Supplementation of Exogenous Bile Acid, Emulsifier, and their Combination Enhance Nutrient Digestibility and Growth Performance Without Negatively Influencing Blood Metabolites of Commercial Broiler Chickens

ABSTRACT

The objective of this experiment was to check the effect of supplementation with exogenous bile acid (BA), emulsifier (Em) and their blend on performance parameters, fat utilization, relative organ weights, and blood lipid profile in growing broilers. A total of 800 day-old broiler chicks were procured from a local hatchery and distributed into four experimental treatments in completely randomized design, with ten replicates with twenty birds each. The treatments were: Control (CON) (basal diet without any Em or BA), CON supplemented with 0.03% Em, CON supplemented with 0.025% BA and CON supplemented with a blend of Em (0.03%) and BA (0.025%). Starter (0-7), grower (8-21) and finisher (22-35) diets were offered to birds for 35 days. Body weight gain, feed intake and feed conversion ratio were recorded weekly. On days 21 and 35, nutrient digestibility was assessed using the acid-insoluble ash method. Blood lipid profiles and internal organ weights were measured at the end of the trial. The results of digestibility assays carried out on days 21 and 35 showed that supplementing BA alone, Em alone or their combination increases (p<0.05) body weight gain and improves (p<0.05) feed conversion ratio on days 7 to 21, days 21 to 35, and days 0 to 35. Results also showed that supplementation of BA, Em and their blend in the diet improve dry matter and fat digestibility (p<0.05), while the level of low-density lipoprotein decreased (p<0.05) in blood. Furthermore, the treatments did not influence carcass and organ weight (p>0.05); however, BA, Em and their blend increased the weight of the spleen (p<0.05). In conclusion, BA, Em and their combination enhance growth performance and nutrient digestibility in broilers without negatively affecting lipid metabolism.

Keywords:
Bile acid; emulsifier; combination; broiler; nutrient; digestibility; growth

INTRODUCTION

In broiler production, the energy of the diet is considered one of the main factors that influence body weight gain (BWG), feed conversion ratio (FCR), blood metabolites and carcass characteristics (Anwar et al., 2023; Liaqat et al., 2023; Rasool et al., 2023). To fulfill the dietary energy requirements, lipids are usually incorporated into the diet of fast-growing broilers (Attia et al., 2021; Oketch et al., 2023). However, it has been reported that lipid utilization is lower in young birds due to inefficient digestion and deficient endogenous emulsifiers and bile acids (BA), which are necessary for proper fat digestion (Abudabos, 2014; Kamran et al., 2020; Shahid et al., 2021a). It has also been reported that at a young age, the gastrointestinal tract of broilers is immature and bile acid and lipase production are insufficient (Noy & Sklan, 1995; Shahid et al., 2020), hindering the digestion of lipids at early ages. To address these issues, exogenous emulsifiers and BA are supplemented in the diets of broilers to support fat digestion, particularly during the initial stages of growth.

Numerous studies have indicated that major emulsifiers have hydrophilic and hydrophobic components that decrease water and fat surface tension, enhance emulsification, and reduce fat chylomicrons, hence increasing fat absorption in the intestine (Singh et al., 2009; Jansen et al., 2015; Hu et al., 2019; Sizova & Ryazantseva, 2022). It has also been reported that adding an emulsifier to the diet of poultry compensates the deficiency of lipase and BA in birds and enhances the utilization of fat (Rovers & Excentials, 2014; Jansen et al., 2015).

Natural as well as synthetic emulsifiers are used in poultry nutrition. Natural emulsifiers include BA and salts such as cholic and chenodeoxycholic acids, lecithin, taurocholate, casein, and phosphatide. These emulsifiers are mainly synthesized in the body of animals and obtained after slaughter processes (Soares & Lopez-Bote, 2002). Bile and its salts are particularly known to decrease oil-water interface tension and help to activate pancreatic lipase, but they also protect against enzyme denaturation as lipase enters into contact with emulsified fat droplets (Boesjes & Brufau, 2014; Xu, 2016). Synthetic emulsifiers, the other type of emulsifiers, include lysolecithin, lysophosphatidyl choline and mono and polyoxyethylene glycol dioleates. These synthetic emulsifiers are recognized as liver function boosters, and they also enhance bile duct function, which increases weight gain, FCR, and nutrient digestibility (Zhang et al., 2011; Viñado et al., 2020).

Nowadays, in broiler diets, various types of emulsifiers and BA are being added to enhance the digestion and utilization of fat (Siyal et al., 2017; Orinetha et al., 2022; Gul & Alsayeqh, 2023). For example, Lammasak et al. (2019) demonstrated that incorporating exogenous BA in broiler diets could enhance their digestibility and poultry performance. Similarly, other studies have examined the impact of BA supplementation of up to 0.04% in growing broilers’ diets and reported that BA supplementation not only improves daily BWG in broilers but also improves the FCR and carcass yield (Lai et al., 2018a; Lai et al., 2018b). Moreover, the result of a recent research by Shahid et al. (2021b) demonstrates that supplementation of exogenous synthetic emulsifier (lysophospholipid) to broilers’ diets between days 0-35 and 21-35 resulted in the highest experimental body weight and improved FCR. Various other studies reported that the addition of exogenous synthetic emulsifiers (lysophosphatidylcholine, lysolecithin, mono oxyethylene glycol dioleates and polyoxyethylene glycol dioleates, etc) in the diet of poultry improves weight gain, FCR and digestibility of nutrients (Upadhaya et al., 2018; Viñado et al., 2020).

Prior studies have examined the impact of either exogenous emulsifiers or BA supplementation on growth, digestion, FCR and bird performance. However, no studies have used both exogenous emulsifier and BA in combination to evaluate their impact on broiler performance throughout productive life. However, a study of Upadhaya et al. (2018) showed that supplementing 0.05 to 0.10 % of emulsifier blends in the diet of broiler diets improves the digestibility of nutrients as well as the weight gain in broilers. Saleh et al. (2020) also showed that supplementation of exogenous emulsifier blends (lysophosphatidylcholine, phosphatidylcholine, and polyethylene glycol ricinoleate) in the diet of broiler improves FCR and positively influences meat and carcass quality and serum lipid profile. Therefore, it could be hypothesized that the simultaneous supplementation of exogenous emulsifier and BA could enhance the nutrient digestibility of a dense diet and the growth performance of broilers. Therefore, the current study aimed to evaluate the influence of the supplementation of exogenous BA, emulsifier, and their combination on the performance parameters, fat utilization, relative organ weights, and blood lipid profile of growing broilers.

MATERIALS AND METHODS

The current study was executed at R&D Farms of Sind Feeds and Allied Products (Pvt. Ltd) Karachi for 35 days. All the procedures and plans of the current experiment related to management, welfare and bird care were reviewed and duly approved by the Synopsis Evaluating Committee of the Faculty of Animal Husbandry, Institute of Animal and Dairy Sciences, University of Agriculture Faisalabad and Director, Directorate of Graduate Studies, University of Agriculture Faisalabad, Pakistan (DGS/25249-52), dated 30/06/2022. Furthermore, birds were provided ad libitum access to feed and water, and the welfare of the birds was carefully considered following the guidelines for adequate care of the animals (Care IoLARCo, 1986). The study was reported according to the guidelines of Percie et al. (2020). Moreover, all birds were reared and transported under conditions specified in the ‘Punjab Animal Health Act 2006’.

Experimental plan, design, birds, and diets

In the current study, a total of 800 one-day-old Ross 308 male birds were randomly distributed into four experimental treatments, with each experimental treatment comprising 10 replicates, and each replicate containing 20 birds. This sample size was selected to ensure adequate representation and reliable results. Four experimental diets were formulated, considering the pre-starter (0-7), grower (8-21) and finisher (22-35) stages. The four treatments were: Control (CON) formulated without any emulsifier and BA, CON supplemented with 0.03% emulsifier, CON supplemented with 0.025% BA, and CON supplemented with a blend of emulsifier and BA (0.03% emulsifier + 0.025% BA). The emulsifier and BA were supplied by Alimentier Pvt. Ltd. Karachi, Pakistan. The BA was a product of Shandong Longchang Animal Health Care Co. Ltd. (Dezhou, Shandong, China), containing 8.00% hyocholic acid, 19.61% chenodeoxycholic acid, and 70.67% hyodeoxycholic acid. The emulsifier (lysolecithin) was a product of Kemin Animal Nutrition and Health, Belgium (Lysoforte®).

Experimental birds were reared in an environmentally controlled house in experimental pens with similar dimensions (1.86 × 1.66 m2). House temperature was maintained throughout the experiment. For the first week, the temperature of the shed was maintained at 33 ± 1°C, with a weekly decrease of 3°C until it reached 24°C. Then the temperature of the house was kept at 24°C for the rest of the experimental period, following NRC (1994). Relative humidity was also maintained at 60% throughout the experimental period. Fluorescent lights provided 24 h/d artificial lighting to promote access the feeder and drinker freely, along with the regulation of feed and water intake. Constant availability of water and feed to the broilers was ensured throughout the experimental trial. Experimental diets were provided in a crumbled form, prepared in a commercial feed mill (Sind Feed and Allied Products (Pvt. Ltd) Karachi) according to the guidelines of NRC (1994). The chemical composition and ingredient levels in the feed are summarized in Table 1.

Table 1
The ingredients and chemical composition of diets (as-fed basis).

Ingredients (%)	Starter feed (0-7 days)	Grower feed (8-21 days)	Finisher feed (22-35 days)
Maize	55.50	62.87	65.50
Soybean meal 46%	30.00	25.00	24.34
Fish meal 55%	0.00	4.00	4.00
Poultry by-product meal 53%	4.00	4.00	4.00
Canola meal 35%	8.00	2.00	0.00
Chips limestone	0.90	0.90	0.50
Bone meal	0.52	0.00	0.00
Poultry oil	0.00	0.25	0.75
Premix*	0.10	0.10	0.10
Lysine HCL	0.24	0.20	0.17
L Met	0.28	0.30	0.27
L Thr	0.08	0.08	0.04
1Phytase (10000 FTU/Kg)	0.02	0.02	0.02
NaHCO3	0.10	0.07	0.11
NaCl	0.26	0.21	0.20
Calculated Composition
Dry matter	88.45	89.25	87.78
Metabolizable energy kcal/kg	3000	3100	3200
Crude protein	23.05	21.50	19.50
Digestible lysine	1.25	1.15	1.02
Digestible Met	0.51	0.47	0.43
Digestible Thr	0.84	0.77	0.68
Calcium	0.96	0.87	0.78
Available phosphorus	0.48	0.44	0.39
Sodium	0.22	0.22	0.20
Chloride	0.23	0.23	0.23

• Premix*= Provided per kg of premix:10,000 IU of retinol, 1100 IU of calciferol,11 IU tocopherol, 1 mg phytonadione, 2 mg B1, 5 mg B2, 12 mg B5, 2.2 mg pyridoxin (B6), 0.11 mg biotin, 1.55 mg folacin, 12 µg vitamin B12, 250 mg choline chloride, 44 mg niacin (B3), 50 mg zinc, 60 mg manganese, 5 mg coper, 0.1 mg cobalt, 0.3 mg Iodin, 30 mg ferrous and 1 mg selenium; 1Phytase was obtained from Qingdao GBW Group Co., Ltd. (Shandong, China).

Experimental procedure, sampling methods and analysis

To calculate feed intake (FI), BWG, and FCR, experimental birds from each replicate and feed from each replicate were weighed on days 0, 7, 14, 21, 28, and 35. For this purpose, the quantity of feed offered to each pen at the onset of the week and after the week was measured. Later, a calculation was executed to evaluate weekly FI by subtracting the total feed left from the whole amount provided (Truong & Van Thu, 2023). The body weight of the day-old chicks was also recorded, and they were weighed again at the end of each week. On days 7, 21 and 35, average weight gain was determined. The data regarding FI and BWG were used to calculate FCR as described by Mehmood et al. (2023) and Yu et al. (2024). Mortality data were recorded for each replicate throughout the experiment. Mortality checks were conducted twice daily, and any dead birds’ weights were recorded.

Two digestibility assays were conducted to determine nutrient digestibility on the 21st and 35th days of the experiment. Acid insoluble ash (AIA) as an external marker was mixed into experimental diets at a concentration of 1% for four days before the collection of ileal digesta. On days 21 and 35, two chicks per replicate were slaughtered, and the fecal samples (ileal content) were picked from the ileum (Meckel’s diverticulum to the ileocecal junction) by gently squeezing them into sealed bags as described in previous studies (Aziz ur Rahman et al., 2021; Satti et al., 2024). These samples were then saved by freezing at −20°C till further chemical analysis. The collected samples (feed and digesta) were ground using a 0.5 mm screen. Moisture, nitrogen, and fat were measured following the methods of AOAC (2000), as described by Anwar et al. (2023) and Boonmee et al. (2024). Nitrogen and fat were quantified using the Kjeldahl and Soxhlet apparatuses, respectively. Acid insoluble ash was determined by ashing the samples and then treating them with boiling HCL (Farooq et al., 2022; Rahman et al., 2023).

Digestibility was calculated according to the equation:

$\%Digestibility=\left(1-\frac{\%mar\ker infeed\times \%nutrientinilealcontent}{\%mar\ker inilealcontent\times \%nutrientinfeed}\right)\times 100$

To assess relative organ weight, 10 birds from each replicate with similar average body weights were selected at 35 days of age. After a 4-hour feed deprivation period, the birds were sacrificed by humane slaughter following Islamic guidelines, as described by Awan & Sohaib (2016). In brief, a quick incision on the anterior part of the neck was performed to sever the carotid arteries, jugular veins, windpipe and esophagus, leading to extensive bleeding and resulting in rapid and painless death in just a few seconds. Abdominal fat, breast muscle, spleen, gizzard, and liver were weighed after slaughter. In brief, immediately after slaughter, the trained attendant extracted the organs and weighed and expressed the weight as a percentage of body weight. On day 35 of the trial, one healthy bird per replicate was randomly selected to obtain blood samples. One bird per replicate was the minimum sample size necessary for statistical significance and to reduce any potential stress or suffering, following ethical guidelines. To collect the blood samples from the brachial wing vein (approximately 1 mL/broiler chicken), sterile syringes in vacuum tubes comprising no additives were used. The samples were then centrifuged at 3,000 × g for 15 min at 4°C to obtain the serum that was then stored at −20°C. Harvested serum was further analyzed for low-density lipoprotein (LDL), total cholesterol, triglyceride, and high-density lipoprotein using spectrophotometry.

Statistical analysis

The data obtained from the current study was analyzed by one way ANOVA employing the SPSS software (version 23, SPSS Inc., Chicago, IL). The data was also examined for normality and homogeneity of variance before statistical analysis. The experimental design was completely randomized, with replicates as the experimental units. Statistical differences between experimental treatments were compared using Tukey’s multiple comparison test. A statistical difference at p<0.05 was considered significant.

RESULTS

Growth performance

The observed results for supplementation of BA and emulsifier alone or blended on FI, and growth performance of broilers are presented in Table 2. Results of FI and growth performance showed that supplementation of the diet with BA, emulsifier, or their blend showed no significant impact on total FI, BWG and FCR during days 0-7 of the experiment (p>0.05). However, incorporating the basal diet with BA, emulsifier, or their blend improved BWG and FCR on the grower period from days 7 to 21, the finisher period from days 21 to 35, and the the overall period, from days 0 to 35 (p<0.05). On the other hand, FI was similar among the control, BA, emulsifier, and their blend groups in starter, grower, finisher and overall phases.

Table 2
Effect of dietary supplementation of bile acid (BA) and emulsifier alone or in combination on the growth performance of broilers.

	Control	Emulsifier	Bile Acid (BA)	Emulsifier +BA	SEM	*p-value
D 1-7
Feed intake	193.98	195.01	195.57	196.21	2.03	0.512
Body weight gain	161.99	162.95	167.00	166.00	1.97	0.069
Feed conversion ratio	1.198	1.196	1.171	1.182	0.023	0.510
D 7-21
Feed intake	920.01	913.11	910.31	889.00	14.98	0.317
Body weight gain	591.31b	607.49ab	621.01ab	630.59a	10.03	0.003
Feed conversion ratio	1.560a	1.503b	1.466c	1.410c	0.017	<0.001
D 21-35
Feed intake	1558.79	1555.99	1541.89	1531.32	14.78	0.233
Body weight gain	944.10b	967.98ab	999.10a	1001.71a	13.98	0.003
Feed conversion ratio	1.651a	1.607ab	1.543bc	1.529c	0.016	<0.001
Overall
Feed intake	2738.90	2721.10	2722.60	2711.99	32.21	0.231
Body weight gain	1759.01c	1799.90bc	1849.01ab	1865.10a	12.98	<0.001
Feed conversion ratio	1.557a	1.512b	1.472c	1.454c	0.011	<0.001

• *^abc indicate significant differences within the same row (p<0.05); N=800

Nutrient digestibility

Table 3 shows the results of the nutrient digestibility of broilers on day 21 and day 35 of the experiment. The outcomes indicated that supplementation of the basal diet with BA, emulsifier or their blend did not affect crude protein digestibility on either day 21 or day 35 (p>0.05). However, supplementation of the basal diet with BA, emulsifier or their combination improved dry matter and fat digestibility on day 21 and day 35 (p<0.05).

Table 3
Effect of dietary supplementation of bile acid (BA) and emulsifier alone or in combination on the nutrient digestibility of broilers.

Items (%)	Control	Emulsifier	Bile Acid (BA)	Emulsifier +BA	SEM	*p-value
D 21
Dry Matter	73.25b	74.09ab	74.77a	75.73a	0.57	0.032
Crude Protein	70.87	71.92	71.19	72.32	0.79	0.443
Fat	79.63c	80.32b	80.13b	82.32a	0.82	0.037
D35
Dry Matter	72.00c	74.39b	74.31b	75.33a	0.31	0.039
Crude Protein	71.78	72.55	72.33	72.26	0.78	0.335
Fat	81.32c	82.89b	82.93b	83.24a	0.93	0.039

• *^abc indicate significant differences within the same row (p<0.05); N=800

Blood serum lipid profile

Table 4 shows the findings on the serum lipid profile of broilers fed the diet supplemented with BA, emulsifier or their combination. Results showed that supplementing the basal diet with BA, emulsifier or their blend did not affect total cholesterol, triglyceride and high-density lipoprotein (HDL) (p>0.05). Moreover, an elevated level of LDL was found in the group fed the emulsifier and BA (p=0.051) blend compared to the other experimental and control diets.

Table 4
Effect of dietary supplementation of bile acid and emulsifier alone or in combination on the serum lipid profile of broilers.

Items	Control	Emulsifier	Bile Acid (BA)	Emulsifier +BA	SEM	*p-value
D 35
Cholesterol (mg/dL)	143.23	146.13	147.91	151.23	3.21	0.104
Triglyceride (mg/dL)	87.21	91.67	92.34	91.92	4.2	0.124
HDL (mg/dL)	100.67	101.07	104.92	106.24	3.81	0.195
LDL (mg/dL)	39.07	36.7	35.30	34.30	2.2	0.051

• HDL high density lipoproteins; LDL low density lipoproteins; N=40

Relative organ weights

Table 5 shows the results of the relative organ weights of experimental birds fed a diet supplemented with BA, emulsifier or their combination. The results show that supplementation of BA, emulsifier or their blend had no significant impact on the breast muscle, gizzard, and bursa of Fabricius of birds (p>0.05). Furthermore, supplementation of BA, emulsifier or their combination had no significant effect on the abdominal fat of birds (p>0.05) between the experimental treatments and control group. Better liver % was observed in birds fed BA and emulsifier alone or in combination (p<0.05) in comparison to the other control group. Supplementation of BA, emulsifier or their combination decreased (p<0.001) spleen weight.

Table 5
Effect of dietary supplementation of bile acid and emulsifier alone or in combination on the relative organ weight of broilers at day 35.

Items (% of BW)	Control	Emulsifier	Bile Acid (BA)	Emulsifier +BA	SEM	*p-value
Breast muscle	18.03	18.32	18.34	18.62	0.33	0.324
Liver	2.70b	2.87a	2.86a	2.85a	0.07	0.037
Bursa of Fabricius	0.08	0.11	0.10	0.07	0.001	0.216
Abdominal fat	3.57	3.49	3.63	3.73	0.08	0.106
Spleen	0.14a	0.07b	0.07b	0.08b	0.01	0.001
Gizzard	1.07	1.00	0.98	0.99	0.03	0.300

• *^ab indicate significant differences within the same row (p<0.05)

DISCUSSION

Various kinds of feed additives are used in livestock production to improve feed intake (Arief et al., 2023; Truong et al., 2023), growth performance (Sengul & Ak, 2024; Boonmee et al., 2024), nutrient digestibility (Ghany et al., 2023; Gul & Alsayeqh, 2023; Pazla et al., 2024), gut health, immunity, overall health and meat quality (Susalam et al., 2024). Among these, emulsifiers and BA have been studied individually in the diet of broilers and reported to enhance growth performance, FCR, nutrient digestibility, and carcass characteristics. However, in our study, both emulsifier and BA were added to broilers’ diets at all three growth phases, either alone or in combination. Results of our study indicated that supplementation of emulsifier and BA, either alone or in combination, did not influence FI, BWG, and FCR during the first week of life, which may be attributed to low endogenous bile secretion in young broilers. However, supplementation of emulsifier and BA either alone or in combination during the grower, finisher, and overall phases had a positive influence on BWG and FCR. This positive change may be due to an enhanced lipid digestion, leading to better energy utilization (Wang et al., 2006). The concept of adding an emulsifier to the diet of broilers at an early age is usually adopted because bile secretion in young broilers is limited, especially during their first week of life, which results in decreased fat digestion, growth, and feed efficiency in birds (Tancharoenrat et al., 2013). Interestingly, in this study, the supplementation of BA, emulsifier and their combination did not affect FI, weight gain and FCR during the first week, contradicting the results of prior studies reporting that broilers at an early age cannot replenish BA as efficiently as older birds, with that low quantity of bile resulting in lower fat absorption and bird performance (Serafin & Nesheim, 1970; Shahid et al., 2021). Other researchers also reported that adding emulsifier and BA into broilers’ diets during early ages enhances fat digestion and absorption (Fedde et al., 1960; Alzawqari et al., 2011; Shahid et al., 2020). The discrepancy in the results during the first weeks of life in our study may be due to the short period (0-7 days) attributed to this category in it, whereas previous research considered early life to be from 0-21 days (Zhao & Kim, 2017). Another possible reason could be the absence of oil in the diet during the first week, as indicated in Table 1, which might have prevented optimal emulsifier and BA activity. However, during days 7 to 21 and 22 to 35, when the diet included oil, these supplements effectively improved BWG and FCR.

Although BA alone showed better results for weight gain and FCR compared to emulsifier, the best results were seen in diets that had a combination of both emulsifier and BA. Alzawqari et al. (2011) reported better weight gain and FCR with bile salt supplementation in a broiler diet. Other research has also reported that the addition of bile salts in the basal diet of broilers increases body weight, supporting our findings that dietary supplementation of BA increases BWG, improves feed conversion, and has no influence on daily FI (Lai et al., 2018a). The improved results with emulsifier supplementation on weight gain and FCR in this study align with Wang et al. (2016) and Zhao & Kim (2017), who reported that exogenous emulsifier enhance these parameters in broilers. Notably, the combination of emulsifier and BA yielded the best results, supporting the notion that emulsifier improves fat digestion’s initial step by breaking down fats into microdroplets, while BA activates lipase and further promotes fat digestion. Despite limited scientific research, our results suggest that combining emulsifier and BA enhances growth performance parameters.

Additionally, this study observed improved dry matter and fat digestibility by supplementing a combination of emulsifier and BA in the diet of the broiler on days 21 and 35. This outcome was anticipated, as both components are known to enhance fat digestion and absorption, thus improving growth performance (Maisonnier et al., 2003; Alzawqari et al., 2011; Shahid et al., 2020; Shahid et al., 2021). Upadhaya et al. (2018) discovered that a blend of emulsifiers not only boosts dietary fat utilization efficiency but also improves dry matter and fat digestibility in broilers. Similarly, another recent study has reported that adding glycerol polyethylene glycol ricinoleate to broiler diets improved fat and nutrient digestion (Roy et al., 2010). Therefore, it could be speculated that BA and emulsifier worked synergistically in the current study and increased fat and dry matter digestibility. Upadhaya et al. (2018) documented a significant association between dry matter and fat digestibility, supporting the current study’s results that both parameters increased with BA and emulsifier combination supplementation in the diet, contributing to better growth performance in broilers. Improved fat digestibility led to high energy (Arshad et al., 2020), which may influence FI. In the current study, FI remained unchanged, which could be explained by the higher body weight of the birds after improved fat digestibility and FCR, which subsequently raises birds’ energy requirements for maintenance, with additional energy availability being primarily redirected to maintenance, thus not influencing the FI. The other possible reason could be that improved fat digestibility, while enhancing energy availability, might not directly affect blood sugar levels, which play a key role in regulating FI (Kumar et al., 2021). High-concentration diets are recommended for fulfilling the nutrient requirements of high-genetic-potential and growing broilers, though they can affect blood glucose and lipid profiles (triglycerides, cholesterol, HDL, LDL) (Upadhaya et al., 2018). These diets can negatively impact performance and health. Supplementation with BA and emulsifier in combination may mitigate these negative effects, as documented in the previous study by Roy et al. (2010), which showed that supplementation of glycerol polyethylene glycol ricinoleate in the diet of broilers reduces LDL and total cholesterol, while improving performance. In this study, supplementation with BA and emulsifier alone or in combination significantly reduced LDL concentration, and the combination had the most pronounced effect. This result aligns with the findings of Upadhaya et al. (2018), who reported that emulsifiers reduce circulating LDL by enhancing fat utilization. Thus, it can be inferred that BA and emulsifier supplementation in combination positively impacts digestibility and overall bird health. Although LDL levels decreased with these supplements, cholesterol, triglycerides, and HDL were not significantly affected, despite increased fat digestibility. The improved growth rate, average daily gain, and FCR observed in the current study suggest an efficient nutrient use, particularly of fats, when exogenous BA and emulsifier are included alone or in combination in the diets of broilers.

Relative body organ weight results demonstrate that the addition of BA, emulsifier or their combination does not influence gizzard, breast muscle, and abdominal fat. However, the current study demonstrated that dietary supplementation of BA, emulsifier, and their combination in the diet of broilers had an influence on the weight of the spleen and liver. Moreover, the relative weight of the spleen was decreased, while the relative weight of the liver was enhanced with the supplementation of BA, emulsifier, and their combination in the diet, which may be due to the higher metabolic activity associated with improved lipid digestion. Roy et al. (2010) and Zhao & Kim (2017) also reported that supplementation of BA or emulsifier has no effect on the internal organs, which is similar to the findings of our study. However, no reduction in spleen weight was observed in any of the previous studies. This finding raises concerns about potential immunological effects, as the spleen plays a crucial role in immune function. The possible reason for decreased spleen weight could be the higher efficiency in utilizing fat for energy, especially with feed containing both fat and emulsifiers (Nobakht, 2012). A previous study by Upadhaya et al. (2018) has demonstrated that a blend of emulsifiers could reduce the weight of the spleen, although the underlying mechanisms remain unclear. It is well known that reduced weight of the spleen may influence spleen functions, resulting in immunosuppression and health problems in broilers. Thus, the reduction of spleen weight in the current study due to the supplementation of BA, emulsifier and their combination may result in immunosuppression. However, we did not study immunity parameters, so additional studies are suggested for exploring the process involved in the reduction of spleen weight when BA and emulsifier are supplemented alone or in combination in broiler diets. As reported previously in the study by Upadhaya et al. (2018), liver weight is increased when fat digestibility is increased in broilers. In the current study, the elevated liver weight might also be a result of increased metabolic activity because of improved fat digestibility. Overall, the findings of this study highlight the synergistic effects of supplementing broiler diets with BA and emulsifiers, improving growth performance, fat digestibility and lipid metabolism. Additional studies are necessary to determine the ideal dosage of these additives for performance optimization without negatively impacting health.

CONCLUSION

In conclusion, supplementation of bile acid and emulsifier alone or in combination during the growing, finishing, and overall phases may improve growth performance, fat and dry matter digestibility without influencing the blood lipid profile. However, the effect of supplementation of a combination of emulsifier and bile acid in the diet was more prominent on the parameters of performance of broilers as compared to supplementation of bile acid or emulsifier alone. Relative organ weights were not influenced by supplementation of bile acid and emulsifier alone or in combination, except for a reduction in the relative weight of the spleen in birds. The reason for this reduction in spleen weight in the current study remains unclear, and future research into the role of supplementation of the emulsifier and bile acid is important for understanding the reason for this influence on spleen weight.

ACKNOWLEDGEMENTS

We acknowledge the hard work of workers, lab staff and technicians at R&D farms.

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• FUNDING
  Funding was provided by Sindh Feed and Allied Products: Industry academic linkage Research Project ORIC 28-10-2021-7800, University of Agriculture, Faisalabad. The funders did not contribute to the study design, collection and analysis of data, the decision to publish, or document drafting.
• DATA AVAILABILITY STATEMENT
  All the data related to current experiment will be made available by the corresponding author.
• DISCLAIMER/PUBLISHER’S NOTE
  The published papers’ statements, opinions, and data are those of the individual author(s) and contributor(s). The editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions, or products referred to in the content.

Edited by

• Section Editor:
  Rodrigo Garófallo Garcia

Data availability

All the data related to current experiment will be made available by the corresponding author.

Publication Dates

• Publication in this collection
  08 Sept 2025
• Date of issue
  2025

History

• Received
  25 Feb 2025
• Accepted
  21 Apr 2025