ABSTRACT
The objective of the present study was to evaluate the effect of bile acids derived from swine on the growth performance, carcass traits, serum lipid metabolites and intestinal enzyme activities in broiler chickens. Four hundred thirty-two 1-day-old Arbor Acres male broilers were randomly assigned to 4 treatments with 6 replicates of 18 chicks each for 42 d. The experimental treatments received a corn-soybean basal diet containing lard and were as follows: 0 (control), 40 mg, 60 mg, and 80 mg bile acids/kg of diet. Dietary of inclusion bile acids significantly increased average daily gain and decreased feed to gain ratio from d 21 to d 42 (P < 0.01). However, average daily feed intake was unaffected by dietary supplementation with bile acids. The dressing percentage and the percentage of thigh muscle in the carcass were notably (P < 0.01) higher for broilers fed diets supplemented with 60 and 80 mg/kg bile acids. In contrast, abdominal fat weight was reduced significantly (P < 0.01). In 42-day-old broilers, serum triglyceride, high density lipoprotein and low density lipoprotein concentrations were unaffected (P > 0.05) by bile acids supplementation. Supplementation with 60 and 80 mg/kg significantly increased the activity of duodenum lipase and lipoprotein lipase on d 21 and d 42, as well as decreased the activity of hormone sensitive lipase on d 42. Supplementation of diets with 60 and 80 mg/kg of bile acid can effectively enhance the activity of intestinal lipase and lipoprotein lipase and improve growth performance and carcass traits of broilers.
Key words: Bile acids, performance, carcass, enzyme activity, broiler.
INTRODUCTION
Bile acids, synthesized from cholesterol exclusively in the liver, are specific and quantitatively important organic components of bile (Marin et al., 2016). Bile acids are the primary pathway for cholesterol catabolism in mammals. Cholesterol converts into bile acids by modification in the ring structure, oxidation, and shortening of the side chain. Before bile acids are secreted into the canalicular lumen, they are conjugated with glycine or taurine. This conjugation process increases the amphipathic nature of the bile acids, making them more hydrophilic as well as less cytotoxic (Bellentani et al., 1987; Russell, 2003; Li and Chiang, 2014; King, 2014). Bile acid chemistry is complex because of the great variety of chemical structures in naturally occurring compounds such as cholic acid, chenodeoxycholic acid, deoxycholic acid, and lithocholic acid. The primary bile acids are synthesized from cholesterol in the liver and the secondary bile acids are formed by bacterial modification of primary bile acids in the colon. Bile acids usually consist of a mixture of individual bile acids. In bovines, cholic acid and deoxycholic acid are the primary bile acids, while α-hyodeoxycholic acid and chenodeoxycholic acid are predominant in swine bile. Avian bile acids mainly consist of chenodeoxycholic acid and cholic acid (Hofmann and Hagey, 2008).
Fats and oils constitute the main energy source of animals and possess the highest caloric value of all nutrients, with almost 3 times higher apparent metabolizable energy (AME) than other feedstuffs (NRC, 1994). With the continuous improvement of genetics, the nutritional requirements of modern broiler strains have increased, especially the need for high intake of energy, which necessitates the feeding of high-energy diets (Blanch et al., 1996). Hence, fats are widely added to poultry diets to meet the energy requirements. Digestion and absorption of dietary fat are poorly developed in young animals due to limited bile secretion (Krogdahl, 1985). Several studies have indicated that supplementation with bile acids or bile salts improve the utilization of dietary fat by chicks because of limited endogenous secretion (Gomez and Polin, 1976; Polin and Hussein, 1982; Kussaibati et al. 1982; Ravindran et al., 2016). Some studies tested on synthetic bile acids or bile salts (Polin et al., 1980), however, the cost limits their application in feed manufacture. Therefore, porcine bile acids have been considered in this study as a possible alternative to bile acids or bile salts due to their lower cost, applicable techniques from the gall bladder, well-defined composition, and chemical stability during storage. The effect of the porcine bile acids on the growth performance of broilers has not yet been ascertained. Therefore, the objective of this study was to evaluate the effects of exogenous porcine bile acids on broiler performance, carcass traits, serum biochemical parameters, and intestinal enzyme activity when lard was used as the only energy source in broiler diet.
MATERIALS AND METHODS
Preparation of Bile Acids
Bile acids used in this study were provided by the Shandong Longchang Animal Health Care Co. Ltd. (Dezhou, China). These were extracted from pig bile paste by a process that includes saponification, decolorization, acidification, purification, and desiccation. The bile acids were composed of hyocholic acid, hyodeoxycholic acid, and chenodeoxycholic acid, as determined by infrared absorption spectroscopy. The quantity of each type of bile acids in this product was determined by high performance liquid chromatograph (Lou, 2015). The content of hyocholic acid, hyodeoxychoic acid, and chenodeoxycholic acid was 8.00%, 70.67%, and 19.61% respectively.
Broiler Type, Diet, and Experimental Design
The Animal Welfare Committee of China Agricultural University (Beijing, China) approved the Animal Care Protocol in this experiment. A total of 432 (average body weight, 43.08 ± 1.94 g) 1-day-old Arbor Acres male broiler chicks were obtained from Arbor Acres Poultry Breeding Company (Beijing, China). On d 1, all birds were randomly assigned to 4 treatments, with 18 per cage and 6 replicate cages per treatment.
All broilers were raised in wire-floored cages in an environmentally controlled room with 23 h of lighting with ad libitum access to feed and water throughout the experiment. The room temperature was maintained at 35?C for the first 3 d, after which the temperature was gradually reduced by 3?C each wk until it reached 24?C and then was maintained at this temperature until the end of the 42-d experiment. All chicks were inoculated with inactivated Newcastle disease vaccine on d 7 and d 21 and inactivated infectious bursal disease vaccine on d 14 and d 28.
The diets consisted of a corn and soybean diet with 0, 40, 60, or 80 mg/kg bile acids, respectively. The bile acids were premixed into corn flour and then added to each diet. Lard was added to the feed as a fat source.
From d 1 to d 21, the broilers were fed the starter diets and fed the growth diets from d 22 to d 42. All diets were fed in mash form. All nutrients contained in the basal diet met or exceeded the requirements suggested by the NRC (1994). The ingredients and chemical composition of the basal diet are shown in Tables 1 and 2, respectively.
Collection of Performance Data
The mortality for each treatment was recorded daily and the average mortality was less than 1%. The body weight and feed consumption of the broilers were recorded in each pen on d 21 and d 42 after fasting for 12 h, and these values were used to calculate the average daily gain (ADG), average daily feed intake (ADFI), and feed conversion ratio of the broilers (F:G) for the periods between d 1 and d 21, d 22 and d 42, over the course of the experiment.
Slaughter and Sample Collection
On d 21 and d 42, one broiler of visually approximating the average weight from each pen was selected for blood sampling. Blood was collected by cardiac puncture using 0.8-mm-diameter needles into 10-mL anticoagulant-free vacutainer tubes. Two tubes of 5-mL blood samples were collected from one broiler chicken. The blood was allowed to clot by leaving it undisturbed at room temperature for 30 min. The blood samples were then centrifuged at 1,500 × g for 10 min in a refrigerated centrifuge. The serum was apportioned into 0.5 mL aliquots and stored at –20°C for later analysis.
After blood sampling on d 21 and d 42, one broiler per pen was bled from the jugular vein and decapitated. They were defeathered and the carcasses were weighed. Then the organs of the trachea, esophagus, crop, intestinal tract, spleen, pancreas, as well as the reproductive organs were removed. The heart, liver (without gallbladder), proventriculus, gizzard (removal of content and the inner membrane), abdominal fat, lungs, and kidneys were left intact for calculation of the semi-eviscerated weight. Further removal of the visceral organs from the semi-eviscerated carcass allowed the eviscerated carcass weight to be obtained. Abdominal fat, breast muscle, and leg muscle were collected and weighed. Abdominal fat comprises fat tissues surrounding the proventriculus and gizzard lying against the inside abdominal wall and around the cloaca (Ricard et al., 1983).
Three segments of the small intestine were collected, immersed in liquid nitrogen, and stored at –80°C for enzyme activity analysis. The segment of the duodenum was excised at the tip of the loop. The jejunum segment was collected proximal to the Meckel’s diverticulum, and the ileum segment was collected from the middle segment between Meckel’s diverticulum and the caecal junction.
Chemical Analysis
Diets were analyzed according to the methods of the Association of Official Analytical Chemists (AOAC, 2000) for total phosphorus (AOAC method 995.11), calcium (AOAC method 927.02), and crude protein (method 988.05). For dietary methionine determination, performic acid oxidation was performed prior to acid hydrolysis with 6 M HCl (AOAC method 994.12). Lysine was determined by AOAC method 994.12.
Serum triglyceride (TG), high density lipoprotein (HDL), and low density lipoprotein (LDL) concentrations were determined with commercial kits (Biosino Biotechnology and Science Inc., Beijing, China). The Automatic Biochemical Analyzer was a Hitachi 7160 manufactured by Hitachi High-Tech Corporation (Tokyo, Japan).
The activity of intestinal enzymes, including lipoprotein lipase (LPL), lipase (LPS), and hormone sensitive lipase (HSL), was determined according to the instructions provided with the commercial assay kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China).
Statistical Analysis
Data were subjected to Analysis of Variance using the GLM procedure of SAS (SAS Institute Inc., Cary, NC). The linear and quadratic effects of bile acids level were assessed using orthogonal polynomials. The pen served as the experimental unit. Results are reported as means. A P-value of less than 0.05 was considered statistically significant.
RESULTS
Performance The effect of graded levels of bile acids in diets on the growth performance is presented in Table 3. Dietary inclusion of bile acids had no effect (P > 0.05) on ADG and F:G in the starter phase, nor did it affect ADFI the starter and grower phases. However, the ADG increased and F:G decreased with an increasing level of bile acids (linear and quadratic effect, P < 0.01) in the grower phase. The inclusion of 60 and 80 mg/kg bile acids improved ADG and F:G throughout the experiment.
Carcass Characteristics
Table 4 lists the effect of graduated levels of bile acids on carcass characteristics in broilers on d 42. The dressing percent of carcass, eviscerated weight and leg weight was higher (P < 0.01) in birds fed 60 and 80 mg/kg bile acids as opposed to the control and 40 mg/kg bile acids groups (P < 0.01). Abdominal fat pad was also decreased linearly and quadratically (P < 0.01) in these treatments.
Serum Biochemical Parameters
As shown in Table 5, there were no differences in serum TG, HDL, and LDL concentrations among all groups on d 21 and d 42.
Intestinal Enzyme Activity
Day 21 duodenal LPL and LPS activities were higher linearly and quadratically (P < 0.01, Table 6) in broilers fed bile acids, regardless of level. As shown in Table 7, 42-day-old broilers supplemented with 60 mg/kg bile acids showed significantly higher activity of duodenal LPL and LPS and lower activity of HSL compared with the control and 40 mg/kg bile acids groups (linear and quadratic effect, P < 0.01). However, there was no difference between broilers supplemented with 60 and 80 mg/kg bile acids. The activity of HSL, LPL, and LPS in the jejunum and ileum on d 21 and d 42 was unaffected by dietary supplemental bile acids.
DISCUSSION
Bile acids are the principal constituents of bile and play an important role in the digestion and absorption of fat and lipid-soluble vitamins (Russell and Setchell, 1992). They are synthesized from cholesterol within hepatocytes, secreted into the bile canaliculi and subsequently stored in the gallbladder in birds and many other mammals. From here, they flow into the duodenum after ingestion of feed to emulsify dietary lipids. About 95% of bile acids are then absorbed by passive diffusion and actively transported from the ileum and to the liver via the portal vein via enterohepatic circulation (Hofmann and Hagey, 2008). In the newly hatched chick, the ability to digest and absorb dietary fat is poorly developed as a result of limited secretion of bile (Tancharoenrat et al., 2013). For this reason, synthetic bile acid and bile salts have been evaluated in diets of young broilers for the improvement of fat digestion.
Alzawqari et al. (2011) reported that dietary supplementation of tallow with ox bile at 5 g/kg resulted in higher weight gain and better feed conversation. Maisonnier at el. (2003) and Parsaie et al. (2007) found that the supplementing diets with bile salts significantly increased body weight gain in broilers. These results are consistent with our observations that dietary supplemental 60 and 80 mg/kg bile acids increased ADG and improved feed conversion in broilers, but had no effect on ADFI.
However, Piekarski et al. (2016) reported that inclusion of 0.5% purified chenodeoxycholic acid (CDCA) reduced feed intake and body weight in 3-week-old chickens. The CDCA-containing diets showed no effects on feed intake and body weight gain in early-weaned piglets when soybean oil and lard were used in the pre-starter phase and the starter phase, respectively (Diego-Cabero et al., 2015). Possibly, some of the components in natural bile acids act synergistically, and elicit an effect not seen in synthetic or purified bile acids. Furthermore, previous studies tested taurine with animal fats in one-week old chickens, but not in older birds where the effect might have occurred (Yuan and Wang, 2010; Huang et al., 2014). The most abundant bile acids in swine include chenodeoxycholic acid and αhyodeoxycholic acid, which is exclusive to swine, while chenodeoxycholic acid and cholic acid predominate in broiler biliary acids. It appears that chenodeoxycholic acid had little effect on performance in pigs, while it can decrease feed intake of broilers.
Dietary bile acids improve carcass characteristics in broilers partly through a reduction of abdominal fat. In the current study, supplementing with 60 to 80 mg/kg bile acids increased carcass weight, eviscerated weight, and leg weight, but decreased abdominal fat weight. It is interesting that bile acids can improve the absorption of dietary lipids which are not stored in abdominal fat. The abdominal fat pad is a reliable parameter for judging total body fat content because it is linked directly to total body fat content in avian species (Becker et al., 1979; Thomas et al., 1983). Suzuki et al. (2014) investigated the relationship between the level of postprandial bile acid and body fat mass after having a 400- kcal meal, and found that the postprandial bile acids are negatively related with body fat mass in healthy people with normal weight. Thus, in our study it was speculated that supplementation of bile acids may decrease fat mass and increase the percentage of the carcass weight.
Serum TG, LDL, and HDL concentrations have been regarded as diagnostic markers in lipid metabolism. The synthesis of adipose tissue, fat deposition, and the formation of yolk in poultry is dependent on available serum TG. Most fatty acids are synthesized in the liver and transported via LDL or chylomicrons for storage in adipose tissue as triglycerides (Hermier, 1997). In contrast, HDL promotes the uptake of cholesterol from peripheral tissues and facilitate the transport of cholesterol to the liver for catabolism (Miller and Miller, 1975). Alzawqari et al. (2011) observed that dietary supplemental ox bile had no effect on serum cholesterol, TG, HDL, and LDL levels during starter and growth periods, which was in agreement with our findings. These results imply that the ability to transport cholesterol from peripheral tissues to the liver was unaffected by supplemental bile acids.
Intestinal lipase activity can be an indicator of lipid utilization in animals. Bile acids are amphiphilic molecules, having hydrophobic and hydrophilic surfaces that act as detergents to reduce surface tension of lipids. These hydrophobic (insoluble) fat particles are broken into microscopic micelles to increase their solubility and hydrolyzed by lipase to convert the triacylglycerols to monoacylglycerols, diacylglycerols, fatty acids, and glycerol. LPS as well as LPL play a critical role in lipid metabolism by catalyzing the hydrolysis of triglycerides. In the present study, the activity of LPS and LPL was increased by dietary levels of bile acids (linear and quadratic effect, P < 0.01) during the starter and growth phase. Therefore, bile acids clearly play a role in lipid metabolism by elevating enzyme activity and facilitating digestion of dietary fats. HSL is an intracellular neutral lipase and is activated when the body needs energy generated from lipid mobilization. It is the rate-limiting enzyme in the degradation of triacylglycerol to diacylglycerol and free fatty acids (Duncan et al., 2007). The activity of HSL was decreased in 42-day-old broilers fed 60 and 80 mg/kg bile acids. This suggests that bile acids are improving the efficiency of fat digestion and absorption, thus decreasing the level of needed dietary fat. The proportional growth of the small intestine is greater than that of body weight within 6 to 10 d posthatch for broiler chicks (Katanbaf et al., 1988; Sklan, 2001). Nitsan et al. (1991) and Nir et al. (1993) also found that in broiler chicks, the relative growth of the intestine plateaued at approximately 8 to 15 d posthatch and a similar age associated pattern of lipase activity was observed through the first 15 d. Additionally, it was reported that the utilization of lard by chicks peaked at 42 d of age (Renner and Hill, 1960). The present study suggests that duodenum LPS and LPL activity was higher in chickens given 60 and 80 mg/kg bile acids at d 21, and 60 mg/kg bile acids at d 42, respectively.
In conclusion, dietary supplementation of bile acids from swine in broiler chicken diets can effectively improve the growth performance, and carcass characteristics, as well as modulate the activity of intestinal enzyme LPL and LPS in the duodenum. The results indicate that 60 to 80 mg/kg bile acids have the potential to improve absorption of dietary fat, carcass characteristics, and growth performance of broiler chickens.
