The effect of desiccated ox bile supplementation on performance, fat digestibility, gut morphology and blood chemistry of broiler chickens fed tallow diets

Abstract

An experiment was conducted to study the effects of different dietary levels of desiccated ox bile on the performance, fat digestibility, gut morphology and blood variables of broiler chickens fed tallow diet. One hundred and fifty one-day-old male broilers (Ross 308) were equally distributed into three treatments (with five replicates each) in a completely randomised design. Desiccated ox bile was added to the experimental diets during starter (7–21 day) and grower (22–42 day) periods (0.00, 0.25 and 0.50% of diet). During the experiment, the measured parameters were average body weight gain, average feed intake, average feed conversion ratio, gut morphology, serum cholesterol (Chol), triglyceride, high density lipoprotein and low density lipoprotein. During the grower and overall periods, ABWG and AFCR were significantly improved (p<0.05). Dietary supplementation of desiccated ox bile in the diet resulted in significant (p<0.05) linear increase in fat digestibility ranged from 51% (0.00% desiccated ox bile in the diet) to 84% (0.50% desiccated ox bile in the diet). Dietary desiccated ox bile supplementation increased (p<0.05) villus height, crypt depth, villus width and villus apparent surface area in the jejunum and ileum. The results of this study indicated that dietary desiccated ox bile can be used as a natural non-nutrient additive to improve broiler performance and tallow digestibility in broiler chickens at least by stimulation of morphological maturation of gastrointestinal tract.

Keywords:

• desiccated ox bile
• tallow
• broiler performance
• gut morphology
• blood variables

Introduction

Bile is a yellow-green aqueous solution in which major constituents include bile acids, cholesterol, phospholipids and the pigment biliverdin (Carey and Duane 1994; Hofmann 1994). Bile acids are synthesised from cholesterol, and conjugated to either glycine or taurine in the pericentral hepatocytes of the liver, stored and concentrated in the gallbladder interdigestively, and released into the intestine (Begley et al. 2005). The bile functions as a biological steroidal detergent that emulsifies and solubilises lipids, thereby playing a critical role in fat digestion and absorption through the intestinal wall (Begley et al. 2005). In vitro supplementation of the bile salt, taurodeoxycholic acid, has been shown to stimulate intestinal cell proliferation and prevent intestinal apoptosis. It also alleviates mucosal damage and improves survival after lipopolysaccharide induced intestinal injury. Therefore, bile acid supplementation may potentially protect the intestine from injury or infection (Perrone et al. 2010). In poultry, fat digestion is influenced by numerous factors such as bile secretion and dietary fat sources (Mossab et al. 2000; Preston et al. 2001). Bile salts secretion, required for emulsification and micelle formation in the intestine, has been found to be influenced by quantity and quality of dietary lipids and by other emulsifiers. Several studies have shown that bile salt secretion and the activity of pancreatic lipase are reduced in young chicks in which bile salt secretion appears to be the first limiting factor for lipid digestion through the first few weeks of post hatch. Atteh and Leeson (1985) concluded that the supplementary cholic acid resulted in better performance in broilers which were fed a diet containing mixture of palmitic and oleic acids through the improvement of dietary metabolizable energy (ME). Orban and Harmon (2000) also showed that the inclusion of desiccated pig bile powder in the diet improved fat digestibility in the weaned pigs. The objective of this study was to investigate the effects of dietary desiccated ox bile (DOB) supplementation in male broiler chickens fed saturated fats.

Materials and methods

DOB preparation

Fresh ox bile was collected and transferred to closed containers at –20°C immediately to prevent decomposition (Coleman et al. 1979). The concentrated DOB was prepared as recommended by Irvin et al. (1939). Typical analysis characteristics of DOB were: form, brownish to yellow powder; solubility, 100%; pH, 5.5–7.5; total ash, 15%; moisture, 5%. After negative test results for Escherichia coli and salmonella, DOB samples were milled to powder form, and then included in the experimental treatments at the rate of 0.00, 0.25 and 0.50% of the diet.

Experimental birds, diets and housing

One hundred and fifty one-day-old male broiler chicks (Ross 308) were obtained from commercial hatchery. The birds were divided into three treatments with five replicates in a completely randomised design. The experimental diets were given to the birds for two periods of the starter (7–21 days) and grower (22–42 days). Corn soybean meal mash basal diets contained 2950 Kcal/kg ME, and 5% tallow as saturated fat in starter and grower periods. Diets were formulated to meet or exceed the requirements suggested by National Research Council (NRC) (1994). Feed and water were provided ad libitum. The house temperature was initially maintained at 32°C and gradually decreased (2.5°C every week) to reach a constant temperature at 42 days of age. Birds were maintained under continuous light.

Performance measurements

The average body weight gain (ABWG), average feed intake (AFI) and average feed conversion ratio (AFCR) were determined during 7–21 and 22–42 days of age. Daily mortalities were recorded and used to correct performance criteria.

Fat digestibility

The chromic oxide marker method (Scott et al. 1976) was used to measure fat digestibility. Chromic oxide was added to the diet at the rate of 3 g/kg. At 19–21 and 40–42 days of age, excreta samples were collected twice daily (6:00 and 18:00) from all chickens. Contaminants such as feathers and scales were removed carefully and then excreta were stored in −20°C immediately to further analysis. Fat content of the diet and excreta samples were determined by Soxhlet extraction (soxtec system HT 1043 Extraction unit) according to AOAC recommendations (2006). Chromic oxide was measured using the method of Fenton and Fenton (1979).

Tissue sampling

At the end of each period, one bird in each experimental unit (five birds per treatment) was euthanised by cervical dislocation. Digesta were removed from the intestinal segments including duodenum, jejunum and ileum. Then, the intestinal segments were fixed in 10% neutral-buffered formalin for histological assay according to Girdhar et al. (2006). Morphometric indices were villus height (VH) from the tip of the villus to the crypt, crypt depth from the base of the villi to the submucosa, villus width (VW; average of VW at one-third and two-third of the villus), muscularis from the submucosa to the external layer of the intestine and the villus-to-crypt ratio (Geyra et al. 2001). Apparent villus surface area (AVSA) was calculated according to Iji et al. (2001). Morphometric measurements were performed on 15 villi chosen from the each segment, using a table of random numbers and a computer-aided light microscope image with DP2-BSW software (DP2-BSW Version 1.3).

Blood chemistry

After 4 hours of starvation, blood samples were obtained from one bird per pen into the syringes at 21 and 42 days of age. Collected blood samples were allowed to clot for 2 hours at 37°C, and then serum was decanted and stored at –20°C for later analyses (Tankson et al. 2002). Serum parameters including cholesterol (Chol), triglyceride (TG), high density lipoprotein (HDL) and low density lipoprotein (LDL) were determined using an auto analyzer system (Autolab, BT 3500, Autoanalyzer Medical System, Rome, Italy).

Statistical analysis

The data were analysed based on a completely randomised design using the GLM procedure of SAS (SAS Institute 2002). Tukey's test was used to separate the means when significant differences were observed (p<0.05).

Results and discussion

Performance

There were no significant differences for AFI, ABWG and AFCR among treatments during 7–21 days of age (Table 1). The highest ABWG was achieved by 0.50% DOB supplementation (p<0.05) at 22–42 and 7–42 days of age. For AFCR, treatments effect was significant at the grower and overall periods and 0.50% DOB treatment had the best value (p<0.05). Similar results were reported by others who showed that the addition of bile salts in diets increased weight gain significantly (Maisonnier et al. 2003; Parsaie et al. 2007). Reinhart et al. (1988) also observed that performance of pigs weaned at 21 days increased linearly with increased levels of bile salt in the diet. Other studies showed that improvements in broiler performance were due to inclusion of 0.2% colic acid in the diet (Atteh and Leeson 1985). It has been shown that the addition of bile acid in trout rainbow fish diet had some positive effects on growth performance and nutrient utilisation (Yamamoto et al. 2007; Li et al. 2008). The significant increase in ABWG of birds fed higher levels of DOB might be due to more availability of energy that is coming from more fat absorption.

Table 1.  Effect of supplemental desiccated ox bile (DOB) on performance of male broiler chickens during 7–42 days of age.

	(Periods (day))
Dietary DOB%	7–21	22–42	7–42
Average feed intake (g/bird/d)
0.00%	52.3	120.9	93.5
0.25%	53.3	123.5	95.4
0.50%	50.3	120.3	92.3
±SEM	1.07	2.67	1.77
p-value	0.16	0.68	0.48
Average BW gain (g/bird/d)
0.00%	31.9	44.4^a	39.4^a
0.25%	31.6	50.6^ab	43.0^ab
0.50%	31.7	56.3^b	46.4^b
±SEM	0.62	1.94	1.24
p-value	0.96	0.003	0.006
Average feed conversion ratio (g:g)
0.00%	1.63	2.75^b	2.39^b
0.25%	1.69	2.45^ab	2.22^ab
0.50%	1.58	2.15^a	2.00^a
±SEM	0.040	0.107	0.080
p-value	0.19	0.007	0.01
Means with no common letters differ significantly (p<0.05).

Fat digestibility

Incremental levels of DOB in the diet linearly increased (p<0.05) fat digestibility (Table 2). The increase in fat digestibility ranged from 51 (0.00% DOB of the diet) to 84% (0.50% DOB of the diet). Similar responses were obtained by others (Fedde et al. 1960; Maisonnier et al. 2003). They observed that the apparent absorbability of beef tallow was increased from 53 to 80%. Orban and Harmon (2000) also demonstrated that the inclusion of desiccated pig bile in the diets of early weaned pigs improves digestion and utilisation of dietary fat as the level of bile in the diets increased. This improvement in fat digestibility by addition of bile salt to diets might be due to insufficient bile salts secreted by the animal, replenishment of the active catabolism of bile salts by the intestinal microflora and more emulsification of saturated fats by DOB. In this regards, Young et al. (1963) demonstrated that addition of antibiotics to diets of chicks appeared to increase their fat utilisation, probably by reducing intestinal microbial populations and thereby reducing their rate of bile salt catabolism. Gomez and Polin (1976) showed that dietary bile salt enhances digestibility of saturated long chains fatty acids.

Table 2.  Effect of supplemental desiccated ox bile (DOB) on fat digestibility of male broiler chickens at 19–21 and 40–42 days of age.

	(Periods (day))
Dietary DOB%	19–21	40–42
Fat digestibility (%)
0.00%	51.92^a	59.25^a
0.25%	68.97^b	79.34^b
0.50%	78.87^c	84.39^c
±SEM	1.093	0.859
p-value	0.0001	0.0001
Means with no common letters differ significantly (p<0.05).

Gut morphology

Villus height (VH), crypt depth, VW, submucosa, muscularis thickness, villus apparent surface area and villus-to-crypt ratio were affected by DOB supplementation (Tables 3 and 4). There were no significant differences among treatments in terms of morphological traits in the duodenum. However, during 7–21 days of age, VH, crypt depth, VW, submucosa, villus apparent surface area and villus-to-crypt ratio for 0.25% DOB treated birds were greater (p<0.05) than those of other treatments in the jejunum, and in the ileum, VH, villus apparent surface area and villus to crypt ratio in the birds fed 0.50% of DOB had the higher values when compared with those of others (p<0.05). During the grower period, some morphological indices of jejunum including VH, crypt depth and submucosa thickness had the highest values for the 0.25 and 0.50% DOB treatments, respectively. However, during 22–42 days of age, VH and submucosa thickness in the ileum of birds fed 0.50% DOB were significantly higher than those of other treatments (p<0.05). Villus is one of the most important sites of nutrient absorption. It has been suggested that long villi result in increased surface area and are capable of greater absorption of available nutrients (Caspary 1992). The better performance in this study might be related to longer villi caused by higher levels of DOB as supported by others (Samanya and Yamauchi 2002; Maneewan and Yamauchi 2004). Bile acids play an important role in the defence mechanism of the macroorganism against bacterial endotoxins (Kocsar et al. 1969). Moreover, bile acids can reduce endotoxin absorbability (Sheen-Chen et al. 2002), repair physical damage to the intestinal mucosa (Kamiya et al. 2004), and inhibit noxious bacteria, such as E. coli and Clostridium botulinum (Huhtanen 1979).

Table 3.  Effect of supplemental desiccated ox bile (DOB) on intestinal morphology^a of male broiler chickens at 21 days of age.

	Villus height	Crypt depth	Villus width	Submucosa	Muscularis thickness	Villus apparent surface area	Villus-to-crypt ratio
Parameter dietary DOB%	µm					µm^2
Duodenum
0.00%	1223.75	250.63	167.50	9.35	275.63	20,619	4.93
0.25%	1255.00	276.25	166.25	10.18	277.50	20,948	4.61
0.50%	1217.50	275.00	155.63	8.44	278.75	19,127	4.46
±SEM	2.24	2.24	2.24	0.80	7.53	19,645.93	0.19
p-value	0.724	0.314	0.774	0.339	0.958	0.787	0.256
Jejunum
0.00%	790.00^a	153.13^c	238.13^c	7.12^c	222.50	12,097^c	3.51^c
0.25%	970.75^b	171.25^b	280.00^b	10.69^b	238.75	15,719^b	3.33^c
0.50%	900.00^c	153.75^c	210.00^c	10.32^b	238.50	13,861^b	4.34^b
±SEM	18.08	4.77	8.85	0.51	6.34	6533.32	0.11
p-value	0.0001	0.032	0.0004	0.0005	0.219	0.007	0.0001
Ileum
0.00%	691.25^c	126.25	212.50	8.25	223.00	7448^c	2.84^ab
0.25%	605.00^c	143.75	225.63	8.50	209.00	8678^ab	2.70^c
0.50%	697.50^b	142.80	213.13	8.73	235.38	9805^b	3.55^b
±SEM	18.49	6.31	10.33	0.61	18.84	4485.67	0.19
p-value	0.003	0.130	0.610	0.855	0.624	0.01	0.02
Means with no common letters differ significantly (p<0.05).
^aAll measurements were done under 5xobjective lens.

Table 4.  Effect of supplemental desiccated ox bile (DOB) on intestinal morphology^a of male broiler chickens at 42 days of age.

	Villus height	Crypt depth	Villus width	Submucosa	Muscularis thickness	Villus apparent surface area	Villus-to-crypt ratio
Parameter dietary DOB%	µm					µm^2
Duodenum
0.00%	1345.00	283.13	145.00	9.70	312.50	28,779	4.80
0.25%	1357.50	290.63	136.88	8.70	305.00	29,007	4.86
0.50%	1346.25	285.63	138.13	8.68	309.38	26,892	4.80
±SEM	25.43	20.66	6.39	0.59	7.21	14,601.66	0.32
p-value	0.770	0.688	0.636	0.401	0.765	0.548	0.808
Jejunum
0.00%	1167.50^a	339.38^b	130.00	8.63^c	323.75	15,045	3.50
0.25%	1362.50^b	313.75^b	131.88	10.25^a	330.63	16,173	3.86
0.50%	1157.50^a	263.75^a	134.378	11.60^b	314.06	15,570	4.41
±SEM	49.06	12.34	6.73	0.29	6.57	12,669.13	0.24
p-value	0.020	0.003	0.899	0.0001	0.241	0.823	0.061
Ileum
0.00%	807.81^a	239.06^a	101.72^a	8.00^a	301.50	8225^a	3.38
0.25%	878.75^a	279.38^b	115.88^b	7.69^a	334.00	10,160^b	3.17
0.50%	992.51^b	273.13^ab	98.13^a	10.38^b	334.70	10,181^b	3.81
±SEM	35.72	10.22	2.96	0.20	10.41	4166.39	0.17
p-value	0.011	0.035	0.003	0.0001	0.071	0.016	0.166
Means with no common letters differ significantly (p<0.05).
^aAll measurements were done under 5x objective lens.

Blood chemistry

No significant differences in blood Cholesterol (Cho), TG, HDLs and LDLs among birds fed diets containing levels of DOB during starter (7–21 days) and grower (22–42 days) periods was observed (Table 5). However, the serum cholesterol (Chol) content was numerically decreased in the birds fed 0.50% DOB during 7–21 days of age (p>0.05). The possible reason for this reduction might be due to lower needs for cholesterol synthesis observed in 0.50% DOB supplementation. Similarly Edwards et al. (1962) reported that inclusion of 0.05 or 0.025% cholic acid or lithocholic acid in the laying hen diet numerically decreased Chol levels. Crespo and Esteve-Garcia (2003) reported that when diets contain higher tallow than those of diets with sunflower and linseed oils, blood cholesterol might be increased due to higher need for bile salt synthesis. Regarding the importance of cholesterol for bile acid synthesis in the liver, both fat digestion and absorption might be dependent on bile secretion rate. Furthermore, higher cholesterol concentration in the serum might be parallel to increase in bile secretion to the duodenum caused by more tallow entered to this area and thus improvement in fat digestibility might be seen. In fact, lower bile secretion may reduce the absorption of particularly dietary saturated long chain fatty acids in the gut of broiler chickens (Ketels 1994). The present study revealed a significant linear relationship (p<0.05) between dietary DOB concentration and fat digestibility in the broiler chickens.

Table 5.  Effect of supplemental desiccated ox bile (DOB) on blood chemistry of male broiler chickens at 21 and 42 days of age.

	(Periods (day))
	21				42
Parameter	Chol	TG	HDL	LDL	Chol	TG	HDL	LDL
Dietary DOB%	(mg/dl)
0.00%	113.3	75.3	68.0	29.7	52.3	36.0	39.7	15.1
0.25%	112.0	80.3	56.0	35.4	54.3	25.7	25.7	17.4
0.50%	104.0	82.7	55.7	29.7	72.7	27.7	55.1	22.7
±SEM	9.67	15.74	3.67	4.88	6.91	6.60	7.84	1.85
p-value	0.770	0.945	0.091	0.657	0.151	0.537	0.098	0.063
Means with no common letters differ significantly (p<0.05).

Conclusions

Under the conditions of this study, it was concluded that DOB can be used as a natural non-nutrient additive in broiler diets to improve performance in broiler chickens. The effects might be achieved through the changes in fat digestibility and gut morphology of broiler chickens fed dietary DOB.

Acknowledgements

This study was supported by the Excellence Center for Animal Sciences and Department of Animal Science, Faculty of Agriculture, Ferdowsi University of Mashhad, Mashhad, Iran.