Hepatic transcriptome analysis reveals altered lipid metabolism and consequent health indices in chicken supplemented with dietary Bifidobacterium bifidum and mannan-oligosaccharides

This study investigated the role of dietary prebiotic mannan-oligosaccharides (MOS), and probiotic Bifidobacterium bifidum (BFD) in lipid metabolism, deposition, and consequent health indices in broiler chicken. The supplementation of 0.2% MOS along with either 106 or 107 CFU BFD/g feed resulted in downregulation of Acetyl-CoA carboxylase, fatty acid synthase, sterolregulatory element binding protein-1, and apolipoprotein B100; and up-regulation of peroxisome proliferator activated receptor-α AMP-activated protein kinase α-1, and stearoyl CoA (∆9) desaturase-1 hepatic expression in broiler chicken. The birds supplemented with 0.2% MOS along with either 106 or 107 CFU BFD/g feed depicted lower body fat percentage, palmitic acid, stearic acid, and saturated fatty acid contents, whereas, higher palmitoleic acid, oleic acid, and MUFA contents were observed. The ∆9-desaturase indices of chicken meat have shown higher values; and elongase index (only thigh) and thioesterase index have shown lower values in birds supplemented with 0.2% MOS along with either 106 or 107 CFU BFD/g feed. The meat health indices such as Polyunsaturated fatty acids (PUFA)/Saturated fatty acids (SFA) ratio, Mono-saturated fatty acids (MUFA)/SFA ratio, unsaturated fatty acids (UFA)/SFA ratio, hypocholesterolemic/hypercholesterolemic fatty acid ratio, saturation index, atherogenic index, thrombogenic index, and hypercholesterolemic fatty acid content were positively improved in birds supplemented with 0.2% MOS along with either 106 or 107 CFU BFD/g feed. Similarly, the birds supplemented with 0.2% MOS along with either 106 or 107 CFU BFD/g feed have shown lower serum triglyceride and total cholesterol levels along with higher high density levels and improved serum health indices cardiac risk ratio, atherogenic coefficient, and, atherogenic index of plasma.


Results
Hepatic gene expression. The relative expression pattern of genes involved in lipid metabolism of broiler chicken under the influence of BFD and MOS are given in Figs. 1, 2, 3, and 4. The expression pattern of ACC, FAS, ME, SERBP-1 (21 days only), and apoB100 depicted a progressive (P < 0.05) decrease from T1 (control) to T6. There were no significant differences between T1 and T2; and T5 and T6. Also, the expression pattern of SERBP-1 in broiler chicken at 42 days of age did not show any significant dietary effects. However, the expression pattern of PPAR-α, AMPKα-1 (21 days only), and SCD-1 revealed an increasing trend from T1 (control) to T6. www.nature.com/scientificreports/ Again, no significant differences were observed between T1 and T2; and T5 and T6. The expression of AMPKα-1 at 42 days of was not affected by dietary treatments.
Fat percentage and fatty acid profile of chicken meat. The results given in Tables 1 and 2 reveal significant effects of BFD and MOS supplementation on fat percentage and some constituent fatty acids of broiler chicken breast and thigh at 42 days of age, respectively. However, it is noteworthy to point out that CARIBRO Vishal tends to deposit more fat compared to other commercial chicken. From T1 (control) to T6 a significant decreasing trend was observed in percentages of fat (P < 0.01), palmitic acid (P < 0.01), stearic acid (P < 0.05), and SFA (P < 0.05). However, increasing trend was observed in percentage of palmitoleic acid (P < 0.01), oleic acid (P < 0.05), and MUFA (P < 0.01) in chicken breast and thigh meat from treatment T1 to T6. But, no significant differences were observed between T1 and T2; and T5 and T6. The other constituent fatty acids and PUFA were not affected by dietary treatments. Similar pattern was observed at 21 days of age (data not shown).
Lipid metabolism and health related indices. The product: precursor ratios were used to assess the activities of various enzymes involved in lipid metabolism. The enzyme activity indices at 42 days of age have revealed a progressive increase (P < 0.01) in ∆9-DI (18), ∆9-DI (16), and total DI; and a progressive decrease (P < 0.05) was observed in thioesterase index and elongase index (thigh only) from treatment T1 (control) to T6 in chicken meat ( Table 3). The ∆5 + ∆6 Desaturase index and elongase index (breast only) did not reveal any significant treatment effect and no significant differences were observed between T1 and T2; and T5 and T6. The health related indices measured in chicken breast and thigh meat were significantly influenced by BFD and MOS supplementation except ω-6: ω-3 PUFA ratio (Table 4). The PUFA:SFA ratio, MUFA:SFA ratio, UFA:SFA ratio (P < 0.01), DFA content, and h/H ratio (P < 0.05) exhibited a progressive increase; and S/P, AI, TI (P < 0.01), and HFA content (P < 0.05) revealed a progressive decrease from treatment T1 (control) to T6. But, no significant differences were observed between treatment group T1 and T2; and T5 and T6. Similar trend in lipid metabolism and health indices pattern was observed at 21 days of age (data not shown).

Discussion
In this study, a nutrigenomic approach was adopted to understand the molecular mechanisms behind the effects of dietary MOS and BFD supplementation on lipid metabolism in broiler chicken. Since modern day broiler chickens are hyperphagic and thus, more prone to obesity they are considered as models of choice to study lipid metabolism and their consequent effects in response to dietary synbiotic supplementation 3 . Liver was chosen for the transcriptome analysis because it is a major metabolic organ of the body involved in lipid metabolism 22 . Lipid deposition is the function of balance between the lipolysis and lipogenesis in the body 14 and alteration in the expression pattern of genes involved in lipid metabolism in response to dietary synbiotics affect the lipid deposition in broiler chicken 3 . The key enzymes involved in lipogenesis are ACC, FAS, ME, and SCD-1 which are regulated by SERBP-1 and the key enzymes involved in lipolysis are PPAR-α and AMPKα-1 2,3,23 . The present study revealed lower fat percentage of chicken meat from birds supplemented with 0.2% MOS along with either 10 6 or 10 7 CFU BFD/g feed. The possible determinants of lower fat percentage of chicken meat in the present study could be decline in the synthesis of lipids due to the down regulation of ACC, FAS, ME, and SERBP-1, lesser transport and deposition of synthesised lipids due to down regulation of apoB100, and enhanced oxidation or inhibition of lipogenesis due to upregulation of PPAR-α and AMPKα-1 expression in chicken liver. The process of lipogenesis is initiated by ACC by catalysing the rate-limiting step of carboxylation of acetyl-CoA to malonyl-CoA followed by a series of repetitive reaction by FAS 3,24 . During this process of lipogenesis FAS requires a continuous supply of reducing agent NADPH which is provided via oxidative decarboxylation of malic acid to pyruvic acid and CO 2 by ME 25,26 . In chicken, synbiotic supplementation down regulates the hepatic expression of SERBP-1c, a lipogenic nuclear transcriptional regulator that directly regulates the expressions of ACC, FAS, and ME 3,25 . On the other hand increased hepatic expression of PPAR-α was observed in broiler chicken due to dietary synbiotic supplementation 3,27 which upregulates the expression of fatty acid catabolism genes, such as Carnitine palmitoyl transferase-1 and Acyl CoA oxidase-1, resulting in enhanced fatty acid β-oxidation www.nature.com/scientificreports/ in mitochondria 28 . Another mechanism put forward by earlier researchers as a cause of reduced body fat deposition is stimulation of farnesoid X receptor-PPAR-α-acyl CoA oxidase pathway in laying hens 14 and mice 29 . Furthermore, the upregulation of AMPKα has been reported to decrease FAS expression by negatively regulating the expression of SERBP-1 in avian species 30 . Therefore, the consequent effect of SERBP-1c down regulation and upregulation of PPAR-α and AMPKα under dietary synbiotic supplementation is lower deposition of fat content in broiler chicken 2,3 as observed in the present study. Another protein involved in lipid metabolism and investigated in this study was apoB100 protein. It is directly involved in transport and deposition of lipids in tissues 31 and upregulation in its expression results in greater fat accumulation in broiler chicken 32 . Similar to the results of present study, synbiotic supplementation has been reported to down regulate the hepatic gene expression of apoB100 protein with the corresponding decline in muscle fat percentage 3 . Therefore, down regulation of hepatic apoB100 expression by synbiotic supplementation can be another possible mechanism of fat reduction in broiler chicken. However, it is noteworthy to state that lipid synthesis and its consequent deposition also depends on the age of birds and older chicken tend to deposit more fat in their body 33 . Corresponding to this age dependence of lipid deposition in chicken present study revealed the up regulation of PPAR-α and AMPKα-1 expression, and down regulation of SERBP-1 expression at 21 days of age but not at 42 days of age. It indicates that in young chicken rate of lipogenesis and lipid deposition is lower compared to adult birds 3,34 .
In the present study, ∆9-DI (18), ∆9-DI (16), and total DI increased significantly in birds supplemented with 0.2% MOS along with either 10 6 or 10 7 CFU BFD/g feed by mediating the up regulation of SCD-1 which catalyses the biosynthesis of MUFA from corresponding SFA 3,35 . Therefore, decrease in palmitic acid, stearic acid, and total SFA content; and increase in palmitoleic acid, oleic acid, and total MUFA content of chicken meat can be associated with up-regulation of SCD-1 expression. Lower thioesterase and elongase indices of chicken meat observed in the present study could be because of increased conversion of palmitic and stearic acids to their unsaturated counterparts under the influence of SCD-1 upregulation. The ∆5 + ∆6 desaturase indexof chicken meat, associated with catalysis of PUFAs synthesis, did not reveal any significant effect of synbiotic supplementation. This non-significant effect of synbiotic supplementation on ∆5 + ∆6 desaturase index and in turn on the PUFA content of chicken meat was also reported by Dev et al. 3 . On the similar pattern, the health indices of chicken meat detailed in this study were maximally improved in birds supplemented with 0.2% MOS along with either 10 6 or 10 7 CFU BFD/g feed because of increased MUFA content at the cost of SFA content of chicken meat which happened under the influence of SCD-1 up regulation. Other genes involved in lipid metabolism www.nature.com/scientificreports/ and studied in this experiment did not reveal any significant effect on health indices of chicken meat because of their non-significant effect on the fatty acid profile 3 . Serum lipid metabolites such as triglycerides, total cholesterol, and other lipoproteins are sensitive indicators of lipid metabolism rate in chicken 3,36 . In the present study, serum TG, and TC decreased, whereas, serum HDL C increased in birds supplemented with 0.2% MOS along with either 10 6 or 10 7 CFU BFD/g feed. This hypocholesterolemic and hypolipidemic effect of synbiotic supplementation in broiler chicken resulted in improved serum health indices -CRR, AC, and AIP which was also reported by 3 in broiler chicken in response to synbiotic supplementation. Improved serum lipid profile has been reported earlier in broiler chicken due to synbiotic supplementation 2,3,37,38 . Various mechanism of lipid lowering effects due to probiotics have been put forward, such as enhancement of deconjugation of bile acids 39 which diverts more cholesterol towards synthesis of bile acids, intestinal conversion of cholesterol to coprostanol 40 , and subsequent excretion via faeces 3 . Also, the inhibition of 3-hydroxyl-3-methylglutaryl-coenzyme (HMG-CoA) reductase has been linked to cholesterol lowering effect of probiotics 9 . On the other hand, the lipid lowering effects of prebiotics have been ascribed to their ability to increase gut viscosity and mucus layer thickness which in turn inhibits cholesterol uptake from intestines; and further has been reported to enhance cholesterol breakdown 3,13 .
In conclusion, this study establishes that reduction in fat content of broiler chicken meat under the influence of synbiotic supplementation in chicken feed occurs by three possible mechanisms-reduction in rate of lipogenesis by down regulation of ACC, FAS, ME, and SERBP-1 expression, reduced transport and deposition of lipids in tissues by down regulation of apoB100 expression, and enhancement of β-oxidation of body lipids by upregulation of PPAR-α and AMPKα-1 expression in chicken liver. The ∆9-desaturation of chicken meat increased significantly under the influence of synbiotic supplementation by mediating the upregulation of SCD-1 expression which consequently resulted in increase of MUFA content at the cost of SFA content and in turn improved certain health indices of chicken meat. Further, the synbiotic supplementation in chicken feed produced hypocholesterolemic and hypolipidemic effect which improved the serum health indices of broiler chicken.        Experimental design. In this study a total of 288 one-day-old CARIBRO Vishal with considerable uniformity in body weight were obtained from the hatchery of Central Avian Research Institute, Izatnagar. The antibiotic bacitracin methylene disalicylate (BMD), prebiotic mannan-oligosaccharides (MOS), and probiotic Bifidobacterium bifidum (BFD) were added to corn-soybean meal basal diet of chicken to form six treatment     www.nature.com/scientificreports/ groups-T1 (negative control diet), T2 (positive control, 20 mg BMD/kg diet), T3 (0.1% MOS + 10 6 cfu BFD/g feed), T4 (0.1% MOS + 10 7 cfu BFD/g feed), T5 (0.2% MOS + 10 6 cfu BFD/g feed), and T6 (0.2% MOS + 10 7 cfu BFD/g feed). The details of ingredients and nutrient composition of basal diet is shown in Supplementary 1. Each treatment group was allotted 48 birds in six replicates with eight birds in each. The diets in all the treatment groups were similar in energy, protein, and fatty acid profile to avoid potential confounding effects on the results of the study. BMD, with a certified 44% bacitracin activity, was purchased from ALPHARMA Animal Health Division New Jersey-USA and MOS was purchased from Kothari Fermentation & Biochem Ltd. India. BFD (UBBB-55), with strain number MTCC 5398, was purchased from Unique Biotech Ltd. India. It is of healthy human fecal origin in the form of cream to brown coloured powder with characteristic odour and certified stable at room temperature. The E. coli and Salmonella species per 10 g powder; and Staphylococcus aureus and Pseudomonas aeruginosa per g powder are certified to be absent with yeast and mould count not more than 100 cfu/g. To ensure the exact dose of probiotic, the concentration of live bacteria in BFD powder was verified by culture-based counting. Bifidobacterium Selective Count Agar Base (BSC Propionate Agar Base) was used for enumeration of BFD by making a serial dilution of BFD powder in sterile phosphate buffer saline. The dilution showing most visible and countable colony forming units on the agar was replicated six times and an average concentration was calculated at this dilution. The birds were fed ad libitum with free access to clean drinking water for an experimental period of 42 days. For the analysis of lipid deposition and the consequent effects in broiler chicken in response to MOS and BFD supplementation sampling was done at 21 and 42 days of age. Six birds were sacrificed (one bird from each replicate with equal number of males and females) from each treatment group after 12 h of fasting and fresh breast and thigh meat samples (5 g) without skin (from same place) were collected in duplicate for fatty acid profile analysis. From the same birds liver samples (1 g) from similar areas were also collected and stored in RNAlater to analyze the expression profile of genes involved in lipid deposition in broiler chicken. Further, blood samples (2 ml) were collected from the same birds in duplicate in test tubes without anticoagulant. The serum was extracted from the blood samples and stored at − 20 °C till lipid profile analysis.
Hepatic RNA extraction and expression of genes involved in lipid metabolism. From the liver samples RNA extraction was done by using Trizol reagent (INVITROGEN, Carlsbad, CA, USA) following the instructions of manufacturer. Nanodrop (NANO DROP 1000, thermo-scientific, Singapore) was used to check RNA sample concentration at 260 nm, ethidium bromide staining was used to check RNA integrity by agarose gel-electrophoresis, and RNA sample purity was checked by UV spectrophotometry (OD 260 /OD 280 ). The reverse transcription of extracted RNA samples (5 µg) was carried out along with a negative control by using 'Revert Aid First strand cDNA synthesis kit' (MBI Fermentas, Hanover, MD, USA) for cDNA synthesis. Again, the concentration of synthesized cDNA samples was determined by using Nanodrop at 260 nm (NANO DROP 1000, thermo-scientific, Singapore) and thereafter, stored frozen at − 20 °C till further use.
The expression analysis of major enzymes involved in lipid metabolism such as acetyl carboxylase (ACC), fatty acid synthase (FAS), malic enzyme (ME), apolipoprotein B100 (apoB100), sterolregulatory element binding protein-1 (SREBP-1), Stearoyl-CoA (Δ9) desaturase-1 (SCD-1), Peroxisome proliferator activated receptor-α (PPAR-α), and AMP-activated protein kinase α-1 (AMPKα-1) was carried out by using their specific primers against β-actin as housekeeping gene. Oligonucleotide primer pairs used for lipid metabolism related gene expression study shown in Table 6. The primers were synthesized commercially by Integrated DNA technologies (New Delhi). The ideal conditions for each gene specific primer pair was worked out through gradient PCR in Gradient thermal cycler (BIO-RAD, USA). Real time qPCR analysis was performed by IQ5 Cycler system (BIO-RAD, Hercules, CA, USA) with SYBER Premix Ex Taq reagent Kit (TAKARA Biotech, Japan). The qRT-PCR conditions were as follows: initial denaturation at 95 °C for 15 min; followed by 40 cycles of subsequent denaturation at 95 °C for 30 s, annealing at 58 °C for 30 s, and extension at 72 °C for 30 s. All the samples were run in triplicate in nuclease-free 8 tube-strips with optically clear flat caps (AXYGEN Scientific, Inc. USA). The gene expression levels were normalized to β-actin and the results were analyzed by 2 -ΔΔCT method 41 . Chromatography and fatty acid profile. Fatty acid methyl esters (FAMEs) were prepared directly from meat samples as described by O'Fallon et al. 42 and C13:0 ME was used as internal standard. The fatty acid composition of FAMEs was analysed by capillary gas chromatograph by following the standardised laboratory protocol 43  www.nature.com/scientificreports/ The conversion of essential fatty acids (EFA)-linoleic acid (LA) and ALA was measure in terms of Δ 5 + Δ 6 desaturase index and the conversion of myristic acid (C14:0) to palmitic acid (C16:0) and further to steric acid (C18:0) was measured by thioesterase and elongase indices, respectively 46 : Health indices of meat. Various health indices, useful for evaluating the nutritional value and healthiness of the chicken meat, were also calculated from the data of fatty acid profile. The saturated fatty acids (SFA) are considered as proatherogenic and prothrombogenic fatty acids, whereas, unsaturated fatty acids (UFA) are considered as antiatherogenic and antithrombogenic fatty acids. The common health indicators of chicken meat ω-6 to ω-3 PUFA, PUFA to SFA, MUFA to SFA, and UFA to SFA ratios were calculated. The other indicators of health value of chicken meat calculated in this study were saturation index (S/P), atherogenic index (AI), thrombogenic index (TI) 46 , desirable fatty acid (DFA) content, hypercholesterolemic fatty acids (HFA), and hypocholesterolemic to hypercholesterolemic fatty acid ratio (h/H) 47 F-TGC GTG ACA TCA AGG AGA AG  R-TGC CAG GGT ACA TTG TGG TA  300  58.2  L08165   FAS   F-CTA TCG ACA CAG CCT GCT CCT  R-CAG AAT GTT GAC CCC TCC  TACC   107  55.9  J03860   ACC  F-AAT GGC AGC TTT GGA GGT GT  R-TCT GTT TGG GTG GGA GGT G  136