Effects of lycopene on abdominal fat deposition, serum lipids levels and hepatic lipid metabolism-related enzymes in broiler chickens

Objective The present study was conducted to investigate the effects of lycopene on growth performance, abdominal fat deposition, serum lipids levels, activities of hepatic lipid metabolism related enzymes and genes expression in broiler chickens. Methods A total of 256 healthy one-day-old male Arbor Acres broiler chicks were randomly divided into four groups with eight replicates of eight birds each. Birds were fed basal diet supplemented with 0 (control), 100, 200, and 400 mg/kg lycopene, respectively. Results Dietary 100 mg/kg lycopene increased the body weight at 21 day of age compared to the control group (p<0.05). Compared to the basal diet, broilers fed diet with 100 mg/kg lycopene had decreased abdominal fat weight, and broilers fed diet with 100 and 200 mg/kg lycopene had decreased abdominal fat percentage (p<0.05). Compared to control, diets with 100, 200, and 400 mg/kg lycopene reduced the levels of total triglyceride and total cholesterol in serum, and diets with 100 and 200 mg/kg lycopene reduced the level of serum low density lipoprotein cholesterol (p<0.05). The activity of fatty acid synthase (FAS) in 400 mg/kg lycopene treated broilers and the activity of acetyl-CoA carboxylase (ACC) in 100, 200, and 400 mg/kg lycopene treated broilers were lower than those fed basal diet (p<0.05). Lycopene increased the mRNA abundance of adenosine monophosphate activated protein kinase α (AMPK-α), whereas decreased the mRNA abundance of sterol regulatory element-binding protein 1, FAS, and ACC compared to the control group (p<0.05). Conclusion Dietary lycopene supplementation can alleviate abdominal fat deposition and decrease serum lipids levels, possibly through activating the AMPK signaling pathway, thereby regulating lipid metabolism such as lipogenesis. Therefore, lycopene or lycopene-rich plant materials might be added to poultry feed to regulate lipid metabolism.


INTRODUCTION
The broiler industry has thrived, and broiler chicks have become one of the common meat species during the past few decades. However, excessive deposition of abdominal and sub cutaneous fat resulting from the intensive production, not only leads to high occurrence of ascites syndrome and sudden death syndrome, but also has a negative impact on con sumers' healthy diet [1]. Studies have shown that dietary supplementation of specific natural or synthetic compounds had lipid lowering effect [24]. Consumers increasingly prefer natural to synthetic compounds in consideration of food safety and health. Thus, plant derived compounds or phytogenic products used as feed additives have received quite a bit of attention [5].
With the increase of natural source additives used in animal diets, carotenoids such as lycopene are gradually drawing the attention of the feed industry. Lycopene, which is a natural isoprene compound belonging to carotenoids, is present in vegetables, fruits, and flowers with red, yellow, or orange colors, especially in tomatoes, carrots, watermelons, pawpaws [6,7]. Further more, lycopene is a natural and efficient pigment used in food processing, and has the bioactivity of antioxidant ac tivity, immunoregulation, anticancer, blood lipids regulation and cardiovascular diseases prevention effects [7]. It has been identified as a class A nutrient by the United Nations Food and Agriculture Organization (FAO) and World Health Organization (WHO) [7]. Although lycopene is known for its antioxidant activity, the blood lipids regulation capacity is not neglectable. Studies in rats, mice or humans have shown that lycopene and lycopene enriched tomato products could reduce the risk of obesity and cardiovascular disease by reg ulating lipid metabolism [810]. Lycopene or lycopene enriched plant materials in poultry diet also showed positive regulation effect on serum lipids levels [1114].
In this study, we investigate the effects of dietary lycopene supplementation on abdominal fat deposition, serum lipids levels, activities of hepatic lipid metabolism enzymes, as well as mRNA expression levels of related enzymes and transcrip tion factors in liver, to elucidate whether lycopene could influence lipid metabolism in broiler chickens. The results of this study will provide information for the application of ly copenerich plant materials in animal feed.

Animal and management
All animal experiments were conducted in accordance with the Institutional Animal Care and Use Committee of Yang zhou University, Yangzhou, China (No. SYXK(Su)20160020).
A total of 256 healthy onedayold male Arbor Acres broilers chicks were obtained from a commercial hatchery (Nantong, Jiangsu Province, P. R. China). All birds were ran domly divided into four groups with eight replicates per group, each replicate comprised eight birds, and kept in an environmentally controlled room. The room temperature was 32°C to 34°C for the first three days, then gradually de creased to 22°C±1°C by 2°C to 3°C per week and maintained until the end of the experiment. Broilers in the control group were offered the basal diet, whereas broilers in the other three groups were fed the basal diet supplemented with 100, 200, and 400 mg/kg lycopene, respectively. Lycopene (80% of purity) was purchased from Shanghai yuanye BioTechnol ogy Co., Ltd (Shanghai, China). The basal cornsoybean meal diet was formulated based on the recommendation of NRC (1994) to meet the nutrient requirement of broilers (Table 1). All birds in the experimental period (1 to 42 day of age) enjoyed free access to feed and water with a lighting cycle of 23 h light and 1 h dark. The management proce dures were conducted based on the commercial chicken farm feeding management procedures. Body weight (BW) and feed intake (FI) were recorded at 21 and 42 day of age by replicate and gain to feed ratio (G/F) was calculated by the ratio between body weight gain (BWG) and FI.

Sample collection
At the end of the experiment, one bird per replicate with an averaged BW of the replicate was selected after 12 h feed de privation. Blood sample was obtained from the wing vein, then the serum was collected after centrifuged at 3,000×g for 10 min at 4°C. The serum samples were stored at -20°C for total triglyceride (TG), total cholesterol (TC), low density li poprotein cholesterol (LDLC) and high density lipoprotein cholesterol (HDLC) analysis. After bleeding, the broilers were euthanized by cervical dislocation. Immediately after death, the abdominal fat of each broiler was harvested and weighed. The liver samples were excised, washed with icecold steril ized saline, then snapfrozen in liquid nitrogen, and stored at -70°C before use.

Determination of abdominal fat deposition
The abdominal adipose deposition was expressed as ab dominal fat percentage (AFP), which was calculated by the percentage of abdominal fat weight (AFW) to final BW.

Measurement of serum TG, TC, LDLC, and HDLC levels
The serum TC, TG, LDLC, and HDLC levels were deter mined using an automatic biochemical analysis system (UniCel Synchron DxC 800 synchron, Beckman Coulter, Inc., Fullerton, CA, USA). The results were expressed as millimole per litre (mmol/L).

Hepatic enzymatic activity
The commercial enzymelinked immunosorbent assay kits of Shanghai Yu Bo Biotech Co., Ltd (Shanghai, China) were used to measure the hepatic activities of fatty acid synthase (FAS), acetylCoA carboxylase (ACC), lipoprotein lipase (LPL), and hepatic lipase (HL), and operated according to the instructions of the corresponding kits. The bicinchoninic acid assay was used to measure the protein concentration of liver [15]. The results were calculated based on the protein concentration of liver and expressed as units per gram of protein (U/g protein).

mRNA quantification
Total RNA isolation of liver sample was performed following the protocol of Uni10 column Trizol total RNA extraction kit (Sangon Biotech Co. Ltd., Shanghai, China). The quality of the RNA was performed by agarose gel electrophoresis detection, and the quantity of the RNA was measured with a microspectrophotometer (NaroDrop 2000c, Thermo Scien tific, Waltham, MA, USA). Immediately following the total RNA isolation, reverse transcription was performed using the PrimeScript RT Mas ter Mix kit (TaKaRa Biotechnology Co. Ltd., Dalian, China) to synthesis cDNA. Realtime polymerase chain reaction (PCR) was performed with the 7500 RealTime PCR System (Applied Biosystems, Foster City, CA, USA) with a TB Green Premix Ex Taq II kit (TaKaRa, China) following the instruc tion of the kit. The primer sequences for broiler adenosine monophosphate activated protein kinase α (AMPKα), sterol regulatory elementbinding protein 1 (SREBP-1), FAS, ACC, and βactin (as a reference gene) are listed in Table 2. The primers were synthesized by Sangon Biotech Co. Ltd (China). The relative expression level of each target gene mRNA was normalized to the mRNA level of βactin, and the results were expressed as the fold difference between the treated groups and the control group using the 2 -ΔΔCt method [16].

Statistical analysis
Data are presented as mean±standard error of the mean, and statistical analyzed by SPSS (version 22 for windows, SPSS Inc, Chicago, IL, USA). Data were subjected to oneway anal ysis of variance using the general liner model procedure of SPSS, and Tukey test was used to assess the differences among the treatment means. Differences were considered signifi cant when p<0.05.

Growth performance
Effects of various dietary lycopene supplementation on growth performance of broiler chickens are shown in Table 3. Com pared to the control group, diet supplemented with 100 mg/kg lycopene increased the BW at 21 day of age (p<0.05). Di etary lycopene addition had no significant impact on FI, G/F, BWG from 22 to 42 days of age and BW at 42 day of age (p> 0.05). Table 4 shows the effects of dietary lycopene inclusion on the AFW and AFP of broiler chickens. The AFW of broilers treated by 100 mg/kg lycopene was decreased in comparison to the basal diet (p<0.05). The AFP of broilers in 100 and 200 mg/kg lycopene supplementation groups were lower than that of the control group (p<0.05).

Serum lipid level
The serum lipid parameters are presented in Table 5. Broilers fed diets supplemented with 100, 200, and 400 mg/kg lyco pene reduced the levels of TG and TC in serum compared to control (p<0.05). Broilers fed diets supplemented with 100 and 200 mg/kg lycopene reduced the level of serum LDLC compared to control (p<0.05).

Hepatic enzymatic activity
As shown in Table 6, the hepatic activity of FAS in the 400 mg/kg lycopene group was lower than control group (p< 0.05). The activity of ACC in the 100, 200, and 400 mg/kg lycopene treated broilers were lower than broilers fed basal diet (p<0.05). However, the activities of LPL and HL were similar among all experimental groups (p>0.05).

Relative mRNA expression level of gene
To investigate the potential mechanism of lycopene in low ering lipid, the gene expression of some key enzymes and factors in the hepatic lipid metabolism were determined. The mRNA expression levels of some genes that significantly changed can be seen from Figure 1. The mRNA expression level of AMPKα in lycopene supplemented groups were increased compared to the control group (p<0.05). Where   as, the mRNA expression levels of SREBP1, FAS and ACC in lycopene supplemented groups were decreased compared to the control group (p<0.05).

DISCUSSION
A previous study suggested that broiler chickens' diet with added 100 mg/kg lycopene from 14 to 35 days of age en hanced the final live weight [11]. According to the study of Lira et al [17], tomato waste might be used as an ingredient of broiler chickens' diet (up to 20%) from 29 to 42 days of age with no negative effect on growth performance. Diet supplemented with 5% dried tomato pomace (lycopene en riched) increased body mass and decreased FI to weight gain ratio of broilers at 28 day of age [12]. Results in the present study showed that lycopene was beneficial to the early growth of broiler chickens. Although some discrepancies exist among these abovementioned studies due to species, ages, doses, or duration, it is convincible that there is no negative effect of lycopene on growth of broilers. Therefore, lycopene or lyco pene enriched materials can be used as additives or ingredients in poultry feed. Abdominal adipose mass is an important index used to measure lipid deposition in broilers. Serum TG, TC, LDLC, and HDLC levels are essential biochemical parameters re flecting the lipid metabolism status. TG, TC, and LDLC have been established as risk factors and elevated HDLC level possesses protective properties. Our present study revealed that dietary lycopene addition decreased abdominal fat mass, and reduced serum TG, TC, and LDLC levels. Similarly, lyco pene enriched tomatowine raised HDLC level and produced antiobesity impact through inhibition of lipid biosynthesis in highfat diet (HFD) fed rats [9]. Rats ingesting virgin olive oil and argan oil with lycopene had reduced serum TG, TC, LDLC concentrations and elevated serum HDLC concen tration, and decreased hepatosomatic index, these indicated that lycopene could fight against hyperlipidemic and hyper cholesterolemicderived disorders [18]. Lycopene and tomato powder (both provided 10 mg lycopene/kg food/d) supple mentation in mice diet reduced serum TG concentration, Values are presented means ± standard error of the means, n = 8. FAS, fatty acid synthase; ACC, acetyl-CoA carboxylase; LPL, lipoprotein lipase; HL, hepatic lipase. a,b Means within a row with different superscripts differ significantly (p < 0.05). prevented HFDinduced hepatic steatosis and hypertrophy of adipocytes [19]. Dietary 200 mg/kg lycopene supplemen tation in Feedlot Bamei Lamb reduced the levels of plasma TG, TC, and LDLC, as well as atherogenic index, and the levels of TG and LDLC were decreased with the feeding time extension [20]. Similar results were observed in poultry. Broilers fed diet supplemented with 5% dried tomato pomace (708 mg lycopene/kg diet) from 1 to 28 days had lower TG and higher HDLC concentrations in serum than the basal diet [12]. Dietary supplementation of 50, 100, and 200 mg/kg lycopene increased the HDLC concentration, whereas de creased TC, TG, and LDLC concentrations in Japanese quails [13]. A previous study of Sun et al [14] revealed that 40 mg/kg lycopene supplementation in chick diet increased serum HDLC level. The results observed in the present study agree with the abovementioned literatures [1214,19,20], sug gesting that lycopene or lycopene enriched tomato product could regulate serum lipids levels in poultry. Diet with to mato juice could inhibit the activity of cholesterol synthesis ratelimiting enzyme, 3hydroxy3methylglutaryl coen zyme A reductase (HMGCR) [21]. The possible mechanism in the effect of lowering cholesterol of lycopene and tomato derivatives possibly via inhibiting the activity and expres sion of HMGCR, modulating the low density lipoprotein receptor (LDLR) and inhibiting the activity of acylcoen zyme A:cholesterol acyltransferase (ACAT) [22]. Therefore, lycopene might have a role in lipid metabolism.
In avian species, the liver accounts for about 95% of de novo fatty acid synthesis, making it the main organ of lipo genesis, and it is generally considered that most of the lipid that accumulate in adipose tissue is synthesized in the liver or provided by diet fat [23]. The liver is often used as target organ in the scientific research of exogenous materials treat diabetes and obesity. Therefore, regulation of the hepatic lipogenesis or steatolysis enzymes in relevant metabolic path ways play essential roles in lipid metabolism. The AMPK is a central regulator of energy metabolism and plays a crucial role in lipid metabolism regulation [24]. AMPK consists of three subunits (α, β, γ), α is the catalytic subunit, β and γ are the regulatory subunits, and α subunit is essential for AMPK activation [25]. In liver, the lipid metabolism regu lation effect of AMPK acts through the regulation of SREBP1 [26]. SREBP1 is a key transcription factor in cholesterol and fatty acid biosynthesis, which is preferentially involved in the regulation of genes related to fatty acid synthesis, can directly stimulate the transcription of genes encoding FAS [27], and modulate the expression of enzymes involved in the synthesis of fatty acid, cholesterol, triacylglycerol, and phospholipid [28]. The activation of AMPK decreased the activity of FAS through downregulating the mRNA expres sion of SREBP1, thereby inhibiting lipogenesis [29]. In addition, the activation of AMPK phosphorylates/inhibits ACC results in reduced malonylCoA, then inhibits the synthesis of fatty acid [24]. Plantderived phytochemicals are indirect activators of AMPK [24]. Zeaxanthin, a kind of carotenoid, could play antiobesity role by activating AMPK, thereby inhibiting lipogenesis [30]. Fenni et al [19] have recently shown that lycopene and tomato powder (both provided 10 mg lycopene/kg food/d) supplementation in HFD fed mice decreased the mRNA expression of hepatic ACC, FAS, and SREBP1c. Similarly, in the present study, lycopene addition in broilers diet decreased the activities of FAS and ACC, upregulated the mRNA expression level of AMPKα, down regulated the mRNA expression levels of SREBP1, FAS, and ACC in liver. Thus, it is convincible that broilers fed diet supplemented with lycopene might acti vate the AMPK signaling pathway, thereby inhibiting lipid synthesis in liver. The reduced abdominal fat deposition and regulated serum lipid level might partly be attributable to the activated AMPK signaling pathway.

CONCLUSION
Compared to basal diet, dietary lycopene supplementation decreased abdominal fat deposition, reduced serum TG, TC, and LDLC levels, and reduced hepatic FAS and ACC activi ties of broilers. Furthermore, lycopene might activate the AMPK signaling pathway, thereby modulating lipid metabo lism such as lipogenesis. The beneficial effects of lycopene appeared to be mainly mediated through inhibition of he patic lipid biosynthesis, and the inclusion level of 100 mg/kg lycopene in broiler diet was recommended in this study. These findings can benefit our understanding on the supplementa tion of lycopene as a potential precaution to protect against hyperlipidemia, excessive abdominal fat deposition and as sociated diseases in poultry, as well as provide information for the application of lycopenerich plant materials in animal feed.

CONFLICT OF INTEREST
We certify that there is no conflict of interest with any financial organization regarding the material discussed in the manu script.