Effects of Dandelion ( Taraxacum officinale ) Supplementation on Productive Performance, Egg Quality Traits, Serum Biochemical Parameters and Liver Fat Rate in Laying Hens

Background: This study was conducted to investigate the effects of dandelion ( Taraxacum officinale ) root supplementation on performance, egg quality and some serum antioxidant enzymes capacity with increasing age, after the peak laying stage in hens. This study, also aimed to overcome diseases that occur due to lipid metabolism in layers in the late laying period. Methods: In total, one hundred and twenty 70-week-old Lohman LSL laying hens were randomly assigned to five groups equally (n = 24). Each treatment was replicated six times (6 replicates  4 birds). The control group was fed basal diet (2650 kcal/kg ME) with no supplementation. The other groups were fed high-energy diets (2850 kcal/kg ME) to induce fatty liver and also 5, 10 and 15 g/kg dandelion (taraxacum officinale) root were supplemented. Result: The study revealed that the addition of 5 g/kg dandelion root to the high-energy feed positively changed the egg production, decreased lipid profiles (triglyceride and VLDL) and liver fat ratio and played a role in preventing lipid peroxidation.


INTRODUCTION
Fatty Liver Syndrome (FLS) is a condition that affects primarily caged layers and is characterized as a nutritional and metabolic disease and is a common cause of death in laying hens.Recent studies have reported that increased age, hormone imbalance, a low-protein high-energy diet, heat stress and cage system of rearing predispose birds to Fatty Liver Hemorrhagic Syndrome (FLHS) (Rozenboim et al., 2016;Gao et al., 2019).The typical characteristics of FLHS are a sudden drop in egg production, which has a devastating effect on the poultry industry (Zhuang et al., 2019;Ürüşan 2021).
Medicinal aromatic plants are one of the treatment and preventive measures against diseases, disorders and environmental factors (such as temperature, stress of settlement density) that adversely affect the health of poultry (Bayraktar and Tekce, 2019;Tekce et al., 2019).Dandelion (Taraxacum officinale) is a medicinal aromatic plant that plays an important role in the treatment of human and animal diseases.It has been used for the treatment of different hepatic disorders and various conditions affecting the gallbladder.The dandelion root, herbs and blossoms constitute the pharmacopeial raw ingredients (Ahmed et al., 2013).
In vivo studies in rat models of non-alcoholic steatohepatitis (NAFLD) treated with dandelion extracts showed significant reductions in hepatic lipid accumulation, liver tissue weight, body weight, serum cholesterol levels and serum malondialdehyde (MDA) levels (Cho et al., 2002).It has been reported that dandelion suppresses lipid accumulation associated with insulin resistance and lipid reduction via AMPK (Davaatseren et al., 2013a).It is known that adenosine monophosphate activated protein kinase (AMPK) reduces fatty acid synthase and acetyl CoA carboxylase (ACC) while suppressing SREBP-1c.After the administration of dandelion leaf extracts, insulin resistance was found to be reduced through activation of the AMPK (50 adenosine monophosphateactivated protein kinase) pathway.Dandelion extract has showed benefits in streptozotocin-induced diabetes (Cho et al., 2002), carbon tetrachloride (CCL4)-induced liver damages and high-fat diet (HFD)-induced fatty liver disease (NAFLD) (Davaatseren et al., 2013a).
Studies to date have shown that older laying hens are more vulnerable to internal and external stimuli than younger birds and that bioactive additives may have curative effects on the performance and physiological function of chickens (Jiang et al., 2020;Tao et al., 2020).However, little is known about the effects of aging on the organ function or metabolism of laying hens.A literature review on the effect Effects of Dandelion (Taraxacum officinale) Supplementation on Productive Performance, Egg Quality Traits, Serum Biochemical... of dandelion on fatty liver in laying hens revealed that such studies have not been undertaken.This study was, therefore, conducted to evaluate liver fat ratio, performance, egg quality and serum antioxidant enzymes capacity with increasing age after the peak laying stage of hens.In addition, this study, also aimed to overcome the diseases that occur due to lipid metabolism in layers in the late laying period.

MATERIALS AND METHODS
This study was guided pursuant to the approval (protocol number: 2020/1) of the Local Ethics Board for Animal Experiments of Directorate of Veterinary Control Centre Research Institute in Türkiye.

Animals and experimental design
In this study, in total, one hundred and twenty Lohman LSL hybrid laying hens at 70 weeks old were assigned to five groups (n=24), replicated 6 times with 4 hens per replicate.Laying hens in the control group were given a corn soybean basal diet (2650 kcal/kg ME) and high energy (HE) groups were given a 2850 kcal/kg ME diet without hipolipidemic additives.The remaining three groups respectively were given the HE diet supplemented with dandelion root powder at the rate of 5 g/kg, 10 g/kg and 15 g/kg.During the experiment (12 weeks), hens were fed and watered as ad libitum.

Performance and egg quality measurements
Hen day egg production (%) was measured.Feed intake (FI) was recorded and feed conversion ratio (FCR) was calculated daily.FCR was calculated as grams of FI per gram of egg mass produced.Egg quality parameters were evaluated every 15-d.Egg quality characteristic included egg weight, yolk index, specific gravity, shell breaking strength and weight of shell, Haugh units (HU), eggshell thickness, albumen weight and yolk weight were measured every two weeks using eighteen eggs from each dietary treatment.

Serum lipid profile and glucose, ALP, AST and estrogen levels
Blood samples were taken with a 23-gauge needle from the vena brachialis and collected into 10 mL biochemistry tubes (VACUETTE ® TUBE 9 mL Z Serum Clot Activator).At the end of the experiment, blood samples (per group, n=6) were centrifuged at 3000 rpm for 10 min at +20C.Serum was obtained following centrifugation at 3000 rpm for 10 min at +20C.Using commercial kits (Roche) in auto-analyzer (Cobas, Japan) with the spectrophotometric method serum cholesterol, triglyceride (TG), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), very low-density lipoprotein (VLDL), total cholesterol, glucose, aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP) and estrogen levels were determined.

Analysis of oxidants and antioxidants
Blood collected in heparin tubes was centrifuged at 3000 rpm for 10 minutes and plasma was removed.Plasma malondialdehyde (MDA) levels, Superoxide dismutase (SOD) activity, glutation (GSH) level, glutathione peroxidase (GPx) activity, catalase (CAT) activity, total proteins (TP) levels and NEFA levels were measured with Biotek Elisa Reader (Bio Single ìQuant MQX200 Elisa reader/USA).Total Protein (TP) levels were used to calculate SOD and GPx activity.

Weight and fat ratio of liver
At the end of the experiment, six laying hens randomly selected from each group were slaughtered.Liver wet and dry weight was calculated.After that, it was kept in an oven at 105C for 24 hours and liver dry weights were determined.By proportioning liver weights to 100 g live weight, the ratio of liver wet and dry weights were determined.Total lipid content from the livers were determined as per Bligh and Dyer (1959).

Feed analysis
The feeds were analysed as per AOAC (2005).

Analysis of dandelion root
The active principles in dandelion root, was identified using GC-MS, as per IUPAC method 2.301.The concentration of phenolic compounds were determined as per Rababah et al. (2011) with slight modifications.

Statistical analysis
Kolomogrov-Smirnov normality test was used to determine whether the data were suitable for normal distribution and Levene's statistics were used to test the homogeneity of variances.
Data were analyzed via one-way ANOVA, according to the following general model: The significance was tested at 0.05 level and SPSS 23.0 program was used in all analyses.

Feed analysis
The ingredient composition and nutrient content of the feeds used in the experiment are given in Table 1.

Analysis of dandelion root
The active principles present in dandelion root is presented in Table 2 and depicts the phenolic compounds present in dandelion root.

Productive performance of birds
The addition of 5 g/kg dandelion to the basal diet significantly increased egg production compared to other groups (Table 3).The reason for this situation is thought to be shaped depending on the dose of dandelion root added to the ration.
Canto Saenz et al. (2021) also observed that the inclusion of the dandelion (1,2,3,4% levels) and CPB (0.20% of sodium butyrate) in the diet of laying hens improved egg production significantly.Herbal extracts have a stimulating effect on animal digestive systems and these effects could be due to the increased production of digestive enzymes and the improved utilization of digestive products through enhanced liver functions (Jamroz, 2003).
Egg production decreased significantly in the group fed with high-energy feed.This result is confirmed by previous studies showing lower egg production in a HE diet (Ürüşan 2021; Ürüşan 2023).High quantities of fat or energy intake cause metabolic burden with increased fat accumulation in the liver, which leads to a decrease in laying rate (Neijat et al., 2014).
Although not statistically significant, the addition of dandelion to the diet decreased feed intake.Smilar findings were reported by Quilcate et al. (2018) they observed that dandelion supplementation (4%) in laying hen diets decreased feed intake.It is possible that a high amount of sesquiterpenic lactones (bitter principle) in dandelion that are responsible for the flavor and bitter taste, may have reduced feed consumption in birds.Schleicher et al. (1998) observed that herbal plants like chamomile and dandelion had negative impacts on FCR and live body weights (LBW) at 52 days of age in broilers.Limin et al. (2016), reported that dietary astragalus polysaccharides and dandelion extracts at the level of 0.1% decreased feed consumption of laying hens.Conversely, Qureshi et al. (2015) revealed that a combination of enzyme-treated fenugreek seeds and dandelion leaves supplementation improved FI in broilers.It is believed that this is due to the difference in the type, dose and duration of administration of dandelion added to the diet as a justification for this condition.
In this study, it was determined that the addition of dandelion to the diet did not affect egg weight.But the highest value was for the HE+dandelion 5 g/kg group (62.85 gr).However, Limin et al. (2016) reported that egg weight in groups fed astragalus polysaccharides and dandelion extracts were higher than those in control group.However, Quilcate et al. (2018) showed that dietary inclusion of dandelion, reduced the weight of the egg.This could be because dandelion was supplemented in the diet at high level.
The addition of dandelion to the diet had no impact on FCR.Consistent with current findings, Quilcate et al. (2018) revealed that the addition of dandelion in the laying hen diet did not affect FCR.Similarly, Maljik (2017) showed that dandelion addition did not affect performance parameters.

Egg quality
The experimental results indicated that shell weight was significantly increased by dandelion in high energy diet (p<0.05)(Table 4).Similarly, Maljik (2017) showed that dietary dandelion in laying hens improved the shell weight of eggs.Dandelion is a good source of calcium and vitamin K, both of which could have contributed to improvement in shell weight.The addition of 5 and 15 g/kg dandelion to the high energy diet increased significantly egg weight compared to other groups.
For consumers, one of the important purchasing criteria of eggs yolk color (Esfahani-Mashhour 2009).Supplementation of Dandelion in layers has been less studied as source of pigment for egg yolk coloration (Ürüşan 2021;Quilcate et al., 2018).This study revealed that supplemental dandelion increased (p<0.05) the yolk color score.This was largely due to carotenoids present in dandelion.Consistent with current findings, Maljik (2017) and Quilcate (2018) also observed that the yolk color index was promoted as the result of dietary addition of dandelion.Dandelion also has high antioxidant activity (Hu and Kitts 2003;Sahingil 2019), suggesting that its supplementation could protect fatty acids in egg yolk and thus affect not only color and carotenoid content but also oxidative stability and sensory properties.Thus, it could be a suitable alternative to improve egg quality and provide beneficial bioactive compounds in the human diet.
Shape index values (%) were significantly affected by all the treatments with the values ranging between 68.50 and 75.10%.An important criterion in determining egg quality is the egg shape index.An egg is always oval in shape (72-76).Round eggs and unusually long eggs do not fit well in egg cartons.Hence, they are much more likely to break during the shipment than the eggs of normal shape (Sarýca and Ersayın 2009).The group fed with high energy diet showed the lowest shell thickness.It has been suggested that especially calcium-binding protein synthesis is impaired due to fatty liver in high-energy diets and as a result, Ca++ absorption from the intestines decreases (Sevinç and Aslan  1998).In the current study, it is thought that there may be a decrease in egg shell thickness due to the decrease in calcium absorption in the HE+group.

Blood serum constituents
Dandelion has many bioactive components, phenolic compounds, glycosides, alkaloids, tannins and flavonoids.It plays a role in reducing cholesterol levels in the blood by intensification of bile secretion (Choi et al., 2010).Data showed that serum triglyceride and VLDL concentrations were lower in the control and HE + 5 g/kg dandelion groups than the other groups (p<0.01).Qureshi et al. (2015) were found that serum cholesterol was decreased significantly in the groups fed a combination of fenugreek seeds and dandelion leaves with or without enzyme as compared to the control.Similarly, Choi et al. (2010) showed that treatment with dandelion root and leaf decreased plasma triglycerides and LDL cholesterol in cholesterol-fed rabbits.
Concerning the hypolipidemic effect, many studies have indicated that dandelion reduces the concentrations of cholesterol and triglyceride in the blood (Qureshi et al., 2015;Jassim et al., 2012;Davaatseren 2013b).The addition of dandelion in the diet of laying hens had no significant differences in ALP, AST, ALT, glucose, total cholesterol, LDL, HDL and estrogen concentration (Table 5).
MDA has been widely used as an indicator of lipid peroxidation.The lowest MDA concentrations were observed in controls, HE 5 g/kg and 10 g/kg dandelion groups.CAT, SOD and GSH-Px are important anti-oxidant enzymes playing a role in reducing toxic effects.They can work together to catalyze free superoxide anion radicals into nontoxic compounds (Xiaohong et al., 2016).The GSH value was significantly increased in the control, HE and 10 g/kg dandelion groups (p<0.01)(Table 6).Consistent with present findings, Choi et al. (2010) observed that GSH activities were significantly improved in both dandelion root and leaf groups, compared to the control group in cholesterol-fed rabbits.Using 10 g/kg dandelion as feed additive in high energy diet had the highest CAT and GPx enzyme value during the experimental period, compared with other treatment groups.
Studies have shown that dandelion has significantly increased hepatic antioxidant enzymes (GPx, GST and GSH) (Choi et al. 2010;You et al., 2010).The results show that dandelion reduced lipidic superoxide damage, improved the antioxidative status and inhibited free radical formation in laying hens.

Weight and fat ratio of liver
The liver weights (dry and wet) of the laying hens were not influenced significantly (p>0.05) by any of the treatments.The liver fat content decreased in HE+5g/kg, HE+15 g/kg dandelion and control diets compared with other groups (p<0.05)(Table 7).Literature review has revealed that dandelion has protective activity in the liver.It has been   reported that the lipid-lowering effect of dandelion is due to the relationship of luteolin with hydroxy-methyl-glutarylenzyme A reductase, liver sterol regulatory element-binding proteins and acetyl-CoA C-acetyltransferase (Gebhardt 2002).
Although there are several studies with dandelion, none of the research has been conducted to appreciate the inûuence of the hepatoprotective effect of dandelion in laying hens.A search of the literature found only the report by Davaatseren et al. (2013a) which recommended that dandelion leaf extract could inhibit hepatic lipid accumulation in high-fat diet-fed mice.In addition, Cho et al. (2002) dandelion water extract addition could be used to improve lipid metabolism in rats.At present, hepatoprotection is used in the integrated therapy of liver diseases.A reduction in relative liver fat in laying hens fed dandelion at 5 g/kg with high energy is promising.

CONCLUSION
The use of dandelion in high energy feed of laying hens improved egg production, shell weight and yolk color.
Dandelion root supplementation at 5 g/kg feed was useful for reducing the plasma triglyceride, VLDL, MDA concentration and liver fat ratio.Further studies are needed to validate these positive effects.

Table 2 :
Phenolic compounds in dandelion root.

Table 1 :
Ingredient composition and nutrient content of diets.

Table 4 :
Effects of dietary supplementation dandelion root on egg quality (n=12).
Column means with no common superscript letters differ significantly; *Significant at p<0.001.

Table 3 :
Effects of dietary dandelion root on the productive performance of laying hens.

Table 6 :
Effects of dandelion root supplementation at different rates in laying hen ration on antioxidant enzyme parameters (MDA, SOD, CAT, GPx) and NEFA levels.

Table 5 :
Effects of dietary supplementation of dandelion root on serum biochemical parameters in laying hens.

Table 7 :
Effects of of dietary supplementation of dandelion root on liver of laying hens.