The effects of β-mannanase supplementation on growth performance, digestive enzyme activity, cecal microbial communities, and short-chain fatty acid production in broiler chickens fed diets with different metabolizable energy levels

Abstract

This study assessed the effects of β-mannanase (BM) supplementation on growth performance, digestive enzyme activity, cecal microbial communities, and short-chain fatty acid (SCFA) production in broiler chickens fed diets with different metabolizable energy (ME) levels. A total of 1,296 male 1-d-old Cobb 500 broilers were randomly distributed in a 3 × 2 factorial arrangement (3 ME levels × 0 or 200 g/ton BM), with 6 replicates per treatment combination. The 3 ME levels were 3,000 (ME1), 2,930 (ME2), and 2,860 (ME3) kcal/kg, respectively, during the 0 to 3 wk-old stages and 3,150 (ME1), 3,080 (ME2), and 3,010 (ME3) kcal/kg, respectively, during the 3 to 6 wk-old stages. Reducing ME levels increased broiler feed intake (P = 0.036) and decreased average daily gain (ADG, P = 0.002) during the entire period. While BM supplementation increased ADG (P = 0.002) and improved the feed conversion ratio (P = 0.001) during the 0 to 3 wk-old stages, with no effect during the 3 to 6 wk-old stages. Overall, reducing ME levels increased pancreatic lipase (P = 0.045) and amylase (P = 0.013) activity and duodenal amylase activity (P = 0.047). Notably, BM supplementation significantly increased pancreatic lipase activity (P = 0.015) and increased lipase (P = 0.029) and amylase (P = 0.025) activities in the jejunal chyme. Although diet or enzyme supplementation did not affect microbial diversity, significant differences in microbial communities were observed. At the genus level, decreasing ME levels significantly affected the average abundances of Tyzzerella (P = 0.028), Candidatus_Bacilloplasma (P = 0.001), Vibrio (P = 0.005), and Anaerotruncus (P = 0.026) among groups, whereas BM supplementation reduced the average abundances of Escherichia-Shigella (P = 0.048) and increased the average abundances of Barnesiella (P = 0.047), Ruminococcus (P = 0.020), Alistipes (P = 0.050), and Lachnospiraceae_unclassified (P = 0.009). SCFA concentrations strongly depended on bacterial community composition, and BM supplementation increased acetic acid (P = 0.004), propionic acid (P = 0.016), and total SCFA concentrations. In conclusion, BM supplementation improved the performance of younger broilers, and both enzyme supplementation and reduced ME levels positively affected digestive enzyme activity and intestinal microflora.

This study found that BM-supplemented diets are effective in improving performance, digestive enzyme activity, and intestinal flora in broilers fed low-ME or normal-ME diets. These results may help achieve higher economic productivity of broiler farms.

Introduction

As a soluble non-starch polysaccharide (NSP), βmannan commonly found in feedstuffs such as soybean meal (SBM), rapeseed meal, sesame meal, and corn. Bmannan exhibits anti-nutritive activities in poultry, leading to changes in the development of the gastrointestinal tract, digestive enzyme activities, nutrient absorption process, and microbiota compositions (Choct, 2015; Nguyen et al., 2022). Previous studies have reported that SBM contains 1.1% to 1.7% βmannan, which are highly resistant to digestive enzymes and increase intestinal chyme viscosity in broilers, thus preventing adequate digestion within the intestinal mucosa (Slominski, 2011; Plouhinec et al., 2023).

Supplementing broiler diets with β1,4-mannanase (BM) is suggested to overcome the anti-nutritional effects in the digestive process (Balasubramanian et al., 2018). Usually, the endocarbohydrase BM can randomly cleave the 1,4- β glycosidic bonds of the main mannan, galactomannan, glucomannan, and galactoglucomannan chains as well as the bonds of the mannan chain itself, yielding mannobiose, mannotriose, and mannose as hydrolysis products (Dhawan and Kaur, 2007). This could reduce the viscosity of the intestinal environment (Lee et al., 2003; Mehri et al., 2010), regulate endogenous digestive enzyme activity (Pinheiro et al., 2004), and increase the nutrient utilization of carbohydrates, resulting in improving growth performance in poultry (Ferreira et al., 2016; Balasubramanian et al., 2018; Mohammadigheisar et al., 2021). A meta-analysis of 28 studies showed that the mean difference in BM improvement on average daily gain (ADG, g/d) was +0.23, while that of feed conversion rate (FCR) was −0.02, and BM improved apparent metabolizable energy (ME) by 47 kcal/kg in broiler chickens (Kiarie et al., 2021). In addition, supplementation with BM enabled reduced-energy diets to be adopted (Plouhinec et al., 2023). It has been reported that broilers fed low-energy diets supplemented with BM even performed slightly better than broilers fed high-energy diets without BM (McNaughton et al., 1998). A study also reported that supplementing NSP-degrading enzymes in the maize–SBM diet does not affect the performance of broiler chicken fed normal-energy diets, but improved ME utilization when fed low-energy diets (Zhou et al., 2009). Additionally, chickens fed a low-energy diet with NSP-degrading enzymes supplementation have greater relative liver and spleen weights (Mohammadigheisar et al., 2021; Hussein et al., 2019). On the contrary, some researchers did not find any positive effects of NSP-degrading enzyme supplementation on performance or relative organ weights for broiler chickens (Bin Baraik, 2010; Alagawany et al., 2018). Therefore, though the BM supplementation of broiler chicken diets might improve the utilization of ME, particularly in rations with lower ME levels, this option needs to be investigated.

Exogenous NSP-degrading enzymes might affect the metabolic activity of the gut microbiota (Plouhinec et al., 2023). Supplementation with enzymes (β-glucanase or xylanase) can significantly decrease the relative amount of Enterobacteriaceae in broiler chicken ceca (Jozefiak et al., 2010). Combining Bacillus subtilis and BM effectively reduced Escherichia coli and Clostridium perfringens numbers in piglets and altered short-chain fatty acid (SCFA) concentrations in feces, which is believed to be related to the production of enzyme products termed β-mannooligosaccharides (Wang et al., 2021; Liu et al., 2023). However, previous studies have primarily focused on the effects of supplemental BM on performance, but information on BM supplementation regarding the microbiome and SCFA production was limited reported in broilers fed low- or normal-ME diets, lacking comprehensive studies. To sum up, the main objective of this study was to assess the effects of low- and normal-ME level diets supplemented with BM on performance, relative organ weights, digestive enzyme activity, cecal microbial communities, and SCFA production of broiler chickens.

Materials and Methods

Ethical approval

The study was approved by the Research Committee of the Jiangsu Academy of Agricultural Sciences and conducted according to the Regulations of the Administration of Affairs Concerning Experimental Animals (Order No. 63 of the Jiangsu Academy of Agricultural Science on July 8, 2014). All experiments were conducted in accordance with the ARRIVE (Animal Research: Reporting of In Vivo Experiments) guidelines.

Experimental design

This study was conducted from March 24 to May 5, 2023, at the Experimental Animal Center of the Zunhua Experimental Poultry Farm (Tangshan, China). A total of 1,296 male 1-d-old Cobb 500 chicks supplied by Beijing Dafa Chia Tai Co. Ltd. (Beijing, China), were used for the experiment. The experiment followed a completely randomized design with a 3 × 2 factorial arrangement consisting of 3 energy (ME) levels and 2 BM levels, making a total of 6 treatments with 6 repeats/treatment, and each pen contained 36 birds housed in 2.0 × 3.0 m wood-shaving lined pens, where each pen was considered 1 experimental unit. According to the previous designs of low-energy diets (Zhou et al., 2009; Hussein et al., 2020; Yaqoob et al., 2022), our 3 ME levels included starter and grower phase diets, which contained 3,000 and 3,150 kcal/kg for ME1, 2,930 and 3,080 kcal/kg for ME2, and 2,860 and 3,010 kcal/kg for ME3, respectively. The 2 BM levels were 0 (BM0) and 200 (BM200) g/ton, according to the manufacturer’s recommendations. This enzyme contained 5,000 IU/g β-mannanase supplied by Beijing Challenge Biotechnology Co. Ltd. (Beijing, China). The experimental period was 6 wk (42 d), with a starter diet (0 to 3 wk), and a grower diet (3 to 6 wk) prepared for the different treatments. Light was continuously supplied with 12 h of natural light (6:00 a.m. to 6:00 p.m.) and 12 h of artificial light (6:00 p.m. to 6:00 a.m.). The broilers had ad libitum access to crumble feed and water.

The basal diet was formulated to meet or exceed the formula feeds for layers and broilers (GB/T 5916-2020, China) and the nutrient requirements of the National Research Council (NRC, 1994). The composition of the experimental diet is shown in Table 1. The average daily feed intake (ADFI) and the live body weight (BW) of the broilers were recorded using an electronic scale (YP60001; Hengji, Shanghai, China) before feeding during the test, and the ADG and feed conversion ratio (FCR) were calculated at the end of the experiment. The relative weights of the breast meat, abdominal fat, and organs were expressed as percentages of live BW.

Table 1.

Ingredients and nutrient composition of the experimental diets ¹

Ingredients (%)	Starter (0 to 3 wk)						Grower (3 to 6 wk)
	BM0			BM200			BM0			BM200
	ME1	ME2	ME3	ME1	ME2	ME3	ME1	ME2	ME3	ME1	ME2	ME3
Corn	57.00	54.58	52.16	57.00	54.58	52.16	58.00	55.59	53.17	58.00	55.59	53.17
Soybean meal, 43%CP	30.47	30.89	31.31	30.47	30.89	31.31	26.99	27.40	27.82	26.99	27.40	27.82
Corn gluten meal, 50%CP	5.00	5.00	5.00	5.00	5.00	5.00	5.40	5.40	5.40	5.40	5.40	5.40
Limestone	1.40	1.40	1.40	1.40	1.40	1.40	1.40	1.40	1.40	1.40	1.40	1.40
Dicalcium phosphate	1.10	1.10	1.10	1.10	1.10	1.10	0.80	0.80	0.80	0.80	0.80	0.80
Salt	0.30	0.30	0.30	0.30	0.30	0.30	0.30	0.30	0.30	0.30	0.30	0.30
l-Lysine, 79%	0.65	0.65	0.65	0.65	0.65	0.65	0.70	0.70	0.70	0.70	0.70	0.70
dl-Methionine, 99%	0.21	0.21	0.21	0.21	0.21	0.21	0.26	0.26	0.26	0.26	0.26	0.26
l-Threonine, 98%	0.17	0.17	0.17	0.17	0.17	0.17	0.20	0.20	0.20	0.20	0.20	0.20
Choline chloride	0.10	0.10	0.10	0.10	0.10	0.10	0.10	0.10	0.10	0.10	0.10	0.10
Soybean oil	3.20	3.20	3.20	3.20	3.20	3.20	5.45	5.45	5.45	5.45	5.45	5.45
Zeolete	—	2.00	4.00	—	2.00	4.00	—	2.00	4.00	—	2.00	4.00
Premixes2	0.40	0.40	0.40	0.40	0.40	0.40	0.40	0.40	0.40	0.40	0.40	0.40
TOTAL	100	100	100	100	100	100	100	100	100	100	100	100
Compositions
Metabolizable energy (kcal/kg)#	3,000	2,930	2,860	3,000	2,930	2,860	3,150	3,080	3,010	3,150	3,080	3,010
Crude protein,%*	21.91	22.12	22.05	21.67	21.88	21.69	19.44	19.62	19.57	19.54	19.41	19.67
Fat,%*	6.00	5.92	6.14	6.12	6.02	5.98	8.20	8.12	8.04	8.02	8.32	8.21
Crude fiber,%*	3.90	4.16	3.98	3.96	4.17	4.08	3.90	3.97	3.93	4.00	3.97	3.93
Calcium,%*	1.04	1.07	1.13	1.05	1.08	0.98	0.89	0.88	0.87	0.86	0.90	0.88
Phosphorus, %*	0.54	0.53	0.54	0.52	0.54	0.53	0.48	0.47	0.47	0.47	0.49	0.48
Non-phytate phosphorus,%#	0.30	0.30	0.30	0.30	0.30	0.30	0.25	0.25	0.24	0.25	0.25	0.24
Digestible lysine, %#	1.19	1.19	1.19	1.19	1.19	1.19	1.05	1.05	1.05	1.05	1.05	1.05
Digestible methionine, %#	0.51	0.51	0.51	0.51	0.51	0.51	0.53	0.53	0.53	0.53	0.53	0.53
Digestible methionine + cysteine, %#	0.85	0.85	0.85	0.85	0.85	0.85	0.77	0.77	0.77	0.77	0.77	0.77
Digestible threonine, %#	0.76	0.76	0.76	0.76	0.76	0.76	0.69	0.69	0.69	0.69	0.69	0.69

¹The 3 ME levels were 3,000/3150 (ME1), 2,930/3,080 (ME2), and 2,860/3,010 (ME3) kcal/kg (Starter/Grower) and the 2 BM levels were 0 (BM0) and 200 (BM200) g/ton. ME, metabolizable energy; BM, β-mannanase.

²Mineral supplement supplied per kg of feed: iron, 55.0 mg; copper, 11.0 mg; manganese, 77.0 mg; zinc, 71.5 mg; iodine, 1.10 mg; selenium, 0.330 mg. Vitamin supplement supplied per kg of feed: vitamin A, 8,250 IU; vitamin D3, 2,090 IU; vitamin E, 31 IU; vitamin B1, 2.20 mg; vitamin B2, 5.50 mg; vitamin B6, 3.08 mg; vitamin B12, 0.013 mg; pantothenic acid, 11.0 g; biotin, 0.077 mg; vitamin K3, 1.65 mg; folic acid, 0.77 mg; nicotinic acid, 33.0 mg.

^#Calculated values;

^*Analyzed values.

Sampling and plasma biochemistry

At the end of the experiment, 36 birds (6 birds/treatment and 1 bird/pen) were randomly chosen and slaughtered (by cutting carotid arteries) for immediate blood collection (5 mL/broiler). All blood samples were collected in blood collection tubes containing an anticoagulant (heparin sodium) and centrifuged at 1,500 × g for 15 min. Plasma was stored in 0.6-mL Eppendorf tubes at −80 °C until analysis. Plasma glucose (GLU), triglyceride (TG), low-density lipoprotein (LDL), high-density lipoprotein (HDL), and alkaline phosphatase (ALP) levels were determined using a fully automatic biochemical analyzer (AU5800, Beckman Coulter Co., Ltd., Brea, CA).

Digestive enzyme activities

According to the procedures described by Pinheiro et al. (2004), the pancreas and the small intestine of broilers were immediately removed and stored at −80 °C until required for analysis. Samples were homogenized using an Omni Prep Multi-Sample Homegenizer (Omni, NC) and were then centrifuged at 1,500 × g for 15 min. The supernatant was collected for the determination of digestive enzyme activities. Amylase (EC 3.2.1.1), lipase (EC 3.1.1.3.), and trypsin (EC 3.4.4.4) activity levels were determined using kits (No. C016, A054, and A080, respectively) obtained from Nanjing Jiancheng Biotechnology Co., Ltd. (Jiangsu, China). The protein concentrations were measured using the Coomassie Brilliant Blue method with bovine serum albumin as a standard. All assays were performed according to the respective manufacturer’s instructions. The samples were tested in triplicate.

SCFAs analysis

The cecum contents of broilers (6 broilers/treatment and 1 bird/pen) were collected on day 42 in 2 mL sterile, internally threaded cryogenic vials and immediately stored in liquid nitrogen until analysis. According to the procedures of SCFA analysis described by Liu et al. (2023), samples were homogenized, diluted, and mixed with 25% (w/v) metaphosphoric acid solution and 210 mmol/L crotonate solution, which was followed by incubation at 4 °C for 30 min. The tubes were then centrifuged at 8,000 × g for 10 min, after which, the supernatant was added to a chromatogen-methanol mixture (1:3 dilution) and recentrifuged at 8,000 × g for 5 min. The levels of SCFAs were analyzed using a gas chromatography system (Agilent Inc., Palo Alto, CA).

16S rRNA sequencing and bioinformatics

DNA from the cecum content samples was extracted using a MicroElute Genomic DNA Kit (D3096-01, Omega Biotek Inc., Norcross, GA) following the manufacturer’s instructions. Total DNA was eluted in 50 μL of Elution buffer and stored at −80 °C until amplification by PCR performed by LC-Bio Technology Co., Ltd, Hang Zhou, Zhejiang Province, China. Forward 343F (5ʹ- TACGGRAGGCAGCAG -3ʹ) and reverse 798R (5ʹ- AGGGTATCTAATCCT-3ʹ) primers were used to amplify the V3 to V4 regions of bacterial 16S rRNA. Amplicon pools were prepared for sequencing and the size and quantity of the amplicon library were assessed using an Agilent 2100 Bioanalyzer (Agilent, Santa Clara, CA) and the Library Quantification Kit for Illumina (Kapa Biosciences, Woburn, MA), respectively. Libraries were sequenced on a NovaSeq PE250 platform (Illumina, San Diego, CA). Paired-end reads were assigned to samples based on their unique barcodes and truncated by cutting off the barcodes and primer sequences. Paired-end reads were merged using FLASH software. Quality filtering of the raw reads was performed under specific filtering conditions to obtain high-quality clean tags using fqtrim v0.94. Chimeric sequences were filtered using Vsearch software v2.3.4. The GenBank accession number for the amplicon sequence variant (ASV) is PRJNA1029764. The reads were filtered using Quantitative Insights Into Microbial Ecology 2 (QIIME2) quality filters. After dereplication using DADA2 (QIIME2-dada2), alpha and beta diversity indexes were calculated by random normalization to the same sequences (Walters et al., 2015). Then, according to the SILVA (release 138) classifier, feature abundance was normalized using the relative abundance of each sample. Alpha diversity was applied to analyze the complexity of species diversity through 4 indexes, including Chao1, Observed species, Shannon, and Simpson. All these indexes in the samples were calculated using QIIME2 (Bolyen et al., 2019). Beta diversity was calculated using QIIME2, and graphs were drawn using R software v3.5.2. BLAST was used for sequence alignment and feature sequences were annotated using the SILVA database for each representative sequence. Other diagrams were constructed using R software v3.5.2.

Data analysis

The experimental data derived from 6 replicates per treatment were analyzed using SPSS Statistics 16 program (IBM Corporation, Somers, NY). Each replicate was considered as an experimental unit and the statistical procedure used was a multivariate analysis of variance using the general linear model procedure (GLM). When significant differences were observed, pairwise comparisons of the treatment means were conducted using Tukey’s multiple-range test. A 2-tailed test was considered statistically significant at a probability level of less than 5% (P < 0.05).

Results

Effects of supplementing BM on growth performance in broiler chickens fed diets with different ME levels

The effects of BM supplementation on the growth performance of broilers fed different ME levels from days 1 to 42 are shown in Table 2. No interaction effects between ME and BM supplementation on growth performance were observed in both the starter and grower stages, as well as the entire period (P > 0.05). For ME, reducing ME levels significantly increased broiler feed intake. Broilers fed the ME3 diets exhibited higher ADFI than those receiving the ME1 diet on both days 21 (P = 0.004) and 42 (P = 0.007) of age, as well as throughout the entire period (P = 0.036). Broilers in the ME3 group had lower ADG both on day 21 of age (P = 0.046) and throughout the entire period (P = 0.002) compared to those in the ME1 group. Moreover, ADG (P = 0.002) in 21-d-old broilers of the BM200 group increased compared to those in the BM0 group, whereas FCR in these groups decreased (P = 0.001). However, ADFI, ADG, and FCR did not exhibit significant differences between broilers with or without BM from days 21 to 42 (P > 0.05). ADG in the BM200 group was higher throughout the entire period compared to that in the BM0 group (P = 0.043).

Table 2.

The effects of supplementing BM on growth performance in broiler chickens fed diets with different ME levels ¹

Treatment		0 to 3 wk			3 to 6 wk			Entire period
BM	ME	ADFI, g	ADG, g	FCR	ADFI, g	ADG, g	FCR	ADFI, g	ADG, g	FCR
ME1	BM0	60.67	51.67	1.174	157.0	92.81	1.692	108.8	72.24	1.507
	BM200	59.33	53.33	1.113	151.3	91.67	1.650	105.3	72.50	1.453
ME2	BM0	58.67	49.67	1.181	155.2	90.62	1.713	106.9	70.14	1.524
	BM200	59.17	50.67	1.168	157.3	92.00	1.710	108.2	71.34	1.517
ME3	BM0	61.00	49.00	1.245	164.8	89.62	1.839	112.9	69.31	1.629
	BM200	60.17	50.17	1.199	162.0	90.29	1.794	111.1	70.23	1.582
SEM		0.453	0.443	0.009	2.565	2.448	0.203	2.161	1.214	0.021
Main effects 2
BM	BM0	60.11	50.11a	1.200a	159.0	91.02	1.748	109.56	70.56	1.553
	BM200	59.56	51.39b	1.160b	156.9	91.32	1.718	108.21	71.35	1.517
ME	ME1	58.92a	52.50b	1.143	154.2a	92.24	1.671	107.08a	72.37b	1.480
	ME2	60.00ab	50.17ab	1.174	156.3b	91.31	1.711	107.59a	70.74ab	1.521
	ME3	60.58b	49.59a	1.222	163.4c	89.95	1.817	111.99b	69.77a	1.605
Significance
ME		0.004	0.046	0.098	0.007	0.064	0.103	0.036	0.102	0.077
BM		0.150	0.002	0.001	0.117	0.117	0.289	0.063	0.043	0.785
ME × BM		0.131	0.259	0.508	0.055	0.130	0.068	0.390	0.257	0.145

¹The 3 ME levels were 3,000/3,150 (ME1), 2,930/3,080 (ME2), and 2,860/3,010 (ME3) kcal/kg (Starter/Grower) and the 2 BM levels were 0 (BM0) and 200 (BM200) g/ton (n = 6 repeats). ME, metabolizable energy; BM, β-mannanase; BW, body weight; ADFI, average daily feed intake; ADG,: average daily gain; FCR, feed conversion ratio; SEM, standard error of mean. Different lowercase letters on the shoulders indicate significant differences (P < 0.05).

The effects of BM supplementation on the relative weight of organs and plasma indicators of broilers with different ME levels on day 42 of age are shown in Table 3. The results showed that different ME content or adding BM to diets did not affect the relative weights of breast muscle, abdominal fat, heart, liver, gizzard, and spleen of broilers on day 42 (P > 0.05). Broilers with BM-supplemented feed exhibited higher GLU (P = 0.010) than those who received diets without BM, whereas plasma TG, HDL, LDL, and ALP concentrations of broilers showed no differences among treatments (P > 0.05).

Table 3.

The effects of supplementing BM on the relative weight of organs and plasma indicators in broiler chickens fed diets with different ME levels¹

Treatment		Relative weight of organs (%)					Plasma indicators
BM	ME	Breast muscle	Abdominal fat	Heart	Liver	Spleen	TG, mmol/L	HDL, mmol/L	LDL, mmol/L	GLU, mmol/L	ALP, U/L
ME1	BM0	24.15	7.844	0.0046	4.097	0.078	0.473	2.243	0.835	7.962	2,618
	BM200	23.48	8.316	0.0046	4.127	0.072	0.532	2.135	0.877	10.43	1405
ME2	BM0	23.83	9.064	0.0043	4.797	0.060	0.576	2.595	1.175	8.667	4,644
	BM200	20.98	7.787	0.0052	4.192	0.068	0.561	2.272	0.994	11.80	3133
ME3	BM0	21.64	9.408	0.0045	3.911	0.068	0.513	2.233	1.016	9.931	2,003
	BM200	19.66	8.677	0.0046	5.316	0.061	0.602	2.364	1.200	11.49	3,357
SEM		3.767	1.305	<0.001	0.392	0.006	0.083	0.119	0.114	1.001	796.6
Main effects
BM	BM0	23.206	8.772	0.0045	4.268	0.069	0.520	2.357	1.009	8.853a	3,088
	BM200	21.369	8.260	0.0048	4.545	0.067	0.565	2.257	1.023	11.24b	2,632
ME	ME1	23.811	8.080	0.0046	4.112	0.075	0.502	2.189	0.856	9.195	2,011
	ME2	22.401	8.425	0.0048	4.495	0.064	0.568	2.433	1.085	10.24	3,888
	ME3	20.649	9.042	0.0046	4.613	0.065	0.557	2.298	1.108	10.71	2,680
Significance (P-value)
ME		0.716	0.766	0.752	0.442	0.142	0.700	0.181	0.065	0.367	0.103
BM		0.561	0.640	0.062	0.402	0.763	0.505	0.337	0.870	0.010	0.512
ME × BM		0.961	0.802	0.172	0.056	0.366	0.794	0.192	0.247	0.744	0.175

¹The 3 ME levels were 3,000/3,150 (ME1), 2,930/3,080 (ME2), and 2,860/3,010 (ME3) kcal/kg (Starter/Grower) and the 2 BM levels were 0 (BM0) and 200 (BM200) g/ton (n = 6 repeats). ME, Metabolizable energy; BM, β-mannanase; TG, triglyceride; HDL, high-density lipoprotein; LDL, low-density lipoprotein; GLU, glucose; ALP, alkaline phosphatase; SEM, standard error of mean. Different lowercase letters on the shoulders indicate significant differences (P < 0.05).

The effects of supplementing BM on digestive enzyme activities in broiler chickens fed diets with different ME levels

The effects of BM supplementation on the digestive enzyme activities of broilers with different ME levels on day 42 are shown in Table 4. No interactions were observed between ME and BM supplementation for digestive enzyme activity in the pancreas, duodenal chyme, or jejunal chyme (P > 0.05). But notably, reducing dietary ME levels increased lipase (P = 0.045) and amylase (P = 0.013) activity in the pancreas and amylase activity in the duodenal chyme (P = 0.047). Supplementation with BM resulted in a significant increase in lipase activity (P = 0.015) in the pancreas. Similar changes were observed in lipase (P = 0.029) and amylase (P = 0.025) activity in the jejunal chyme.

Table 4.

The effects of supplementing BM on digestive enzyme activities in broiler chickens fed diets with different ME levels ¹

Treatment		Pancreas			Duodenal chyme			Jejunal chyme
BM	ME	Lipase, U/g	Amylase, U/mg	Protease, U/mgprot	Lipase, U/g	Amylase, U/mg	Protease, U/mgprot	Lipase, U/g	Amylase, U/mg	Protease, U/mgprot
ME1	BM0	321.5	2.336	2,698	8.929	1.076	476.4	116.7	3.344	17,96
	BM200	398.2	2.364	3,429	10.89	1.300	476.6	152.0	3.345	1,587
ME2	BM0	386.3	2.317	3,123	14.18	1.588	494.8	132.6	4.256	1,901
	BM200	445.2	2.496	3,724	13.08	1.885	419.5	165.7	6.053	1,631
ME3	BM0	408.8	2.700	2,848	13.99	1.982	495.2	164.0	2.611	1,556
	BM200	467.0	2.877	3,705	31.51	1.921	471.6	182.4	4.254	1,220
SEM		30.65	0.224	508.8	7.892	0.385	148.9	31.22	1.184	294.6
Main effects
BM	BM0	372.2a	2.451	2,890	12.37	1.549	488.8	137.8a	3.404a	1,751
	BM200	436.8b	2.579	3,619	18.50	1.702	455.9	166.7b	4.551b	1,479
ME	ME1	359.9a	2.350a	3,063	9.907	1.188a	476.5	134.4	3.345	1,691
	ME2	415.8ab	2.406a	3,424	13.63	1.737ab	457.1	149.2	5.155	1,766
	ME3	437.9b	2.788b	3,277	22.76	1.952b	483.4	173.2	3.433	1,388
Significance (P-value)
ME		0.045	0.013	0.796	0.273	0.047	0.985	0.467	0.281	0.408
BM		0.015	0.470	0.106	0.358	0.635	0.795	0.029	0.025	0.268
ME × BM		0.944	0.922	0.972	0.462	0.890	0.970	0.958	0.730	0.977

¹The 3 ME levels were 3,000/3,150 (ME1), 2,930/3,080 (ME2), and 2,860/3,010 (ME3) kcal/kg (Starter/Grower) and the 2 BM levels were 0 (BM0) and 200 (BM200) g/ton (n = 6 repeats). ME, metabolizable energy; BM, β-mannanase; SEM, standard error of the mean. Different lowercase letters on the shoulders indicate significant differences (P < 0.05).

The effects of supplementing BM on cecal microbial communities in broiler chickens fed diets with different ME levels

In the analysis of cecal microbiota by 16S rRNA (V3-V4) gene sequencing, after quality control and chimera removal, 2,504,427 valid tags were retained, with an average of 69,567 tags per sample, and were identified as being of bacterial origin. After dereplication using DADA2, these sequences were assigned to 13,261 ASV feature sequences of bacterial species. Microbial taxa present among different ME levels (core microbiome) were represented by 905 ASVs without BM and 927 ASVs with BM (Figure 1A). The key components of biodiversity, including species richness, evenness, and rarity, were measured using Shannon, Simpson, and Chao1 indexes (Figure 1B). On day 42, the addition of BM to the diet did not affect the observed species or alpha diversity indexes (P > 0.05). Interactions were also not observed between ME and BM supplementation in broiler diets (P > 0.05). The overall composition of the microbiota at the phylum and genus levels in the cecum on day 42 is shown in Figure 1C. Among these groups, the dominant phyla were Firmicutes, Bacteroidetes, and Proteobacteria, and the dominant genera were Faecalibacterium, Clostridia_UCG-014_unclassified, Lachnospiraceae_unclassified, Ruminococcaceae_unclassified, Alistipes, and Negativibacillus. Importantly, many subtle differences were observed among the microbial communities of broilers fed different ME and BM diets. At the phylum level, the average abundance of Bacteroidetes in broilers fed the BM-supplemented diets increased with decreasing ME levels (P = 0.045; Figure 2A). The average abundance of Proteobacteria decreased in diets with decreasing ME levels without BM supplementation (P = 0.042), and their average abundance in the ME1 group was greater than that in the ME3 group (P < 0.01). In addition, at the genus level, decreasing ME levels significantly affected the average abundance of Tyzzerella (P = 0.028), Candidatus_Bacilloplasma (P = 0.001), Vibrio (P = 0.005), and Anaerotruncus (P = 0.026) among the experimental groups (Figure 2B). The average abundance of Tyzzerella showed an upward trend with decreasing ME levels in the diets without BM supplementation, whereas the average abundances of Candidatus_Bacilloplasma and Vibrio declined as ME levels decreased in the diets with BM supplementation. Moreover, BM supplementation reduced the average abundances of Escherichia-Shigella (P = 0.048), whereas the average abundances of Barnesiella (P = 0.047), Ruminococcus (P = 0.020), Alistipes (P = 0.050), and Lachnospiraceae_unclassified (P = 0.009) increased when supplementary BM was given. There were no significant differences among Eubacterium, Butyricicoccus, Ruminococcus_torques_group, Bacteroides, Faecalibacterium, and Clostridia_UCG-014_unclassified in the groups on day 42 (P > 0.05).

Figure 1.

The effects of supplementing BM on intestinal microbial communities in broiler chickens fed diets with different ME levels. (A) the Venn diagram; (B) the Alpha diversity indices (the observed species, Chao1, and Shannon indices); n = 6 repeats; (C) the overall composition of microbiota and average relative abundance among the experimental groups at phylum and genus levels; (D) analysis of the dominant phyla and genera with Sankey plots. ME, metabolizable energy; BM, β-mannanase. *: P < 0.05; **: P < 0.01.

Figure 2.

The effects of supplementing BM on the average relative abundance in phylum and genus levels in broiler chickens fed diets with different ME levels. (A) the average abundance of Bacteroidetes and Proteobacteria among groups; (B) the average abundance of the dominant and significant genera among groups; n = 6 repeats. ME, Metabolizable energy; BM, β-mannanase. *: P < 0.05; **: P < 0.01.

Effects of supplementing BM on SCFA production in cecum contents of broiler chickens fed diets with different ME levels

The effects of BM supplementation on SCFAs production in cecum contents of broilers with different ME levels on day 42 are shown in Figure 3. The concentrations of acetic acid increased in the diets with decreasing ME levels (P = 0.006), while the use of BM significantly increased the acetic acid concentration (P = 0.004). The average concentration in the ME3 group was greater than that in the ME1 (P < 0.01) and ME2 (P < 0.05) groups. Propionic acid concentrations increased in the diets supplemented with BM (P = 0.016), whereas the concentrations of butyric acid, isobutyric acid, pentanoic acid, and isopentanoic acid did not change among broilers fed different ME levels or diets supplemented with BM (P > 0.05). Based on these results, concentrations of total SCFAs increased with decreasing ME levels (P = 0.002) or diets supplemented with BM (P = 0.006). Furthermore, SCFA concentration in the ME3 group was greater than that in the ME1 (P < 0.01) and ME2 (P < 0.01) groups with BM supplementation.

Figure 3.

The effects of supplementing BM on the production of short-chain fatty acids in broiler chickens fed diets with different ME levels. n = 6 repeats. ME, Metabolizable energy; BM, β-mannanase; SCFAs, the short-chain fatty acids. *: P < 0.05; **: P < 0.01.

Discussion

Scientific and economic considerations with regard to feedstuff formulation have become important factors for reducing the costs and enhancing the benefits, which already is a prevalent requirement of the poultry industry (Chen et al., 2023). Low-energy diets have been increasingly suggested. However, lower nutrient concentrations may result in reduced performance, thereby increasing attention toward the potential use of feed enzymes (Mohammadigheisar et al., 2021; Chen et al., 2023). In the present study, reducing ME levels increased broiler feed intake and decreased ADG, and FCR also showed an increased trend with the ME levels reducing in the starter stage (P = 0.098) or throughout the entire period (P = 0.077), implying a loss of production performance. In line with our results, decreased ADG and increased FCR in broilers fed with low-energy diets without enzymatic supplementation have been observed in many previous studies (Li et al., 2010; Ferreira et al., 2016). However, an increase in ADFI was observed as ME levels decreased when the broilers reached 3 or 6 wk of age in our study, indicating that broilers require higher feed intake to meet the demand for rapid growth when reducing the energy density of the diets (Pinheiro et al., 2004). Thus, regardless of the stage analyzed, lower energy levels can compromise broiler performance, as well as an increase in feed intake can lead to higher feed costs. In our study, BM supplementation improved ADG and FCR in the starter stage of broilers, which corroborated the findings of Balasubramanian et al. (2018) and Mohammadigheisar et al. (2021). Interestingly, BM supplementation increased ADG value and improved FCR during the 0 to 3 wk-old stage, but these effects were not observed in 3 to 6 wk-old broilers in the present study. These findings suggest that younger broilers respond better to BM supplementation in cereal-based diets than older broilers. This may be attributed to BM’s role in enhancing the efficiency of the digestion process and may help achieve the animals’ genetic potential when the physiological state of young animals is immature (Jackson et al., 2004; Zou et al., 2006; Mohammadigheisar et al., 2021). As the gastrointestinal system matures, the effects of enzymatic supplements may become limited (Gao et al., 2008). The interaction reactions between ME and BM were not observed, which is consistent with the results reported by Ferreira et al. (2016). This shows that the supplementation of broiler chicken diets with BM widely improved energy utilization, which is not limited to lower ME levels. Certainly, feeding broilers on low-energy diets while simultaneously supplemented with BM may be a more economical approach.

Moreover, our results showed that the relative weights of the organs were not influenced by changes in ME levels or BM supplementation. Similarly, it was reported that BM also did not influence blood proteins, including albumin, globulin, and urea nitrogen (Mohammadigheisar et al., 2021). Meanwhile, we did not observe any effects on plasma TG, HDL, LDL, or ALP concentrations, indicating that the addition of BM to the diets did not influence lipid metabolism or liver function, which is consistent with the results of the previous study (Kiarie et al., 2021). Interestingly, broilers fed BM-supplemented feed exhibited higher GLU levels than those who received diets without BM, which has not been reported in other studies so far. This discrepancy warrants further investigation, and it should be determined whether BM is involved in the regulation of blood glucose levels.

Generally, digestive processes are highly dependent on endogenous enzyme activity (Pinheiro et al., 2004). Our study revealed that reducing dietary ME levels increased the activities of lipase and amylase in the pancreas, along with amylase activity in the duodenal chyme. This finding was similar to previous research suggesting that increased enzyme activity may be an adaptative response to energy restriction, contributing to growth compensation (Zubair and Leeson, 1994). Additionally, our findings showed that the addition of BM to the diet resulted in a significant increase in pancreatic lipase activity and lipase and amylase activities in the jejunal chyme. The results partially agree with those reported by Zhu et al. (2014) and Yuan et al. (2017) and support the notion that BM increases the availability of nutrients in the small intestine, promoting increased digestive enzyme activity (Pinheiro et al., 2004).

Some research groups have speculated that the growth-promoting effect of BM may be related to the oligomers produced by endogenous or exogenous enzymes, with the gut microbiota playing a crucial role (Gao et al., 2008). In the present study, although BM supplementation did not affect the observed species and alpha diversity indices in the cecum, a noticeable change in the microbial communities was noticed. Specifically, BM reduced the average abundance of the genus complex Escherichia-Shigella in broilers. In general, the reduction in abundance of Escherichia-Shigella is beneficial for maintaining the intestinal health of broilers. This result from our study is in agreement with a previous study of growing pigs fed with diets with or without BM and they reported that the addition of BM reduced the count of excreta E. coli (Upadhaya et al., 2016). The genera Ruminococcus and Alistipes and the family Lachnospiraceae are believed to be involved in carbohydrate catabolism (Parker et al., 2020; Vacca et al., 2020; Crost et al., 2023). Dietary and host carbohydrates typically shape the gut microbiota by providing a major source of nutrients for gut microbes inhabiting the gut. In the present study, BM supplementation increased the average abundances of Barnesiella, Ruminococcus, Alistipes, and Lachnospiraceae_unclassified in the cecum of broilers, indicating that the use of BM may promote carbohydrate metabolism. Specifically, the genus Ruminococcus is typically a strict anaerobe in the gut of healthy animals and can grow on fermentable carbohydrates, producing acetate and formate, but not butyrate, as fermentation products (Crost et al., 2023). Alistipes are anaerobic gram-negative bacteria found mostly in healthy human gastrointestinal tract microbiota, and its major acid product involves succinic acid, with minor amounts of acetic, propionic acid, isobutyric, and isovaleric (Rautio et al., 2003; Parker et al., 2020). Lachnospiraceae are potentially beneficial bacteria that participate in the metabolism of various carbohydrates and exhibit strong abilities to degrade pectin and produce acetic and butyric acids through fermentation. Furthermore, our findings regarding SCFA concentrations also support these results, as BM supplementation led to significant increases in the levels of acetic acid, propionic acid, and total SCFAs, which are essential for maintaining gut health in animals. A negative correlation between the molar proportion of SCFAs and ileum mucosa-associated opportunistic pathogens and a positive correlation between the molar proportion of SCFAs and colon mucosa-associated beneficial bacteria were observed previously (Plouhinec et al., 2023). These findings support the idea that BM supplementation promotes intestinal health and enhances carbohydrate digestion by regulating the gut microbiota, which is consistent with previous research on weaned piglets (Liu et al., 2023) and broiler chickens (Munyaka et al., 2016).

Changes in dietary energy levels also affected the intestinal microbiota. We found that decreasing ME levels increased the average abundance of the genus Tyzzerella and decreased that of the genera Candidatus_Bacilloplasma and Vibrio in the cecum chyme. Tyzzerella is 1 of the 5 prominent genera of Firmicutes (Sugiyama et al., 2022). While only limited research exists on the biological functions of Tyzzerella, recent studies have reported that Tyzzerella may be involved in the regulation of glucose in animals (Li et al., 2022). An overwhelming abundance of Candidatus Bacilloplasma and Vibrio has been identified in species cultured in freshwater or saltwater systems, and these 2 species have been identified as the main core genera for decapod crustaceans (Foysal 2023). However, they are considered opportunistic pathogens in poultry and can increase the risk of bacterial gastroenteritis in broilers (Šimunović et al., 2020). Therefore, our microbiota results suggest that decreased dietary energy levels are beneficial for inhibiting the growth of harmful bacteria in the cecum. Moreover, as SCFAs are dietary fermentable carbohydrate products, their concentrations are highly dependent on bacterial community composition. Thus, we further examined the changes in the SCFA content of the cecum chyme and noticed increased levels of acetic acid and total SCFA contents with decreasing dietary ME levels. These findings align with a recent study that reported a negative correlation between the molar proportion of SCFAs and intestinal pathogens (Plouhinec et al., 2023).

In summary, our study demonstrates the positive impact of BM supplementation on the growth and digestive efficiency of younger broiler chickens. Although organ weights remained unaffected, BM enhanced GLU levels and digestive enzyme activity. Supplementation with BM or decreasing ME levels affected the intestinal microbial community compositions, especially in inhibiting the growth of harmful bacteria. Additionally, SCFAs concentrations strongly depend on the microbial community, and BM supplementation led to an increase of the acetic acid, propionic acid, and total SCFAs levels, which are essential for maintaining gut health in animals.

Glossary

Abbreviations

ADFI

average daily feed intake

ADG

average daily gain

ALP

alkaline phosphatase

ASV

amplicon sequence variant

BM

β-mannanase

BW

body weight

FCR

feed conversion ratio

GLM

general linear model

GLU

glucose

HDL

high-density lipoprotein

LDL

low-density lipoprotein

ME

metabolizable energy

NSP

non-starch polysaccharides

SBM

soybean meal

SCFA

short-chain fatty acid

TG

triglyceride

Conflict of interest statement

The authors have no competing interests to declare.