Impact of dietary supplemental bile salts on growth performance, carcass, immunity and antioxidant parameters and bacteriology of broiler chicks

Abstract The aim of the present study was to evaluate the effect of bile salts on the growth performance, carcase estimates, immunity and antioxidant measurements, some blood biochemical parameters, intestine enzyme activities and microbiology of broiler chickens. One hundred twenty 1 day old of Ross 308 broiler chicks were randomly assigned to 4 treatments with 5 replicates of 6 chicks each for 42 days. The first group was the basal diet only and served as control. The second, third and fourth groups were fed the basal diet plus 0.5, 1 and 1.5 ml bile salts/kg diet, respectively. Body weight and daily body weight gain were linearly (p < .01) increased with increasing bile salt levels compared to control. Addition of bile salts to broiler diets significantly (p < .01) decreased feed intake and improved feed conversion than control. The addition of 1.0 ml bile salts to broiler chicks' diets increased plasma proteins concentrations, total antioxidant capacity and superoxide dismutase compared with the control group. The cholesterol and triglyceride levels, liver enzymes and intestinal enzymes (amylase, protease and lipase) activities were significantly (p < .05 or 0.01) increased in bile salt groups. While, malondialdehyde was significantly (p < .01) decreased in all treated groups compared to control. Total bacteria, total fungi, Escherichia coli and Salmonella were decreased in broiler chicks fed diet supplemented with bile salts. It could be concluded that bile salts especially levels (0.5 and 1.0 ml/kg diet) have a positive impact on growth performance and carcass traits, blood biochemical measurements, intestinal enzymes activities, digestibility of nutrients and microorganism's content of broiler chicks. Highlights Addition bile salt to broiler diets improved growth rate and feed conversion. Addition bile salt to broiler diets improved nutrients digestion. The addition of 1.0 ml bile salts in broiler diets increased total antioxidant capacity and superoxide dismutase. Total fungi, Escherichia coli and Salmonella were decreased in chicks fed diet supplemented with bile salts


Introduction
The diversity of oils and fats must be taken into account when evaluating their nutritive value (Mohamed et al. 2019;Alagawany, El-Hindawy et al. 2020;Reda, El-Kholy et al. 2020;. Quantity and quality of dietary fats play an important role in the formation of micelle in the intestine. The secretion of gall bladder acts as a natural emulsification . The low digestibility of fat in young broiler chicks may be due to the reduced amount of bile salts or the process of recycling inactive bile salts (Leeson and Summers 2005).
Whereas, secretion of bile salts with small amount in young chicks is considered, along with a decrease in the activity of pancreatic lipase during the first 3 weeks of age. Therefore, several efforts have been made to enhance digestion of fat in poultry. Bile salts as an exogenous source play an important role in improving digestion and absorption of fatty acids.
Bile acids are composed of cholesterol and complemented with glycine or taurine during the liver pericentral hepatocytes, concentrated and stored in the gallbladder, and secreted into the duodenum where, playing an important role in improving the digestion of fat thorough bile salts dissolvable fats and due to absorption increasing (Abudabos 2014). In poultry nutrition, the worth of the several fat origins builds on adipose acids tenor, power contents, digestible coefficient and assimilation. There are many factors that affect the digestion of fats such as dietary fat sources and level of bile secretion (Reshetnyak 2013). Azman et al. (2005) reported that broiler performance was improved with improving dietary metabolisable energy (ME) when nutrition containing mishmash of palmitic and oleic acids supplemented with cholic acid. Viscosity plays an important role in hypothesis reabsorption of bile salts with no mediation the activity of microflora (Azman et al. 2005). The previous studies on bile salts and their effects on growth, antioxidant and immune measurements, digestive enzymes microbiological aspects in the caeca of broiler chickens are scarce. Thus, the present study was designed to investigate the effect of bile salts at levels of 0.5, 1.0 and 1.5 ml/kg diet, on performance, carcase traits, blood parameters, antioxidant and immune measurements, digestive enzymes, digestibility of nutrients and caecum microbiology of broiler chickens.

Design and diets
One hundred twenty 1 day old of Ross 308 broiler chicks were randomly assigned to 4 treatments with 5 replicates of 6 chicks each for 42 days. The first group was the basal diet only and served as control. The second, third and fourth groups were fed the basal diet plus 0.5, 1 and 1.5 mL bile salts/kg diet, respectively. The basal diet or the other diets contained the requirements suggested by the NRC (1994). The basal diet of different phase of experimental period is presented in Table 1. Bile salts were purchased from ICC Industrial Comercio Exportacao e Import Lemma, Sao Paulo Brazil (purchased from Total Vet Company for vet medicine, Qalyubia Governorate, Egypt). Bile salts were gently mixed with a portion of the basal diet then, mixed with all the experimental diets. The composition of bile salts is 70% bile salts, 46.9% cholic acid, 17.8% desoxycholic and 4.06% chenodeoxycholic.
Chicks were individually weighted at 1, 14, 28 and 42 days of age. Feed intake was determined weekly until marketing age on a replicate basis. Consequently, feed conversion (g feed/g gain) and body weight gain were estimated during each experimental period (1-14, 15-28, 29-42 and 1-42 days).

Blood measurements
At the end of trial period, 10 birds from each group were weighed and slaughtered to obtain blood samples. Blood samples were collected into tub with EDTA to determine some blood constituents, and then noncoagulated blood used for estimating (WBCs) White blood cells count (x10 3 /mm 3 ), lymphocyte (%), Monocytes (%) and Heterocytes (%) (Stoskopf et al. 1983).
Plasma was obtained by centrifugation of the whole blood at 3000Âg for 20 min and stored at À20 C until analysis. Total protein (g/dL) was estimated by the Biuret method according to Armstrong and Carr (1964). The concentration of albumin (g/dL) was estimated calorimetrically according to Wise (1965). Globulin concentration (g/dL) was calculated. Creatinine (mg/dL) and urea (mg/dL) levels were determined spectrophotometrically using commercial kits from Biodiagnostic Company (Giza, Egypt). Triglycerides, cholesterol were determined according to Allain et al. (1974) high-intensity lipoprotein (HDL; Myers et al. 1994), low-intensity lipoprotein (LDL; Friedewald et al. 1972) and liver enzymes activity alanine-aminotransferase (ALT) and aspartate-aminotransferase (AST) were determined by Reitman and Frankel (1957). Digestive enzymes amylase activity was estimated by the method of Somogyi (1960). Lipase activity was examined by the technique described by Tietz and Fiereck (1966). Protease activity was measured as dissected by Lynn and Clevette-Radford (1984). Total antioxidant capacity (TAC) was measured according to Koracevic et al. (2001), superoxide dismutase (SOD) was determined spectrophotometrically as described by Nishikimi et al. (1972) malondialdehyde (MDA) was measured according to Mihara and Uohiyama (1978). Gama immunoglobulin (IgG) was determined by ELISA technicality as demonstrated by Bianchi et al. (1995).

Digestibility coefficient
At the end of the study, 10 birds were used to determine the apparent digestion coefficients of each dietary nutrient and to calculate the nutritive values of the experimental diets. Birds were individually settle in metallic cage and fed their respective trail diets. The preliminary period continued for 3 days and the collection period extended to 5 days in which excreta were quantitively collected every 24 h and daily feed intake was recorded. The excreta were clean out from feathers and samples diet then weighed and dried in oven at 70 C for 36 h. Finally, grind and placed in screw top glasses till analysis. The direct analysis of diets and excreta was executed according to AOAC (2006). Faecal nitrogen also was estimated . Nutritive values were calculated as total digestible nutrients (TDN) and metabolisable energy (ME). The ME was thoughtful as 4.2 kcal per gram TDN according to Titus (1960).
The following calculations were used to determine digestibility of nutrients. The apparent faecal DM digestibility was calculated as follows: DM digestibility ¼ 100 À [100 Â (manure excretion/feed consumption)], where DM digestibility ¼ the apparent faecal digestibility of the DM contents of the diet (%). OM digestibility ¼ 100 -[100 Â (OM excretion/OM consumption)], where OM digestibility ¼ the apparent faecal digestibility of the OM contents of the diet (%).CF digestibility ¼ 100 À [100 Â (CF excretion/CF consumption)], where CF digestibility ¼ the apparent faecal digestibility of the CF contents of the diet (%). EE digestibility ¼ 100 À [100 Â (EE excretion/EE consumption)], where EE digestibility ¼ the apparent faecal digestibility of the EE contents of the diet (%). Apparent faecal N digestibility was calculated as follows: N digestibility ¼ 100 À [100 Â (N excretion/N consumption)], where N digestibility ¼ the apparent faecal N digestibility of the N contents in the diet (%). All calculations were made on a DM basis, and results are expressed on per bird, per day basis (Roberts et al. 2007;Alagawany et al. 2014).

Microbiology traits
The authors collected the caecal fresh contents and subjected to a stream of CO2 in bottles and transported to the lab. The count of total bacteria was determined via plate count agar at 30 C for 2 days. Salmonella and E. coli bacteria were determined on Eosin Methylene Blue agar plates and on XLD agar plates after incubation at 37 C for 1 day, respectively ).

Statistical analysis
Data were statically analysed by using the GLM procedure of SAS (package program, SPSS 2008). Orthogonal polynomial contrasts (linear and quadratic) were used to test the significance of the different levels of dietary bile salts.

Growth performance
Live body weight and body weight gain At 28 and 42 days old, body weight (BW) was linearly increased with the bile salt supplementation (Table 2). However, daily body weight gain (DBWG) through 14-28, 28-42 and 1-42 days old was linearly improved in chick groups fed diet plus bile salt when compared with control. On the other hand, the results of average BW at 14 days of age and DBWG during 1-14 days old were not significantly affected due to bile salts supplementation (Table 2). It could be noticed that the highest values of BW and DBWG were recorded for birds having 1.5 ml bile salts.
These results agreed with the observation obtained by Alzawqari et al. (2016) and Lai et al. (2018) who indicated that BWG improving significantly in broiler chicks fed diet plus bile acid matched with the nontreated group. However, Rezaeipour et al. (2016) indicated that bile salts preparation failed to take out any increased significantly in broiler chick's performance (LBW and BWG). The improvement in live body weight and body gain due to bile salts supplementation in broiler diets may be attributed to higher digestion coefficients of nutrients. Brz oska et al. (2013) indicated broiler growth enhanced when birds fed diet containing 0.3-0.9% organic acids. Improving digestion and absorption of lipid due to bile salts improves the bioavailability of fatty vitamins (A, D, E and K) (Stamp and Jenkins 2008).
Feed intake (FI) and feed conversion ratio (FCR) Results in Table 2 show that FI was linearly reduced (p < .0261) through 1-28 and 1 À 42 days with increasing levels of bile salts. On the other hand, during 1-42 days of age, FI was quadratically (p < .01) decreased by the levels of dietary bile salts supplementation (Table 2). FCR was linearly improved  (p < .01) throughout overall period of the trail by levels of dietary bile salts supplementation. The improvement in FCR was associated with gradually increasing bile salts from 0.5 to 1.5 ml/kg diet. It could be noticed that chickens feed diet plus 1.5 ml bile salts/kg diet recorded the best value of FCR: while, chicks fed the control diet recorded the poorest value. The reduction in feed intake and improvement in feed conversion may be related to the fatty acid content in the bile salts (Xie et al. 2013).
Our results agree with Alzawqari et al. (2016) who stated that feeding broiler on diet supplemented with bile salts improved feed conversion from 21 to 42 days of age. The chenodeoxycholic acid and cholic acid predominate in broiler biliary acids, it appears that chenodeoxycholic acid can decrease feed intake of broilers (Lai et al. 2018). Also, our findings are partially agree with El-Katcha et al. (2019) who found that laying hens fed on diet supplemented with 400 g/ton of dried bile acids with biotin increased significantly (p < .05) daily FI and improved FCR compared to the control group. On the contrary, our results disagreed with those obtained by Maisonnier et al. (2003) and Karimi et al. (2011) who stated that utilise bile salts in broiler feeds did not progress feed efficiency rate. Lai et al. (2018) found that average feed consumption did not affected by dietary inclusion bile acids. Also, Parsaie et al. (2007) showed an increase significantly in feed consumption of broiler chicks fed a diet plus bile acids.

Digestion coefficients and nutritive values
Chicks received diets contained 0.5, 1.0 and 1.5 ml bile salts/kg diet had higher and linearly (p < .0009 or p < .0001) improved digestion coefficient for OM and DM than the control (Table 3). The digestion coefficient of NFE, TDN and ME was improved linearly (p < 0.00001) with increasing bile salts level. The digestion coefficient of EE and CF was quadratically increased in chicks given 0.5, 1.0 and 1.5 ml bile salts compared with the control. The digestion coefficient of CP was linearly and quadratically improved with 0.5, 1.0and 1.5 ml bile salts. The amelioration in fat assimilation ability by extension of bile salts might be towards deficient gall salts excretion by the animal or regeneration process catabolism of gall salts by illumes bacteria (Adrizal and Yayota 2002;Maisonnier et al. 2003). Other investigators suggested another characteristic of dietary gall salts in improving the digestion of appeased fatty acids characterised long bonds (Gomez and Poplin 1976). Mostly, the enhancement of digestion coefficients by adding bile salts to the feed placed in order to non-fat nutrients turn off minimal preserved by fat droplets and after that there no need to add extra digestion enzymes. Ndelekwute et al. (2019) observed that digestibility of protein, fibre, and ether extract were significantly improved by addition of organic acids than control. Birds feeding on bile salts due to decrease intestines pH consequence the environment unsuitable for Salmonella and Kelly Basil which need pH about 7 and decrease  the damage of intestinal wall, and due to renewing intestinal epithelial cells decreases and increase villi length (Sarangi et al. 2016). The present results harmonise with Kussaibati et al. (1982) who observed that fat absorption enhanced in broiler chicks fed on diets plus bile salts. The improvement in FCR may be due to organic acids whereas, the acidic anions have been found to promote the cation absorption of minerals such as calcium, phosphorus, magnesium and zinc (Edwards and Baker, 1999;Abdel-Latif et al. 2020;Pearlin et al. 2020). Alzawqari et al. (2016) found that fat digestibility increased in broilers fed diet supplemented with bile salts.

Carcase traits
The effect of experimental additives on carcase traits is presented in Table 4. It was observed the relative weights of carcase and dressing were linearly (p < .05) increased while, giblets and liver percentages were quadratically (p < .01) enhanced by adding bile salts levels in broiler chicken's diets. The highest values of the aforementioned relative weights were recorded with 1.0 and 0.5 ml bile salts comparison with the control, respectively. The results in Table 4 showed tested bile salts levels did not affect heart and gizzard weight. The addition of bile acids to broiler diet may decrease body fat mass and increase the percentage of the carcase (Lai et al. 2018), these observation harmonised with our finding. Lai et al. (2018) found that percentages of dressing and thigh muscle were higher (p < .01) for broiler fed diets plus 60 and 80 mg of bile acids/kg diet. Bile salts added to broiler chickens diet decreased feed transit rate, improved nutrients digestion and absorption and provided amino acids in optimal position to improve carcase weight (Poorghasemi et al. 2018). Alzawqari et al. (2016) illustrated that the diets enriched bile acid did not affect carcase dressing and abdominal fat percentage of male broiler chicks at marketing age. However, adding dried bile acids (DBA) due to decrease weight of liver. On the contrary the results disagreed with those obtained by Youssef et al. (2017) who reported that percentages of carcase yield did not affected significantly of broilers chickens fed diet treated by organic acids.

Protein and lipid fractions
The effect of bile salts on total protein and its fractions are presented in Table 5. Globulin concentrations and A/G % were quadratically (p < .01 and 0.05) enhanced with bile salts trail. Total protein and albumin concentrations were numerically increased without statically significant by bile salts addition. Our finding agreed with those obtained by Natsir et al. (2017) who reported that organic acids had better effects on serum total protein and albumin compared with the control in broiler drinking acidifying water. The triglycerides (TG) values were quadratically (p < .0211) higher in groups fed diet plus various levels of bile salts than the control group, also LDL values were linearly (p < .05) increased with bile salts addition than control. While, total cholesterol (TC) and high density lipoprotein (HDL) concentrations were insignificantly increased in all experimental groups than control birds (Table 5). Similarly, Hegsted et al. (1960) showed that the serum cholesterol content of young chickens increased when fed diet containing 0.8% of cholesterol plus cholic acid (which a serious ingredient in the gall acids). Our results may be due to increase the digestion and absorption of lipids; whereas, bile acids can improve the absorption of dietary lipids which are not stored in abdominal and the ability to transport cholesterol from peripheral tissues to the liver was unaffected by supplemental bile acids fat Lai et al. (2018). Alzawqari et al. (2010) demonstrated that birds received diet inclusion 0.5% DBA had significantly higher serum triglycerides, cholesterol, LDL and HDL concentrations at 21 and 42 days old as compared 0.0%. However, Lai et al. (2018) reported that broilers at 42-day old fed diet plus bile acids unaffected (p < .05) serum concentrations of triglyceride, HDL and LDL, the synthesis of adipose tissue and fat deposition in birds is dependent on available serum triglycerides. The most of fatty acids are synthesised in the liver and transported via LDL or chylomicrons for storage in adipose tissue as TG (Hermier 1997). In the contrast, LDL elevates the uptake of cholesterol from peripheral tissues and expedites the transport of cholesterol to the liver for catabolism (Miller and Miller 1975). Contradicting results were obtained by Edwards (1962) who illustrated that laying hen fed diet plus cholic acid or lithocholic acid at grades of 0.05 or 0.025% decreased numerically cholesterol concentrations. El-Katcha et al. (2019) concluded that serum total cholesterol, TG and LDL concentrations insignificantly reduced with addition (400 g/ton) of dried bile acids with vitamin B 12 or biotin to laying hen diet compared with control.

Liver and kidney functions
Results in Table 6 indicated that liver enzymes (ALT and AST) were increased significantly in bile salts groups than the control group. Whereas, AST values were linearly and quadratically increased (p < .0043 and 0.0094), respectively, while ALT and creatinine concentrations were increased quadratically only in chicks treated with bile salts compared to the control. Dietary supplementation of bile salts did not affect total bilirubin, direct bilirubin and uric acids of broiler chicks in comparison with control. Liver as a master organ in poultry metabolism is sensible to nutritional adjustment and actions of ALT and AST in serum are generally looked as a serious index for perception the liver health. It is purified that when liver doing healthy, liver enzymes concentrations in serum will be minimise in broiler chickens (Corduk et al. 2007). Passive diffusion and active transport are principle pathways for absorption of nearly 95% of bile acids from the ileum (Hofmann and Hagey 2008). Diets contain high fat could modify the concerning enzyme picture in liver tissue and serum (Ishii et al., 2010). Our finding showed a significant increase of LDL values which elevate the uptake of cholesterol from peripheral tissues and easily the transport of cholesterol to the liver for catabolism (Miller and Miller, 1975). This mechanism may be makes fatty liver and effect on liver function especially with bile salts or fat treatments that due to increased liver enzymes concentrations. The results cleared that broiler fed diets contained bile salts increased concentricity of ALT and AST as compared to control birds. On the other hand, El-Katcha et al. (2019) stated that serum ALT and AST activities insignificantly increased in laying hens fed on diet plus 400 g/ton of dried bile acids with vitamin B12 or biotin compared with control birds. Our results were contrast with Rezaeipour et al. (2016) who found that concentrations of liver enzymes including ALT and AST were not affected by dietary bovine bile salts.  Relative lymphoid organs, some immune measurements and oxidative parameters The relative weight of spleen and thymus gland was quadratically (P ¼ 0.0162 and 0.0439) increased in birds fed diet supplemented with bile salts ( Table 7). The highest value of relative spleen weight was recorded with birds fed diet supplemented by 0.5 and 1.0 ml/kg diet, while use of 1.0 ml in broiler chick's diet enhanced the thymus relative weight compared with the untreated birds. But, tested bile salts levels insignificantly effect on bursa relative weight, WBCs, lymphocytes, monocytes and heterocytes percentages when compared to control birds (Table 7). Our results agreed with the findings obtained by Alzawqari et al. (2016) who indicated that bursa and spleen at 42 days of did not affected broiler fed diets plus bile acid, the stimulatory effect of the bile salts and acids on evolution and growth of the lymphoid organs may be perfect of improving immunity. The immune system plays important role in maintain the bird's health (Yang et al. 2018). Data in Table 7 showed that Gama immunoglobulin (IgG) was linearly and quadratically enhanced in broiler groups fed diet enriched with bile salts compared to control birds. While, white blood cells and their fractions insignificantly affected by bile salts supplementation. Bile salts which used in the present work enriched with organic acids that may be improved immune system as supported by Devi et al. (2016) found that WBC, Gama immunoglobulin level and lymphocyte % were improved in lactating sow treated with protected organic acid. Lee et al. (2017) found that organic acids had a role in the regulatory T cells of broilers chickens.
The results presented in Table 7 show improvement of MDA in chicks fed diet plus bile salts, SOD values were enhanced linearly and quadratically while, TAC values were increased quadratically (p < .0006) only in broilers fed diet inclusion different levels of bile salts compared with control birds. Our results were supported by Awaad et al. (2018) who stated that use of organic acids in broilers drinking water improved antioxidant parameters.

Digestive enzymes
Amylase enzyme activity was linearly and quadratically enhanced in groups treated with dietary bile salts, however, lipase activity only quadratically (p < .0077) increased in treated groups. The activities of protease enzymes was linearly (p < .0225) decreased in bile salts groups than the control group (Table 8). Liu et al. (2017) found that trypsin and chymotrypsin activities of intestinal tract were higher of broiler chickens fed diet enriched with organic acids. Increase activity of digestive enzymes improving nutrients digestibility. Ndelekwute et al. (2018) reported that digestibility of protein, fibre, and ether extract were significantly improved in broiler by addition organic acids to drinking water. Addition of bile salts to broiler diet increased dietary oils improved digestive enzymes activities. Jang et al. (2007) indicated that the higher activation of pancreatic trypsin, a-amylase and intestinal maltase may be due to oil supplementation. Our results harmonised with those found by Lai et al. (2018) who observed that the activity of duodenum lipase and lipoprotein lipase on 42 days old significantly increased by adding 60 and 80 mg bile acids/kg diet. Also, bile salts play an important role in digestion and absorption of lipid in small intestine and enhanced ME (Stamp and Jenkins 2008). Lipase activity in intestinal can be an indicator of utilisation of lipids in animals whereas, lipase plays a critical role in lipid metabolism. The insoluble fat molecules are broken into microscopic micelles to higher their solubility and hydrolysed by lipase to convert the triacylglycerols to monoacylglycerols, diacylglycerols, fatty acids, and glycerol by catalysing the hydrolysis of triglycerides (Lai et al. 2018).

Bacteriology
Total bacteria was linearly and quadratically reduced in broiler chicks fed bile salts diets as compared to the untreated group; while, total fungi, E. coli and Salmonella were only linearly (p < .01) decreased in broiler groups fed diet enriched with bile salts compared with the untreated group (Table 9). Bile salts can use as an antimicrobial substance, whereas Means within the same row with different common superscripts differ significantly. CFU/gm: colony for unit/gram microorganisms count was significantly decreased in broiler chickens fed bile salt diets in our experiment. Kocsar et al. (1969) stated that bile acids had a substantial function in the protection technique of the microorganism versus bacterial endotoxins. The aggregation of bile salts in the intestine related to reduce the pH intestine which inappropriate of salmonella and another microorganisms (Sarangi et al. 2016). Hassan et al. (2010) reported that broiler chicks treated by a combination of organic acids or salts decreased intestinal E. coli and Salmonella spp compared with control. Furthermore, the ability of bile acids decreased the ability of endotoxin absorption (Sheen-chen et al. 2002).

Conclusions
From our results, it could be concluded that bile salts especially levels (0.5 and 1.0 ml/kg diet) have a positive impact on growth performance and carcase estimates, blood biochemical measurements, intestinal enzymes activities, digestibility of nutrients and microorganism's content of broiler chicks.

Animal welfare statement
This work was carried out at Department of Poultry, Faculty of Agriculture, Zagazig University, Egypt. Animal care and maintenance were performed in accordance with guidelines of Egyptian Research Ethics Committee. This study was approved by the institutional committee.

Ethical approval
Animal care and maintenance were performed in accordance with the guidelines of the Egyptian Research Ethics Committee and the guidelines specified in the Guide for the Care and Use of Laboratory Animals (2011).

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