Effect of Dietary Supplementation with Different Proportions of Amaranthus hypochondriacus Stem and Leaf Powder on Intestinal Digestive Enzyme Activities, Volatile Fatty Acids and Microbiota of Broiler Chickens

Abstract

Amaranthus hypochondriacus is rich in nutrients and represents a feed resource with significant potential. This experiment aimed to study the effects of different proportions of Amaranthus hypochondriacus stem and leaf powder (AHSL) on intestinal digestive enzyme activities, cecal volatile fatty acids, and the microbiota of broilers. A total of 288 one-day-old male broilers (Ross 308) were randomly assigned to the control diet group and the 3%, 6%, and 9% AHSL diet group for days 0–21. Subsequently, the 3%, 6%, and 9% AHSL diets were switched to 5%, 10%, and 15% AHSL diets for days 22–42. The results demonstrated that the dietary inclusion of AHSL significantly affected sucrase activity in the jejunal mucosa (p < 0.05). The 5% AHSL group exhibited the highest sucrase activity, followed by the control group, both of which showed significantly higher activity compared to the 10% and 15% AHSL groups (p < 0.05). The cecal pH in the 5%, 10%, and 15% AHSL groups was significantly lower than that in the control group (p < 0.05). The contents of acetate, propionate, butyrate, and valerate in the cecum of the 10% and 15% AHSL groups were significantly higher than those of the control group (p < 0.05). The addition of AHSL had no significant effect on the alpha diversity of cecum microorganisms. The relative abundance of Ruminococcaceae_UCG_005 and Lactonifactor was significantly higher in the 10% AHSL group compared to the control group, whereas the 15% AHSL group had a significantly higher relative abundance of Clostridium_sensus_tricto12, Peptoclostridium, Anaerofilum, and Peptococcaceae. In summary, the inclusion of 5% AHSL in the diet enhances sucrase activity in the jejunum of broilers, while 10% or 15% AHSL increases the volatile fatty acid content and reduces the pH value in the cecum, without adverse effects on the cecal microbiota.

1. Introduction

In recent years, there has been a growing demand for animal feed ingredients, placing significant pressure on raw materials such as maize and soybean, which are vital both for animal feed and human diets. Consequently, exploring alternative sources of energy and protein feeds has become imperative [1]. One crop that could fulfill this role is amaranth as it offers a balanced combination of energy and protein, making it a potential substitute or complement to these cereals. In fact, amaranth (Amaranthus spp.) has been consumed for centuries as both a green leafy vegetable and as a grain [2] but has received little attention until recent studies described its high nutritional value and agricultural potential. Currently, three primary species of amaranth are cultivated globally as cereals: Amaranthus cruentus, Amaranthus caudatus, and Amaranthus hypochondriacus. Amaranthus hypochondriacus has been cultivated worldwide and is characterized by its rapid growth, adaptability, high yield, and tolerance of high temperatures [3], as well as its regenerative capacity, which allows it to be harvested several times. The energy provided by grain amaranth is comparable to other cereals (millet, sorghum, rice, wheat, and corn), while the protein content is twice as high. On average, amaranth grain comprises 13.1–21.0% of crude protein [4], while the leaves can contain between 17.2% and 32.6% on a dry weight basis [5]. Moreover, amaranth grain has a balanced content of essential amino acids (rich in lysine and sulfur-containing amino acids) [6,7] and is rich in bioactive compounds (tocopherols, flavonoids, polyphenols, and squalene). In addition, amaranth also contains 6.24% to 9.57% (with an average of 7.89%) of dietary fiber, which contributes significantly to the gut health, digestibility, and palatability of animals [8]. These characteristics make amaranth a good source of high-quality protein and a substitute for some cereals in the feed mixtures of animals.

The grain, leaves, and stems of amaranth have been utilized as a forage or silage crop for numerous animals, such as cattle, sheep, chickens, pigs, and rabbits [9]. In poultry, Mbugua et al. [10] reported that diets comprising 20% or 40% inclusion levels of Amaranthus hypochondriacus grain were beneficial in improving the body weight, feed intake, feed efficiency, and carcass fat in 8-week-old broiler chicks compared to a maize–soybean meal control diet. Písaříková et al. [11] examined the impacts of different forms of amaranth (raw grain, heat-processed grain, dried above-ground biomass) as substitutes for animal protein (fish meal) on the body weight, feed efficiency, carcass traits, and meat quality of broiler chickens and observed no adverse effects of the diets with amaranth. Broiler chickens fed diets supplemented with 10% amaranth also exhibited superior meat characteristics including taste, tenderness, texture, and color compared to diets containing fish meal [12]. Moreover, the inclusion of raw amaranth grain in the diet had a beneficial impact on egg quality traits, which could reduce cholesterol levels in eggs and improve the health status of laying hens [13]. Thus, amaranth as a feed ingredient has a positive effect on improving the performance and product quality of poultry.

Dietary components and microbiota are deeply interconnected with the nutritional state and overall health of the host [14]. As dietary elements traverse the gastrointestinal tract, they can be metabolized by bacteria before being absorbed [15]. In particular, the microbiota residing in the cecum and distal colon play a crucial role in the fermentation of dietary fibers, leading to the production of end products like volatile fatty acids (VFAs) [16]. Amaranth is rich in polyphenols and dietary fibers; these components have been demonstrated in other studies to have positive effects on the gut microbiota of animals and humans [17,18]. To date, there are few reports regarding how amaranth can modify the profile of cecal microbiota and influence the generation of VFAs and whether this potential change could have positive effects on digestive enzymes in poultry. Therefore, the aim of this study was to assess the effects of varying proportions of Amaranthus hypochondriacus stem and leaf powder (AHSL), included in experimental diets, on broiler gut health, specifically focusing on intestinal digestive enzyme activities, cecal volatile fatty acids and microbiota composition.

2. Materials and Methods

2.1. Experiment Design and Diets

A total of 288 one-day-old healthy male broilers (Ross 308) with similar body weights (46.63 ± 0.33 g) were selected for the experiment. These broilers were randomly assigned to four dietary treatment groups. Each group had eight replicate cages (1.5 m × 1.2 m × 0.4 m), with 9 chickens per cage. The AHSL contained a high proportion of crude fiber (34.20%) [19]. To prevent adverse effects on the digestion and health of chicks due to excessive dietary fiber, we first add a low level of AHSL to allow the chicks to adapt to the experimental diets. The control group was fed a basal diet, and the three experimental groups were fed 3% AHSL, 6% AHSL, and 9% AHSL diets, respectively, for 0–21 days. Then, the 3%, 6%, and 9% AHSL diets were changed to 5%, 10%, and 15% AHSL diets, respectively, for 22–42 days. Prior to diet formulation, the stem and leaf of Amaranthus hypochondriacus (Fuxian Agricultural Technology Co., Ltd., Xiaogan, China) was ground into a fine powder. According to our previously reported chemical composition of AHSL (ME: 3.52 MJ/kg; CP: 16.7%; CF: 34.20%; EE: 1.70%) [19], the experimental diets containing AHSL that met the nutritional requirements of broiler chickens were formulated. The ingredient and nutrient compositions of the diets are shown in

Table 1. Ingredient and nutrient compositions of experimental diets.

Items	0–21 d				22–42 d
	Control	3% AHSL	6% AHSL	9% AHSL	Control	5% AHSL	10% AHSL	15% AHSL
Ingredient composition (%)
Corn	55.6	52.17	48.58	44.95	59.88	54.1	48.16	42.06
Soybean meal	38.57	38.08	37.61	37.15	33.38	32.56	31.78	31.03
Soybean oil	2.61	3.7	4.95	6.16	3.5	5.38	7.37	9.5
AHSL	0	3	6	9	0	5	10	15
CaHPO4	1.3	1.34	1.38	1.415	1.05	1.1	1.2	1.26
Limestone meal	1.33	1.12	0.92	0.705	1.45	1.12	0.75	0.41
NaCl	0.35	0.35	0.35	0.35	0.35	0.35	0.35	0.35
Multi vitamin 1	0.04	0.04	0.04	0.04	0.04	0.04	0.04	0.04
Methionine	0.2	0.2	0.2	0.2	0.15	0.15	0.15	0.15
Mineral meal					0.2	0.2	0.2	0.2
Total	100.00	100.00	100.00	100.00	100.00	100.00	100.00	100.00
Nutrient level
ME, MJ/kg	12.20	12.17	12.18	12.19	12.58	12.55	12.55	12.57
CF, %	2.16	2.69	3.22	3.75	2.06	3.65	5.24	6.83
CP, %	21.51	21.51	21.51	21.51	19.51	19.51	19.51	19.51
Ca, %	1.00	1.00	1.00	1.00	0.95	0.95	0.96	0.96
P, %	0.69	0.68	0.68	0.68	0.61	0.60	0.61	0.60
Lysine, %	1.29	1.26	1.24	1.22	1.14	1.10	1.07	1.03

¹ Multi vitamin supply of full-price feed per kg for broilers: Vitamin A 12500 IU, Vitamin D3 2500 IU, Vitamin E 30 IU, Vitamin K3 2.65 mg, Vitamin B12 mg, Vitamin B2 6 mg, Vitamin B12 0.025 mg, Biotin 0.0325 mg, folic acid 1.25 mg, niacin 12 mg, niacin 50 mg, b: nutrient level is calculated value.

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2.2. Animal Management and Sampling

The animal handling and management procedures were reviewed and approved by the Ethics and Research Committee of Wuhan Polytechnic University (Hubei, China; approval number: 2010-0029). Before the broilers’ arrival, the experimental facility, along with the feed and water troughs, was thoroughly cleaned and disinfected. The broilers were housed in steel floor cages within a climate-controlled environment that provided continuous lighting, and they had ad libitum access to both feed and water. The initial room temperature was set at 35 °C and was reduced by 3 °C each week until it reached 23 °C. For the first three days, lighting was provided for 23 h followed by 1 h of darkness and was subsequently adjusted to 18 h of light and 6 h of darkness. The relative humidity in the birdhouse was maintained at 55%. During the first week, all chicks were vaccinated against broiler coccidiosis through their drinking water and were vaccinated against the Newcastle disease virus on approximately days 7 and 14.

On day 42 of the experiment, following a 4 h fasting period, eight broilers were randomly chosen from each group based on their average body weight (one broiler per replication) and were subsequently slaughtered. After slaughter, the jejunum and cecum were separated, and the internal pH value of the cecum was determined using a pH meter (FE28-Standard, METTLER TOLEDO; Zurich, Switzerland), and then the jejunal and cecal chyme were collected for freezing and preservation. The jejunal segments were rinsed with saline, after which the mucosa was peeled off with a slide into a 2 mL freezing tube and stored at −80 °C for further analysis.

2.3. Digestive Enzyme Activity

After the samples were thawed, 100 mg of mucosa or chyme samples were accurately weighed, and 9 times the volume of homogenization medium was added at the ratio of weight (g)/volume (mL) = 1:9. The samples were mechanically homogenized in an ice-water bath, followed by centrifugation at 2500 rpm for 10 min, and the supernatant was taken for measurement. The maltase (A082-3-1, Njjcbio; Nanjing, China) and sucrase (A082-2-1, Njjcbio; Nanjing, China) activities of the small intestine mucosa and the trypsin (A080-2-1, Njjcbio; Nanjing, China) and amylase (C016-1-1, Njjcbio; Nanjing, China) activities of the jejunal chyme were determined using commercially available assay kits. The methods were strictly in accordance with the operating instructions for processing.

2.4. Cecal Volatile Fatty Acids

After the thawing of the cecum chyme, 0.5 g of the sample was accurately weighed in a 2 mL centrifuge tube, and 1.5 mL of distilled water was added and mixed for 30 min and then centrifuged at 4 °C and 15,000 rpm for 15 min. Then, 1 mL of supernatant was taken and 0.2 mL of 25% metaphosphoric acid was added, mixed well, and left to incubate at 4 °C for 30 min and centrifuged at 4 °C and 15,000 rpm for 15 min, and the supernatant was filtered through a 0.22 μm filter membrane. The contents of acetic acid, propionic acid, isobutyric acid, butyric acid, isovaleric acid, and valeric acid were determined by gas chromatography–mass spectrometry (Agilent 7890, Palo Alto, CA, USA).

2.5. 16S rDNA Amplicon Sequencing

DNA from cecum digesta was extracted using the E.Z.N.A. ^® Stool DNA Kit (D4015, Omega, Inc.; Norcross, GA, USA) according to the manufacturer’s instructions. The extracted DNA was stored at −20 °C until further analysis. The DNA concentration and quality were assessed using a NanoDrop spectrophotometer (ND-1000, Thermo Fisher, Waltham, MA, USA) and agarose gel electrophoresis.

The V3-V4 region of 16S rRNA genes was amplified using the 338F-806R primer set (338F: 5′-ACTCCTACGGGAGGCAGCA-3′. 806R:5′-GGACTACHVGGGTWTCTAAT-3′). The sample specificity 7-bp barcode was added to the primer for multiplex sequencing. The thermal cycle included an initial denaturation of 98 °C for 2 min, followed by 25 cycles consisting of a denaturation of 98 °C for 15 s, annealing at 55 °C for 30 s, an extension of 72 °C for 30 s, and a final extension of 72 °C for 5 min. PCR amplicons were purified using Agencourt AMPure Beads (Beckman Coulter, Indianapolis, IN, USA) and quantified using the PicoGreen dsDNA Assay Kit (Invitrogen, Carlsbad, CA, USA). After the individual sample was quantified, the amplicon was collected in equal amounts, and pair-end 2 × 300 bp sequencing was performed using the Illumina MiSeq platform with MiSeq Reagent Kit v3 at Hangzhou Mingke Biotechnology Co., Ltd. (Hangzhou, China).

Paired-end reads were assigned to samples based on their unique barcodes and truncated by cutting off the barcode and primer sequence. These paired-end reads were merged using FLASH (v1.2.8). Quality filtering on the raw reads was performed under specific filtering conditions to obtain high-quality clean tags according to the FQTRIM (v0.94). Chimeric sequences were filtered using Vsearch software (v2.3.4). After dereplication using DADA2 (v1.8), we obtained the feature table and feature sequences. Alpha diversity and beta diversity were calculated by Quantitative Insights into Microbial Ecology (QIIME), in which the same number of sequences were extracted randomly by reducing the number of sequences to the minimum of some samples, and the relative abundance (X bacteria count/total count) was used in bacteria taxonomy. Alpha diversity and beta diversity were analyzed by the QIIME2 (version 2020.11) process, and pictures were drawn by R (v3.5.2). The sequence alignment of species annotation was performed by BLAST, and the alignment database was SILVA and NT-16S. LEfSe (linear discriminant analysis effect size) was performed to detect differential abundant taxa across groups using the default parameters (LDA score log10  ≥  2.0).

2.6. Statistical Analysis

The experimental data were analyzed using a one-way ANOVA with IBM SPSS Statistical 26.0 (SPSS Inc., Chicago, IL, USA). Duncan’s multiple range tests were used to determine statistically significant differences, with the significance level set at p < 0.05. The results are presented as means with the standard error of the means (SEM).

3. Results

3.1. Digestive Enzyme Activity in the Jejunum of Broilers

The results for jejunal digestive enzyme activity are summarized in

Table 2. Effect of Amaranthus hypochondriacus stem and leaf powder on digestive enzyme activity of jejunum in broilers.

Items	Control	5% AHSL	10% AHSL	15% AHSL	SEM	P
						Treat	Linear	Quadratic
Jejunal mucosa, U/mg protein
Maltase	531.91	547.64	541.92	568.23	36.852	0.989	0.944	0.972
Sucrase	186.89 ab	212.92 a	142.66 b	154.25 b	9.495	0.026	0.221	0.477
Jejunal chyme, U/mg protein
Amylase	1.21	1.03	1.04	1.07	0.099	0.925	0.977	0.625
Trypsase	3470.73	3204.83	3263.79	3316.08	76.379	0.664	0.446	0.749

Note: different lowercase letters in the same row indicate significant differences (p < 0.05), and the same letters or no letters indicate no significant differences (p > 0.05).

. The inclusion of different proportions of AHSL in the feed did not induce any significant changes in the activity levels of amylase and trypsin in the jejunal contents (p > 0.05). Additionally, there was no discernible variation in maltase activity within the jejunal mucosa (p > 0.05). However, the inclusion of AHSL in the diet significantly impacted the activity of sucrase in the jejunal mucosa (p < 0.05). The highest sucrase activity was observed in the 5% AHSL group, followed by the control group; both groups displayed significantly greater sucrase activity relative to the 10% and 15% AHSL groups (p < 0.05).

3.2. Fermentation Parameters of the Cecum in Broilers

As presented in

Table 3. Effect of Amaranthus hypochondriacus stem and leaf powder on cecal pH and volatile fatty acids in broilers.

Items	Control	5% AHSL	10% AHSL	15% AHSL	SEM	p Value
						Treat	Linear	Quadratic
pH	7.32 a	7.06 ab	6.65 b	6.66 b	0.084	0.005	0.001	0.002
Acetate, μg/g	1150.00 b	1419.73 b	2616.83 a	2633.51 a	147.81	0.001	0.001	0.001
Propionate, μg/g	298.72 b	493.47 a	447.2 a	442.70 a	18.72	0.001	0.414	0.004
Iso-butyrate, μg/g	98.00 a	94.93 a	97.18 a	76.96 b	2.6	0.008	0.056	0.113
Butyrate, μg/g	261.31 b	336.93 b	461.65 a	439.30 a	22.34	0.001	0.004	0.001
Iso-valerate, μg/g	143.55 a	151.64 a	141.05 a	109.73 b	4.69	0.005	0.021	0.021
Valerate, μg/g	102.42 b	112.15 b	115.50 a	115.28 a	1.87	0.042	0.133	0.049

Note: different lowercase letters in the same row indicate significant differences (p < 0.05), and the same letters or no letters indicate no significant differences (p > 0.05).

, the cecal pH was significantly reduced in the 5%, 10%, and 15% AHSL groups compared to the control group (p < 0.05). In terms of volatile fatty acids, the concentrations of acetate, propionate, butyrate, and valerate were significantly elevated in the 10% and 15% AHSL groups compared to the control group (p < 0.05), demonstrating a quadratic linear correlation with the levels of AHSL inclusion (p < 0.05). The levels of iso-butyrate and iso-valerate were significantly diminished in the 15% AHSL group compared to the control group (p < 0.05), with iso-valerate also illustrating a quadratic linear correlation with the AHSL inclusion levels (p < 0.05).

3.3. Cecum Microbiota

The composition of cecal microbiota was further analyzed using 16S rRNA sequencing. Alpha diversity indices, including observed OTUs, Shannon, Simpson, and Chao1, were employed to assess the diversity of the cecal microbiota. The results showed that the inclusion of AHSL in the diets did not significantly alter the diversity of the cecal microbiota (p > 0.05, Figure 1A). The principal component analysis (PCA) results showed no difference in the microbial composition of the cecum microbiome between the control and AHSL groups (p > 0.05, Figure 1B). At the phylum level, the broilers’ cecal biota predominantly consisted of Firmicutes, Bacteroidetes, Tenericutes, Proteobacteria, and Cyanobacteria. Notably, Firmicutes, Bacteroidetes, and Tenericutes constituted the majority, contributing to over 95% of the total microbial community abundance (Figure 2A). At the genus level, the most abundant dominant bacterium was found to be Lachnospiraceae_uncultured. The other dominant genera, which included Bacteroides, Parabacteroides, Ruminococcaceae_uncultured, [Ruminococcus] torques group, Faecalibacterium, Ruminococcaceae UCG-005, Subdoligranulum, Lactobacillus, and Christensenellaceae R-7 group (Figure 2B), collectively accounted for more than 55% of the total microbial community abundance (Figure 2B).

The differentially abundant microbes between the control group and the AHSL groups were analyzed by linear discriminant analysis (LDA) effect sizes (LEfSe). A total of 12 taxa displayed a significant difference in their abundance between chickens in the control and AHSL groups at a stringent cutoff value (absolute LDA score log10  ≥  2.0). These taxa are depicted in Figure 3A,B. The relative abundance of Ruminococcaceae_UCG_005 and Lactonifactor was significantly higher in the 10% AHSL group compared to the control group. The relative abundance of Clostridium_sensus_stricto12, Peptoclostridium, Anaerofilum, Peptococcaceae, Defluviitaleaceae_UCG_011, and Defluviitaleaceae was significantly elevated in the 15% AHSL group compared to that of the control group. On the other hand, the abundance of Lachnospiraceae_NK4A136_group, Shuttleworthia, Ruminococcus_gauvreauii_group and Klebsiella was significantly lower in the 10% and 15% AHSL group than in the control group.

4. Discussion

In our previous findings, we demonstrated that varying AHSL inclusion levels had no impact on the growth performance of broilers at both 21 and 42 days of age [19]. The findings align with those of other researchers who reported no negative impact on growth performance when broiler chickens were fed an amaranth-based feed mixture [20,21,22,23]. However, another study reported that including amaranth pellets in the diet improved the body weight, feed intake, feed efficiency, and carcass fat of 8-week-old broiler chickens [10]. These results suggest that amaranth can partially replace grains in broiler feed while achieving production performance comparable to traditional grain-based diets.

Digestive enzyme activity contributes to the improved feed utilization and availability of enteral nutrition, which plays a vital role in enhancing the growth performance of animals [24,25]. The important disaccharidases in the small intestine of animals are sucrase, maltase, and lactase, which are key enzymes in carbohydrate digestion and absorption and play an important role in sugar absorption [26]. Trypsin and amylase levels reflect the absorption and utilization of protein and starch, respectively [27,28]. The results of this experiment showed that diets with AHSL inclusion had no effect on jejunal maltase, amylase, and trypsin, but the activity of sucrase showed a trend of increasing and then decreasing with an increase in the AHSL inclusion level in the diet, and its activity was highest in the 5% AHSL group. The increased activity of sucrase suggests that AHSL may enhance the digestion and absorption of carbohydrates in broilers. Shang et al. [29] reported that diets supplemented with 3% wheat bran (high dietary fiber) could increase the activity of intestinal digestive enzymes (amylase and trypsin) in broilers. Chen et al. [30] demonstrated that elevating dietary fiber levels increased ileal sucrase and maltase activities in pigs. These data demonstrated the role of dietary fiber in promoting digestive enzyme activity in the small intestine of animals. Amaranth is an excellent source of insoluble and soluble fiber, and both are known to promote gut health [31]. Our previous measurements also showed that AHSL contains 34.2% crude fiber [19]. Therefore, the increase in jejunal sucrase activity in broilers may be related to the dietary fiber provided by AHSL, but AHSL levels need to be controlled. Khokhar [32] reported that dietary fibers from different sources (at levels of 5% or 10%) had a negative impact on the activity of disaccharidases (sucrase and maltase) in the small intestine of rats, and the activities of these enzymes decreased further with the increase in dietary fiber. In poultry, the inclusion of fiber such as cellulose at levels of 3–5% in the diet has been shown to enhance nutrient utilization and stimulate pancreatic enzymatic activity, but the excessive supplementation of fiber can disrupt normal digestive processes by forming coating structures that inhibit the activity of digestive enzymes and their accessibility to nutrients [32,33]. When the inclusion level of AHSL in the diet exceeds 10%, the crude fiber content in the broiler diet already exceeds 5%. Consequently, the groups with 10% and 15% AHSL exhibited reduced sucrase activity due to the inhibitory effects of excess crude fiber.

Microorganisms residing in the cecum can ferment dietary fibers into volatile fatty acids (VFAs), which play a crucial role in poultry wellbeing by promoting energy regulation, microbial homeostasis, mucosal integrity, and immune stability [34]. In broiler and laying-hen chicks, the feeding of a fiber-rich diet has been shown to affect the cecal microbial population and increase the production of VFAs [35,36]. Amaranth is a good source of dietary fiber, of which around 28% is soluble and consists mainly of branched xylans, dominated by disaccharide and trisaccharide side chains and pectic polysaccharides [18]. In addition, its fiber contains more than 25% water-soluble β-(1,3)-D-glucan [37]. In the present study, we observed that a diet with AHSL inclusion decreased cecum pH and increased the content of acetate, propionate, butyrate, and valerate in the cecum of broiler chickens. These findings suggest that the dietary fiber within AHSL is continuously fermented in the cecum, which leads to an increase in the VFAs produced by microbial fermentation in the cecum. VFAs have the capacity to release hydrogen ions (H⁺) and lower the pH in the hindgut, which can prevent the invasion and colonization of pathogens [34]. Butyrate can also consume luminal oxygen to create an anaerobic environment, subsequently limiting the proliferation of aerobic pathogens such as Salmonella within the gut lumen [38]. Moreover, the intake of dietary fructooligosaccharide (dietary fiber) has also been shown to enhance the production of VFAs and reduce the colonization of harmful bacteria such as Salmonella spp., Clostridium perfringens, and Escherichia coli in broiler chickens [39]. Therefore, amaranth, a good source of dietary fiber, could increase the production of VFAs and promote intestinal health in broilers when added to their diet.

The gastrointestinal microbiota exists in a symbiotic relationship with its host through the regulation of digestion, intestine development, nutrient absorption, as well as the innate and adaptive immune system [40]. In reverse, the composition of microbiota is also affected by feed ingredients. In the current study, no significant differences in the richness of cecum microbes between the control and AHSL groups were observed. We thus speculated that induced microbe perturbation relating to AHSL inclusion in the diet is very limited, resulting in a recovery to the initial state instantly. However, it is noteworthy that the abundance of certain microorganisms concerned with fiber degradation and VFA production was significantly up-regulated, including Ruminococcaceae_UCG_005, Clostridium_sensus_tricto12, Peptoclostridium, and Peptococcaceae. Ruminococca-ceae_UCG-005 are very important probiotics in animal intestines, which can secrete cellulase and hemicellulase enzymes to degrade dietary fiber, and produce large quantities of VFAs to provide energy for the animal body [41]. Clostridium sensu stricto help to maintain the structural integrity of the gut and promote the regeneration of the intestinal crypts, and their reduced abundance may cause an inflammatory response in the intestines, resulting in disorders of glucolipid metabolism [42]. In addition, Clostridium sensu stricto and Peptoclostridium are dominant VFA-producing bacterium, which can utilize some macroorganisms to produce volatile fatty acids, mainly acetic acid, butyric acid, and a small amount of propionic acid [43]. Therefore, the addition of AHSL to broiler diets can promote the growth of certain specific beneficial bacteria to increase VFA production in the cecum and further improve the gut immunity and energy metabolism of broilers.

5. Conclusions

The addition of 5% AHSL to diets can increase jejunal sucrase activity in broiler chickens, whereas additions of 10% or 15% decrease sucrase activity. The inclusion of 10% or 15% AHSL in the diet can increase volatile fatty acid content and decrease the pH of cecal chyme in broilers, without adversely affecting the cecal microbiota. The recommended inclusion level of AHSL in broiler diets is between 5% and 10%, but further research is needed to find the optimal addition level that can balance digestive enzyme activity and cecal fermentation.