Korean Journal of
Veterinary Service

Fatty liver hemorrhagic syndrome (FLHS) is an intractable metabolic disease frequently occurred following supply of high- or excessive-energy diets for improvement of egg production in laying hens (Meijering, 1979). The mechanism of FLHS induction remains still unclear; but increased abdominal fat, multiple hemorrhages with overt fatty liver, decreased egg production and high mortality are its characteristic clinical signs (Walzem et al, 1993). High energy but low calcium diet, hot weather and high endogenous estrogen output are known to be inducing factors of FLHS (Polin and Wolford, 1977; Simonsen and Vestergaard, 1978; Haghighi-Rad and Polin, 1981; Roland et al, 1985). Organic selenium, vitamin E or ethoxyquin are provided via feed admixture to protect FLHS, but once FLHS occurred in laying hens, it is difficult to be cured (Squires and Leeson, 1988; Crespo, 2019).

Dandelion is a world-widely growing perennial plant and classified into approximately 2,000 species (Kang and Kim, 2001). Taraxacum coreanum (TC) is a species of dandelion that grows predominantly in the Korean peninsula and several regions of China. Whole parts of TC including roots, leaves, flower and flower stem was found to present anti-diarrheal, fever relieving, diuretic and anti-inflammatory effects (Kang and Kim, 2001; Choi et al, 2012). Choi et al (2012) demonstrated that TC exerts anti-oxidative stress effects via suppressing ONOO-. Polyphenols such as chlorogenic acid, chicoric acid and hydrocinnamic acid and flavonoids derivatives including luteolin and quercetin are components extracted from TC flower and leaves and taraxacin, innnulin, taraxanthin, phytosterol and phenol compounds from TC roots (Williams et al, 1996; Schütz et al, 2006).

Cirsium japonicum var. ussuriense (CJ) named milk thistle is also a perennial wild plant belonged to Asteraccac family. 250∼300 species are reported in the world and 13 species and 6 mutants are found in Korea. It contains much amount of flavonoids including apigenin, luteolin, myricetin, kaemferol, pectolinarin and etc and releases effects of anti-inflammation, anti-carcinogenicity, anti-mutagenicity, anti-fungi, nervous system protection and immune-stimulation (Lee et al, 2003). The effects come from its key component, hispidulin-7-o-neohesperidoside inhibiting lipid peroxidation through increased glutathione in liver (Park et al, 2004).

Either ethanol or hot water extract or dried powder of TC and CJ each or mixture are approved as functional food products for protection effects against alcoholic liver disease, gastric inflammation and skin aging (KMFDS, 2016).

This study was performed to investigate alleviate effects of mixture powder of dried TC and CJ on fatty liver in laying hens and to search the most appropriate dose adding to feed for cure of fatty liver.

All procedures involving animal care and management were in accordance with and approved by the Animal Care and Use Committee of Hoseo University (HTRC-15-47).

Test materials

Whole parts of each TC and CJ including flower, stem, leaves and roots excepting deep underground roots, which grew naturally in Kangwon province region in Korea, were collected and washed with clean water three times immediately after collection. They were dried thoroughly under outdoor with natural wind and sun light and milled separately by grinder (QCG Systems, USA) to make powder of particle sizing 80 mesh. Powder mixture of TC and CJ was prepared at the ratio of 6:4 (w/w), respectively. The powder mixture was added to enforced energy feed for laying hens (San-Ran Ace, Daeju Co., Korea) at 5.0, 10.0, 20.0 and 40.0 g/kg feed.

β-estradiol (Sigma Aldrich Co.) was prepared by solving in corn oil for induction of fatty liver.

Experimental design and animals treatment

Forty-two healthy laying hens of Hy-line brown 33∼35 weeks old with average body weight (BW) of 2.01±0.23 kg were used. Animals were placed in each pens (6 animals per pen) and let access to water and feed (enforced energy feed for laying hens, San-Ran Ace, Daeju Co., Korea) ad libitum. The animals were kept in a room controlled under 14 h/10 h light and dark cycle, at temperature 18∼22℃ and humidity 40∼60%.

The hens were divided randomly into seven groups of C, FL-1, FL-2, M-5, M-10, M-20 and M-40. C group was control group without any treatment. FL-1 (fatty liver-1) group was treated with β-estradiol 2 mg/kg bw via intramuscular injection into breast area twice per week for 3 weeks and then without any treatment for 6 weeks. FL-2 (fatty liver-2) group was treated with β-estradiol 2 mg/kg bw via intramuscular injection twice per week for whole 9 weeks of experiment period. Each M-5, M-10, M-20 and M-40 group was treated orally with TC and CJ mixture (6:4 (w/w)) at 5.0, 10.0, 20.0 and 40.0 g/kg feed, respectively, for 3 weeks after 3 weeks β-estradiol treatment and followed by 3 weeks of non-treatment. Six hens were allocated into each group and observed for total 9 weeks including 3 weeks or 9 weeks of fatty liver induction by β-estradiol, 3 weeks of herbal mixture treatment after β-estradiol injection for 3 weeks and then 3 weeks of non-treatment period (Table 1).

Table 1 . Experimental groups to investigate the effects of TC+CJ mixture on fatty liver of laying hens.

Group	Number of animals	β-estradiol treatment (for 3 weeks or 9 weeks)			TC+CJ treatment (for 3 weeks after stop of β-estradiol treatment)		Total experiment period (weeks)
		Dose (mg/kg bw)	Treatment		Dose (g/kg feed)	Treatment
C	6	0	-		0	-	9
FL-1	6	2.0	IM, 2 times/week for 3 weeks		0	-	9 (3 weeks β-estradiol treatment, 6 weeks observation)
FL-2	6	2.0	IM, 2 times/week for 9 weeks		0	-	9 (9 weeks β-estradiol treatment)
M-5	6	2.0	IM, 2 times/week for 3 weeks		5	Via feed admixture for 3 weeks after stop of 3 weeks β-estradiol treatment	9 (3 weeks β-estradiol treatment, 3 weeks TC+CJ treatment and then 3 weeks observation)
M-10	6	2.0	IM, 2 times/week for 3 weeks		10	Via feed admixture for 3 weeks after stop of 3 weeks β-estradiol treatment	9 (3 weeks β-estradiol treatment, 3 weeks TC+CJ treatment and then 3 weeks observation)
M-20	6	2.0	IM, 2 times/week for 3 weeks		20	Via feed admixture for 3 weeks after stop of 3 weeks β-estradiol treatment	9 (3 weeks β-estradiol treatment, 3 weeks TC+CJ treatment and then 3 weeks observation)
M-40	6	2.0	IM, 2 times/week for 3 weeks		40	Via feed admixture for 3 weeks after stop of 3 weeks β-estradiol treatment	9 (3 weeks β-estradiol treatment, 3 weeks TC+CJ treatment and then 3 weeks observation)

IM, Intramuscular injection..

Observation of clinical signs, body weight, feed consumption and egg production rate

Clinical signs induced by treatment of β-estradiol or TC and CJ mixture were checked daily during 9 weeks of experiment period. Body weights and feed consumption were measured weekly and total body weight gain was calculated per each group by subtracting initial body weights from final ones at 9 weeks after the beginning of β-estradiol treatment. Amount of remained feed and numbers of eggs produced were measured daily at the same time of day. Daily egg production rate was measured with sum numbers of eggs collected at 10 A.M. daily divided by number of animals per group and mean±SD of daily egg production was presented per week.

Blood collection and blood biochemistry analysis

Blood was collected from wing vein weekly and serum was separated after blood coagulation and centrifugation at 3,000 × g for 15 mins. Alanine aminotransferase (ALT, U/L), aspartate aminotransferase (AST, U/L), alkaline phosphatase (ALP, U/L), total bilirubin (T-Bil, mg/dL), total cholesterol (T-Chol, mg/dL) and triglycerides (TG, mg/dL) were measured by automated clinical chemistry analyzer (Ciba-Corning Diagnostics Corp., Medfield, USA).

Analysis of antioxidant enzymes

Enzyme activities of superoxide dismutase (SOD, U/L) and glutathione peroxidase (GPX, ng/mL) in serum were measured with each ELISA kit (Bioassay Technology Laboratory, Shanghai, China).

Gross and histopathological observation

All animals were anaesthetized with carbon dioxide gas, followed by blood sampling. They were then sacrificed by exsanguination prior to necropsy. Liver was gross examined and excised for fixation in neutral buffered 10% formalin. Fixed tissues were embedded in paraffin, micro-sectioned, stained with hematoxylin and eosin (H&E), and examined microscopically.

Statistical analysis

All data were expressed as mean±SD and analyzed by one-way ANOVA, followed by Duncan’s multiple range test at a significance of P<0.05 and P<0.01 using STATISTICA program version 7.0 (StatSoft Com.).

Clinical signs, body weight gain and daily feed consumption

No specific clinical signs were observed by the treatment of β-estradiol for 3 (Group FL-1) or 9 (Group FL-2) weeks or by the treatment of TC and CJ mixture for 3 weeks (M-5, M-10, M-20 and M-40) after β-estradiol treatment. There were no significant changes in body weight, body weight gains and daily feed consumption between each group. However total or daily body weight gain tended to be higher by the treatment of TC and CJ mixture 5, 10, 20 and 40 g/kg feed for 3 weeks compared to that of FL-1 or FL-2 group (Table 2).

Table 2 . Change of body weight, body weight gain and feed consumption by the treatment of TC and CJ mixture after β-estradiol injection in laying hens.

Group	Body weight (kg)				Total body weight gain (kg) (Day 63∼Day 0)	Daily body weight gain (g/animal/day) (Day 63∼Day 0/63)	Daily feed consumption (kg/animal/day) (From Day 0 to Day 63/63)
	Day 0 (Initiation of β-estradiol treatment)	Day 21 (Initiation of TC+CJ treatment)	Day 42 (Stop of TC+CJ treatment)	Day 63 (End of experiment)
C	2.04±0.23	2.01±0.19	2.11±0.18	2.10±0.20	0.08±0.12	1.3±3.0	0.12±0.03
FL-1	2.18±0.39	2.06±0.36	2.01±0.19	1.86±0.13	−0.20±0.47	−4.9±11.2	0.12±0.07
FL-2a	2.00±0.19	1.97±0.20	1.99±0.21	1.94±0.23	−0.03±0.07	−0.7±8.3	0.12±0.03
M-5	2.02±0.15	1.99±0.24	2.06±0.23	2.10±0.17	0.04±0.11	1.1±2.6	0.10±0.02
M-10	2.01±0.14	1.99±0.16	2.06±0.29	2.12±0.12	0.09±0.06	2.0±1.3	0.11±0.03
M-20	2.02±0.17	1.91±0.19	1.95±0.13	2.05±0.10	0.04±0.19	1.0±4.4	0.08±0.04
M-40	2.04±0.34	2.04±0.32	2.04±0.32	2.08±0.39	0.04±0.34	0.6±2.8	0.09±0.02

Data are mean±SD (n=6). Control group was provided with commercial enforced energy feed for laying hens for 9 weeks. FL-1 group was intramuscularly administered with 2 mg/kg bw β-estradiol twice per week for 3 weeks with supply of commercial enforced energy feed for laying hens and then supplied with only feed for 6 weeks. FL-2^a group was intramuscularly injected with 2 mg/kg bw β-estradiol twice per week for 9 weeks with supply of commercial enforced energy feed for laying hens. M-5, M-10, M-20 and M-40 groups were injected intramuscularly with 2 mg/kg bw β-estradiol twice per week for 3 weeks and then provided with each dose of 5, 10, 20 and 40 g/kg feed of TC+CJ admixture, respectively, with commercial enforced energy feed for 3 weeks followed by supplied with only commercial feed 3 weeks..

Hepatic lipogenesis is reported to be markedly enhanced by estrogens in order to meet the demand for vitellogenesis in laying hens (Polin and Wolford, 1977; Squires and Leeson, 1988; Hermier, 1997). High energy diets are usually provide to laying hens to match the increased lipogenesis. This high energy diets can be a reason of fatty liver syndrome in layers and enhances the symptom under high environmental temperature, condensed housing like battery cages, and inflammatory challenges (Simonsen and Vestergaard, 1978).

In this study, animals were provided enforced energy feed for laying hens composed of crude protein 16%, crude fat 3.4%, crude fiber 6.0%, crude ash 15%, Ca 3.2%, P 0.7%, D/L- methionine and cystine 0.6%, methionine hydroxyl derivatives 0.6%, and metabolic energy 2.7 Mcal/kg. Extraneous surplus estrogen can stimulate and intensify fatty liver syndrome in laying hens having high energy diet and expressing naturally increased endogenous estrogen for egg production (Hermier, 1997).

Though there were none significant changes in body weights between each group, tendency of body weight gain decrement in FL-1 and FL-2 groups observed in this study implies that there were metabolism disorders in laying hens by extraneous estradiol. In this study, it was found that body weight loss was weakened by the supplement of antioxidant herbs of TC and CJ mixture, which indicates the herbs may alleviate metabolic disorder induced by extraneous β-estradiol.

Egg production

Mean daily egg production rate of control group during experiment period (9 weeks) was 77.3±24.5%. By the intramuscular injection of 2 mg/kg bw/day β-estradiol twice per week, egg production rate was decreased significantly from 1 or 2 weeks and continued decreased to 31.0∼35.7% on 3 week of β-estradiol treatment. When the treatment of β-estradiol was continued for 9 weeks, the egg production rate was further lowered to 22.9∼25.0% with the lowest rate on 7 and 8 weeks of treatment. When the treatment of β-estradiol was stop on 3 weeks, the decreased egg production was not recovered until 2 weeks after the stop and then turned to be increased a little but still remained lower compared to control group.

Supplement of TC+CJ dried powder 5, 10, 20 and 40 g/kg feed via feed admixture tended to recover egg production rate from 1 or 2 weeks after the supplementation and apparent recovery showing similar level of control was found by 5 or 10 g/kg TC+CJ after 3 weeks of treatment. The recovery was continued even after the stop of the supply of TC+CJ powder (Fig. 1).

Figure 1. Weekly change of daily egg production rate by the treatment of TC and CJ mixture after β-estradiol injection in laying hens. Data are mean±SD (n=6). Control group was provided with commercial enforced energy feed for laying hens for 9 weeks. FL-1 group was intramuscularly administered with 2 mg/kg bw β-estradiol twice per week for 3 weeks with supply of commercial enforced energy feed for laying hens and then supplied with only feed for 6 weeks. FL-2a group was intramuscularly injected with 2 mg/kg bw β-estradiol twice per week for 9 weeks with supply of commercial enforced energy feed for laying hens. M-5, M-10, M-20 and M-40 groups were injected intramuscularly with 2 mg/kg bw β-estradiol twice per week for 3 weeks and then provided with each dose of 5, 10, 20 and 40 g/kg feed of TC+CJ admixture, respectively, with commercial enforced energy feed for 3 weeks followed by supplied with only commercial feed 3 weeks. *, **: P<0.05 and P<0.01 respectively compared to control group (C). #, ##: P<0.05 and P<0.01 respectively compared to FL-1 group.

The fatty liver hemorrhagic syndrome (FLHS) in caged laying hens is notified by excessive accumulation of fat in the liver and abdominal cavity with subsequent liver hemorrhage and fragileness and decreased egg production or sudden death within a relatively short time (Meijering, 1979; Whitehead, 1979; Crespo, 2019).

In this study, 33∼35 weeks old Hy-Line brown breed with average egg production 77.72% were used, which breed was reported to achieve their peak egg production at 27∼31 week of age (Hy-Line Int., 2018).

When β-estradiol 2 mg/kg bw was injected intramuscularly twice per week with high energy feed, egg production was apparently decreased approximately by 20% from 1 week and got more deteriorated over time of treatment in our study. Furthermore, the decreased egg production was not recovered until 4 weeks after stop of β-estradiol treatment. These results indicate that higher estrogen loading in laying hens is a critical cause of decreased egg production and the egg production loss is difficult to be restored. However, the mixture of TC and CJ restored the decreased egg production to almost normal level in three weeks of continuous supply with feed admixture in our study. The finding indicates that decreased egg production is mainly caused by lipid peroxidation and timely regimen including antioxidative herbals such as TC and CJ is critically important for stopping deterioration and restoring.

Body weight and feed consumption were not significantly different between groups whatever 2 mg/kg β-estradiol was injected continuously or not and mixture of TC and CJ were supplemented or not, however egg production was sensitively affected by β-estradiol and recovered by TC and CJ in this study. When FLHS is occurred, decreased egg production is apparent by 25∼30% comparing normal condition with increased mortality (Nelson and Carlson, 1975). This study indicates that vitellogenesis is so highly controlled by the level of estrogen that egg production is affected by additional extraneous β-estradiol. Zou et al. (2007) reported that laying hens tend to exhibit FLHS because of large quantities of free radicals formed by stress of continuous ovulation and accumulation of excessive fat in liver. Large amount of fat in liver destroys normal metabolic function and affects the development and maturation of ovarian follicle (Banerjee and Redman, 1984). So, scavenge of over-formed free radicals and mobilization of lipid for egg production are key factors to cure fatty liver syndrome and egg production loss in laying hens. TC’s effect of scavenging free radicals and CJ’s effect of suppressing lipid peroxidation explain the remedy for decreased egg production.

Blood biochemistry parameters

ALT was significantly increased from 1 or 3 weeks of the injection of 2 mg/kg bw β-estradiol comparing to control group and then kept increased till 6 weeks after stop or continuous treatment of β-estradiol.

AST, T-Chol and T-Bil were also increased from 3 weeks of the β-estradiol injection and kept increased until 6 weeks regardless of stop or not of β-estradiol treatment. Increment of ALP appeared from 1 week after stop of β-estradiol, which change was slower than other indices. TG was not significantly changed excepting FL-2 group on 9 weeks after continuous treatment of β-estradiol.

The mixture of TC and CJ (5, 10, 20 or 40 g/kg feed) decreased gradually ALT, AST and T-Bil and showed significant decrease on 2 or 3 weeks after stop of TC and CJ supplement comparing FL-1 group. For T-Chol, TG and ALP, mixture of TC and CJ presented no significant reduction comparing FL-1 group (Fig. 2).

Figure 2. Change of blood biochemistry values in serum by the treatment of TC and CJ mixture after β-estradiol injection in laying hens. Data are mean±SD (n=6). Control group was provided with commercial enforced energy feed for laying hens for 9 weeks. FL-1 group was intramuscularly administered with 2 mg/kg bw β-estradiol twice per week for 3 weeks with supply of commercial enforced energy feed for laying hens and then supplied with only feed for 6 weeks. FL-2a group was intramuscularly injected with 2 mg/kg bw β-estradiol twice per week for 9 weeks with supply of commercial enforced energy feed for laying hens. M-5, M-10, M-20 and M-40 groups were injected intramuscularly with 2 mg/kg bw β-estradiol twice per week for 3 weeks and then provided with each dose of 5, 10, 20 and 40 g/kg feed of TC+CJ admixture, respectively, with commercial enforced energy feed for 3 weeks followed by supplied with only commercial feed 3 weeks. *, **: P<0.05 and P<0.01 respectively compared to control group (C). #, ##: P<0.05 and P<0.01 respectively compared to FL-1 group.

ALT, AST and ALP are surrogate indicators for diagnosis of FLHS but they are not apparently increased under only steatosis because those enzymes leak due to cellular membrane disruption (Shini and Bryden, 2009; McGill 2016). Generally, biomarker enzymes of liver disease increases dramatically during hepatocytes necrosis. Our study noted that ALT and AST are sensitive and rapid indicators comparing ALP in response to extraneous β-estradiol. 3 weeks supplement of TC and CJ powder made gradual recovery of the level of ALT and AST and significant amelioration effects occurred later on 3 weeks of treatment or on 3 weeks after the stop of TC+CJ treatment, which indicates liver damage induced by extraneous β-estradiol is reversible but the recovery by the supplement of TC and CJ takes time rather than immediate response.

Bilirubin, a major breakdown product of hemoglobin, is mainly excreted through bile duct in liver. Increased serum bilirubin indicates malfunction of lymphatic or liver-gallbladder, bile duct occlusion or dysfunction of liver metabolism (Silva et al, 2007). In this study, serum level of total bilirubin was increased by extraneous β-estradiol 2 mg/kg bw from 3 weeks of treatment and remained higher level for 6 weeks after stop of β-estradiol and got more increased by continuous treatment of extraneous β-estradiol 2 mg/kg bw for 9 weeks. TC and CJ mixture 20 and 40 g/kg feed lowered the level of total bilirubin from 3 weeks of treatment and made recovery to normal level on 3 weeks after the stop of TC and CJ. The recovery was accompanied with ALT and AST decrement.

In this study T-Chol was significantly increased almost 2 times than that of control by 3 weeks treatment of β-estradiol and the recovery was so slow that still remained significantly higher even on 6 weeks after stop of β-estradiol treatment. While, TG was only increased by 9 weeks continuous treatment of β-estradiol.

Estrogen induces hypertriglyceridemia by making increment of triglyceride-rich very low density lipoprotein (VLDL) particles and impaired VLDL catabolism (Packard et al, 1984; Walsh et al, 1991). However, serum TG level is fluctuated by egg production condition and the level of lipids in the liver in laying hens. The diagnostic power of serum TG for fatty liver is weak since similar high levels can occur in hens that show no signs of liver hemorrhage (Butler, 1976). In this study, even high fat droplets in hepatocytes were apparent, serum TG was not different between groups, which indicates serum TG cannot fully reflect fatty liver status in laying hens since it is well modulated by egg production and compensated by various factors including diets.

Formation of cholesterol is regulated by nutritional factors, hormones and intracellular proportions of ATP/AMP and NAD(P)H/ANDP⁺ (Walsh et al, 1991; Han et al, 1993; Kurtoglu et al, 2004). The increase of hepatic fat in hens is naturally occurred because more lipids storage in liver is required for egg yolk synthesis and which is influenced by estrogen (Leeson et al, 1995). Estradiol upregulates hepatic peroxisome proliferator-activated receptor gamma (PPARγ), ATP citrate lyase (ACLY), FAS, and ApoB that regulate lipid metabolism in laying hens (Sato et al, 2009; Lee et al, 2010).

This study presented that TC and CJ mixture did not make significant recovery in serum T-Chol increased by β-estradiol however decreased ALT and lipid vacuoles in liver tissue was apparent by TC and CJ supply, which indicates TC and CJ act for recovery of damaged hepatocytes and improvement egg production by modulation of lipid from liver storage to egg yolk synthesis.

Antioxidant enzymes

SOD and GPX were decreased by 3 weeks treatment of 2 mg/kg bw β-estradiol and remained to be lower until 6 weeks or further decreased by continuous treatment for 9 weeks. TC and CJ mixture recovered the reduced SOD and GPX from the 1 week of the mixture supply with the highest effect by 5 or 10 g/kg feed of TC and CJ mixture (Fig. 3).

Figure 3. Change of antioxidative enzymes activities in serum by the treatment of TC and CJ mixture after β-estradiol injection in laying hens. Data are mean±SD (n=6). Control group was provided with commercial enforced energy feed for laying hens for 9 weeks. FL-1 group was intramuscularly administered with 2 mg/kg bw β-estradiol twice per week for 3 weeks with supply of commercial enforced energy feed for laying hens and then supplied with only feed for 6 weeks. FL-2a group was intramuscularly injected with 2 mg/kg bw β-estradiol twice per week for 9 weeks with supply of commercial enforced energy feed for laying hens. M-5, M-10, M-20 and M-40 groups were injected intramuscularly with 2 mg/kg bw β-estradiol twice per week for 3 weeks and then provided with each dose of 5, 10, 20 and 40 g/kg feed of TC+CJ admixture, respectively, with commercial enforced energy feed for 3 weeks followed by supplied with only commercial feed 3 weeks. *, **: P<0.05 and P<0.01 respectively compared to control group (C). #, ##: P<0.05 and P<0.01 respectively compared to FL-1 group.

Estradiol is known as a potent antioxidant by activating anti-oxidative defense mechanism, scavenging ROS and limiting mitochondrial protein damage under normal or estrogen-deprived condition. However, high oxidative environment in cells stimulates production of catechol estrogen metabolites, and those oxidation products undergo reactive oxygen species-producing redox cycles that upregulates a self-generating toxic cascade and pro-oxidant effects and then result in further lipid peroxidation (Belous et al, 2007; Nilsen, 2008).

Reduction of antioxidative enzymes implies the level of oxidative stress is not overcome and led to liver diseases including cirrhosis, hepatitis and steatosis that is associated with notable changes in cellular lipid metabolic homeostasis. Fatty liver haemorrhagic syndrome (FLHS) found in laying hens is due to overload of free radicals and uncontrolled fat metabolism in the liver (Zou et al, 2007). When a certain amount of fat has accumulated, the normal function of liver and the development and maturation of ovarian follicle is affected (Banerjee and Redman, 1984). Uncontrolled free radicals composed of reactive oxygen or nitrogen species including superoxide anion (O2⁻), hydrogen peroxide (H₂O₂), hydroxyl radical (·OH), nitric oxide (NO), NO₂ and peroxynitrite (ONOO⁻) induce lots of disorders such as inflammation, cardiovascular disease, fatigue and infertility (Phaniendra et al, 2015).

At present, there were many studies to find out natural antioxidant to remove these kinds of free radicals and to treat FLHS. Choi et al. (2012) reported that ethyl acetate fraction from Taraxacum coreanum inhibited lipid peroxidation against ONOO⁻ itself and its precursors of NO and O₂⁻ in porcine renal epithelial cells (LLC-PK1). Water extract of dandelion (Taraxacum officinale) ameliorated D-galactosamine induced acute hepatitis via antioxidative activities inducing antioxidative enzymes such as caltalase, GSH peroxidase, GSH reductase and Mn-superoxide dismutase in rats (Park et al, 2008). Also, orally treated dandelion (Taraxacum coreanum) water extract 3 g/kg bw decreased liver damage induced by mercury chloride in mice (Choeng et al, 2008). Meanwhile, roots, leaves, flowers, stems and seeds of Cirsium japonicum var. ussuriense have been used for the treatment of hemorrhage, blood congestion, alcoholic hepatitis and inflammation in human via its antioxidative and anti-inflammatory activities (Park et al, 2008). Mok et al. (2011) reported that water extract of leaves of Cirsium japonicum var. ussuriense suppressed inflammatory mediators including NO, prostaglandin E2, INOS and COX-2.

In this study, mixture (6:4) of dried powder of whole TC and CJ showed remedy effects against egg production drop. The amelioration effects on laying performance were accompanied by lower ALT, AST, and total bilirubin in serum and higher activities of antioxidant enzymes such as SOD and GPX with less accumulation of fat in liver comparing FL-1 and FL-2 groups. These results indicate that TC and CJ powder mixture scavenges free oxidative radicals by induction of antioxidative enzymes and subsequently restores damaged hepatocytes.

Gross and histopathological lesions

The liver tissues were still pale, swollen and friable with weak hemorrhages by 3 weeks extraneous β-estradiol treatment with supply of high energy diets for laying hens and then 6 weeks only supply of high energy diets without β-estradiol treatment. When 2 mg/kg bw β-estradiol was treated continuously for 9 weeks with high energy diets, liver tissues were severely pale, more friable and showing dull edges almost ruptured. By the treatment of TC and CJ mixture, the damaged liver tissues were ameliorated and looked almost close to normal in color with a little sharp edge and resilience (Fig. 4).

Figure 4. Gross lesions of liver of laying hens. (A) Control. (B) Yellowish pale colored with swollen, friable and slight hemorrhage in FL-1 group treated with 2 mg/kg β-estradiol for 3 weeks. (C) More yellowish pale colored with severe friable and dull edges almost ruptured in FL-2 group treated with 2 mg/kg β-estradiol for 9 weeks. (D) Dark yellowish pale and almost close to normal colored with a little sharp edge and resilience in 10 g/kg feed of TC and CJ mixture for 3 weeks following 3 weeks treatment of β-estradiol. Autopsy was performed on 9 weeks after the beginning of β-estradiol treatment.

2 mg/kg bw β-estradiol treatment for 3 or 9 weeks resulted in increased massive infiltration of fat in liver tissue and hepatocytes with the presence of small or large fat droplets or large vacuoles of fat filling the cytoplasm and distending hepatocytes. TC and CJ mixture made recovery with decreased fat infiltration and smaller vacuoles (Fig. 5).

Figure 5. Histopathological findings of liver tissues of laying hens. (A) Control. (B) Massive accumulation of small or large droplets and large vacuoles of fat in the cytoplasms of swollen hepatocytes in FL-1 group treated with 2 mg/kg β-estradiol for 3 weeks. (C) Massive accumulation of more small or large droplets and large vacuoles of fat in the cytoplasms of swollen hepatocytes in FL-2 group treated with 2 mg/kg β-estradiol for 9 weeks. (D) Decreased accumulation of small or large droplets or large vacuoles of fat in the cytoplasms of hepatocytes in 10 g/kg feed of TC and CJ mixture for 3 weeks following 3 weeks treatment of β-estradiol. Autopsy was performed on 9 weeks after the beginning of β-estradiol treatment. Bar: 100 μm

The paleness of liver color is in correlation with the total liver fat. More severe liver paleness in hens treated with estrogen injections indicates that estrogen is a potent factor accumulating fats in the liver. This study showed that damaged liver were not well recovered by the finding of still remained hepatic lesions on 6 weeks after the stop of β-estradiol treatment. These results indicate that liver fat accumulation and related diseases are not well controlled during laying period under physiological or environmental conditions of increased estrogen level and high energy feeding. In this study, mixture (6:4) of dried powder of whole TC and CJ showed remedy effect against egg production drop and FLHS. The amelioration effects on laying performance and FLHS were accompanied with lowering ALT, AST and total bilirubin in serum and restoring activities of antioxidant enzymes such as SOD and GPX.

Therefore, the dietary supplementation of TC and CJ powder remedies FLHS and egg production reduction in laying hens by their antioxidative effects.

Conclusively, we found that 1) Intramuscular injection of 2 mg/kg bw β-estradiol twice per week for 3 weeks in laying hens supplied with enforced energy feed induce fatty liver syndrome with egg production loss. 2) The dietary supplementation of mixed dried powder of TC and CJ (6:4) recovered liver damages as well as egg production decrement in laying hens via increased antioxidative enzymes with the best effects at the dose of 5 g/kg feed TC and CJ mixed dried powder in terms of recovered egg production rate. 3) Scavenge of over-formed free radicals and mobilization of lipid to be used for egg production are key strategies to cure fatty liver syndrome and egg production loss in laying hens. TC and CJ mixture can be regarded as a good remedy that cure FLHS in laying hens. Further research is required to determine direct amelioration mechanism of TC and CJ against fatty liver syndrome in laying hens.

This work was supported by the Hoseo University research grant in 2021∼2022.

No potential conflict of interest relevant to this article was reported.