.04^b	3.64^a	4.00^a	0.066	<0.001
RYW/ (%)	27.40	28.16	28.51	29.26	0.263	0.099
RESW/ (%)	14.28	13.82	13.66	13.64	0.130	0.279
RAW/ (%)	58.33	58.03	57.84	57.11	0.306	0.544

Note: OV: Orychophragmus violaceus; CC: chicory; OVC: Orychophragmus violaceus +chicory; ESS: eggshell strength; EST: eggshell thickness; ESC: eggshell color; ESI: egg shape index; EW: Egg weight; AH: albumen height; YC: yolk color; HU: Haugh unit; EG: egg grade; RYW: relative yolk weight; RESW: relative eggshell weight; RAW: relative albumen weight. Values in the same line, with no letter or the same letter superscripts are not significantly different (p > 0.05), while those with different letter superscripts are significantly different (p < 0.05).

Sensory evaluation

Table 5 shows that there were no significant differences in egg aroma, yolk fineness, albumen tenderness, and egg flavor as an effect of OV, CC and OVC (p > 0.05). There was a trend for increased YC by OV, CC and OVC (p = 0.089). The YC of CC group was the darkest (yellow to red), followed by the OVC group, OV group (see Fig. 2). Four of the 16 participants complained that the eggs from the control group had a fishy smell of eggs, while those from the forage supplemented groups did not.

Table 5. Sensory evaluation of eggs from Orychophragmus violaceus and chicory forage groups.

Item	Control	OV	CC	OVC	SEM	p-Value
Egg aroma	3.588	3.941	3.941	3.765	0.110	0.627
Yolk color	2.765^b	3.059^ab	3.529^a	3.353^ab	0.112	0.089
Yolk fineness	3.588	4.000	3.706	3.882	0.109	0.551
Albumen tenderness	3.529	3.882	3.588	3.824	0.092	0.450
Egg flavor	4.000	4.059	4.059	3.941	0.097	0.968
Total	18.824	18.765	18.941	17.471	0.341	0.388

Note: OV: Orychophragmus violaceus; CC: chicory; OVC: Orychophragmus violaceus +chicory. Values in the same line, with no letter or the same letter superscripts are not significantly different (p > 0.05), while those with different letter superscripts are significantly different (p < 0.05).

Serum biochemical indexes

Table 6 shows that the serum glucose, HDL-C, LDL-C, TC, TAOC, POD, and T-SOD were not affected by OV, CC and OVC (p > 0.05), but serum TP was significantly lower in OV group than that in the control and CC group (p < 0.05); serum albumin content was significantly decreased in OV, CC and OVC groups (p = 0.006). The GSH-Px activity of OV group was not significantly different to that of the control group, but significantly higher in the CC and OVC groups, compared to the control group (p < 0.05), the GSH-Px activity of CC group was the highest, followed by OVC group.

Table 6. Effects of Orychophragmus violaceus and/or chicory forage on serum antioxidative properties of BYC laying hens.

Item	Control	OV	CC	OVC	SEM	p-Value
TP (g/L)	37.65^a	28.76^b	36.46^a	34.91^ab	1.021	0.042
Albumin (g/L)	17.72^a	13.27^b	14.96^b	15.24^b	0.326	0.006
Glucose (mmol/L)	14.45	14.18	12.54	13.53	0.658	0.742
HDL-C (mmol/L)	0.93	0.89	0.95	0.79	0.088	0.917
LDL-C (mmol/L)	0.46	0.56	0.54	0.64	0.091	0.927
TC (mmol/L)	3.16	2.75	2.65	3.10	0.162	0.629
T-AOC (U/mL)	0.24	0.40	0.40	0.37	0.034	0.371
POD (U/mL)	1.50	1.83	2.57	1.88	0.213	0.392
GSH-Px (μmol/L)	1627.03^c	1964.87^bc	2569.37^a	2402.70^ab	82.317	0.008
T-SOD (U/mL)	143.33	174.15	232.06	223.08	15.031	0.172

Note: OV: Orychophragmus violaceus; CC: chicory; OVC: Orychophragmus violaceus +chicory; TP: total protein; HDL-C: high-density lipoprotein cholesterol; LDL-C: low-density lipoprotein cholesterol; TC: total cholesterol; T-AOC: total antioxidant capacity: POD: peroxidase; GSH-Px: glutathione peroxidase; T-SOD: total superoxide dismutase. Values in the same line, with no letter or the same letter superscripts are not significantly different (p > 0.05), while those with different letter superscripts are significantly different (p < 0.05).

Discussion

With the improvement of people’s living standards, more and more consumers are paying attention to animal welfare and are willing to pay for animal derived products from animals with high welfare standards.^24,25 The range use of hens can be used as an indicator of the impact of a free range system on animal welfare. The high proportion of hens using the range area was associated with the brown genotype, smaller flock size, having roosters in the flock, etc.²⁶ Pettersson et al.²⁷ summarized ranging percentages from 14 studies and showed that the average percentage of hens on the range was 9–38%. In the present study, we observed that more than 80% of the hens grazed outdoors, the reason for which may be due to the small flock size, which is consistent with Bestman and Wagenaar,²⁸ who reported that more than 75% of the hens were observed to be outdoors.

Providing a proper amount of forage can not only provide various nutrients, such as minerals, vitamins, etc., but also reduce feeding cost and improve the performance and economic benefit of poultry. The CC contains many important medicinal compounds such as alkaloids, inulin, sesquiterpene lactones, coumarins, chlorophyll, chlorophyll pigment, unsaturated sterols, flavonoids, saponins, and tannins.^15,29 Dietary CC fructan increased the weight gain and feed conversion rate of broilers.³⁰ Dietary supplementation of 1.0% inulin can increase egg production and feed egg ratio of laying hens.³¹ The water-soluble extract of CC had no adverse effects on performance of laying hens when used in the diet.³² Zheng et al.¹⁰ supplemented 0%, 5%, 8% and 10% fresh CC forage in the basal diet of female BYC aged from 16 wks to 29 wks, and found an increase in average feed intake, feed conversion ratio and mortality with increasing supplementation of CC forage in the diet. This present study found that OV or CC can increase the EM, but the FER of CC group was significantly lower than that of OV group, indicating that the effect of CC on performance of laying hens was better than that of the OV, which may be due to different composition of the two forages, and different utilization of nutrients by the native laying hens.

It is interesting to observe that in the pens with OVC, the hens always preferred to eat OV first rather than CC, probably because the OV leaves contain less tannin than CC leaves. Sesquiterpene lactones in CC leaves (about 0.2% of dry matter) contribute to the bitter taste,⁴ and the purple flower of OV may also be an attraction for hens to peck first.

Exogenous nutrients can change the energy metabolism in poultry, thus affecting the egg quality.³³ The ESS and EST are indicators for evaluating eggshells, and the eggshell quality affects the transportation and storage of eggs, as well as the hatching performance of breeder eggs.³⁴ Dietary supplementation of CC has been shown to decrease ESS,¹⁰ while in this study, the EST was reduced by OV or CC which was opposite to the previous report that dietary 1.5% inulin and 0.5% CC extract could increase the EST.³⁵ KopBozbay et al.³⁶ showed that the eggshells of the control hens with access to non-vegetated areas were thicker than those of the hens with free access to CC vegetated areas, clover vegetated area, and the mixed CC and clover vegetated areas. The reason may be the lower dietary protein intake of laying hens due to the forage supplement. Iqbal et al.³⁷ reported that access to feed supplements can dilute the formulated nutrient intake significantly, sometimes with severe consequences.

The AH and HU are indicators of the freshness of the eggs, reflecting the albumen quality. The larger the AH and HU, the higher the protein content and viscosity, and the fresher the eggs. Xie et al.³⁸ found that Lonicera confusa (LC) and Astragali radix (AR) extracts (LC-AR) increased HU of eggs, while in the present study, AH and HU were decreased in the OV and CC group, the reason may also be related with the lower dietary protein intake of laying hens, but not decreased in OVC group, which need to be verified in future.

The YC is one of the important indicators of internal egg quality. Though it is not directly related to the nutritional value, most consumers associate a well-colored egg yolk (golden yellow to orange) with healthiness and quality, and the pale or non-uniform colored egg yolks are considered as inferior products from sick and unhealthy hens.³⁹ Carotenoids, which are not endogenously synthesized and must be obtained through the diet, are mainly responsible for the YC. In conventional layer rations, corn gluten, alfalfa or saponified extracts from marigold flower, paprika fruit or synthetic carotenoids are included for egg yolk coloration.³⁹ According to Montefusco et al.,⁴⁰ the contents of lutein (8.0–30.1 μg/g f.w.), β-carotene (3.3–14.2 μg/g f.w.) varied in cultivated and wild CC cultivars, while the average carotene content was about 16.2 μg/g f.w. in fresh shoots of OV.¹¹ Horsted et al.⁴¹ reported that when the laying hens were fed with CC and clover separately for 23 days, the YC of the CC group was darker. Zheng et al.⁴² reported that when the 16-wk-old BYC was fed a diet supplemented with CC pulp for 60 consecutive days, the YC was improved. In agreement with these studies, the present study indicated that the YC of the CC group was significantly darker than that of control, OV, and OVC groups, and the reason may be that some carotenoids in CC, such as lutein, are more easily transferred to the yolk than those from the OV. The present study only lasted one month and different coloration effects were already observed.

Sensory evaluation is a dynamic field concentrating on using humans to measure sensory perception and/or their effect on food and taste acceptance,⁴³ which is generally a combination of sensory traits including aroma, flavor, off-flavor and overall difference.¹⁷ Xie et al.³⁸ used LC and AR extracts in 52-wk-old Lohmann pink-shell hens and showed that the scores of overall acceptability of eggs were lower (p = 0.018) in the LC-AR group than that in the control, LC or AR group, by using a sensory 9-point evaluation scale (where 1 = like extremely, 5 = neither like nor dislike, and 9 = dislike extremely). In this study a 5-point scale was used (1 was the worst, 5 was the best), the results showed that there were no differences in egg aroma, flavor, off-flavor except for YC in OV and/or CC forage groups. The YC of CC group was the darkest, the YC of control group was the lightest, which was consistent with previous egg quality measurement. The OV or CC forage had no significant effects on the improvement of egg sensory quality, which may be also due to lack of professional training of the participants. Hayat et al.¹⁷ reported that only trained panelists could detect differences in egg sensory attributes in eggs.

Serum biochemical indicators can reflect the function of various tissues and organs and the nutritional metabolism of the body and indirectly reflect the health status of poultry. The serum total protein and albumin contents reflect the metabolic status of protein in the body to some extent.⁴⁴ This present study showed that the serum total protein and albumin contents in OV group were significantly lower than that in the control group, and the total protein content in the OVC group was higher than that in the OV group and lower than that in the CC group. The possible reason was that the crude protein intake of laying hens in OV group was lower than that in control group.

T-AOC can reflect the antioxidant capability of the body to a certain extent. The POD, GSH-Px, and T-SOD activities are the main members of the antioxidant defense system, forming a protection system against oxidative damage. Cichoric acid was first identified in chicory leaves and was found in at least 63 genera and species of plants.⁴⁵ It is the main component of caffeic acid derivatives of CC and echinacea purpurea extracts and possesses antioxidant properties.⁴⁶ The antioxidant activity of CC has been the subject of investigation in many studies.¹³ Dalar and Konczak⁴⁷ studied the antioxidant activity of roots, stems, leaves, flowers, and the whole plant of CC, and found that the leaves contained the highest level of total phenols and exhibited the highest antioxidant capacities. Jurgoński et al.⁴⁸ reported that CC leaves extract showed higher antioxidant activity (210.1 nmol/g f.w.) compared to the seed extract (505.1 nmol/g f.w.). But there are no reports about antioxidant activity of OV. The present study showed that GSH-Px activity in CC group was higher than those in OV group and control group, and GSH-Px activity in OVC group was higher than that in control group. There was no significant difference between control group and OV group, indicating that the OV has no effect on serum antioxidant capability of laying hens, but CC and OVC can improve the serum antioxidant capability of laying hens, which also provided a possibility for further research on the antioxidant capability of OVC.

Conclusion

The present study indicated that OV and CC forage improved the EM, and CC decreased FCR, indicating a better effect of the CC on the performance of native laying hens than the OV. The OV and CC affected egg quality. The EST, AH and HU were decreased in OV and CC groups, while YC was increased in CC and OVC groups. CC and OVC had higher serum antioxidant capability. The OVC improved YC and serum antioxidative properties of native laying hens without affecting the performance.

Author contributions

Zhao W. Y., Zhang Q. Q. and Zhao Y. F. conducted animal experiment and performance data collection, Zhao W. Y. and Chang C. conducted performance and serum biochemical parameters measurement, Zhao W. Y., Chang C., Wang X. and Geng A. L. performed egg quality measurement, Zhao W. Y. performed statistical analysis, Geng A. L. proposed ideas, wrote and edited papers, and provided financial support.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Data availability statement

The data sets are available upon request from the corresponding author.

Additional information

Funding

The authors wish to thank the earmarked fund for CARS [CARS-41-Z04], BAAFS Capacity Building Project [KJCX20200421], Reform and Development Project of BAAFS, Beijing Innovation Consortium of Agriculture Research System [BAIC06-2023] for providing financial supports, and the staff from Lvdudu farm for feeding and management of the experimental birds.