1. Introduction
The gastrointestinal tract is composed of an intricate ecosystem of microbiota which plays a fundamental role in nutrient digestion and absorption. The gut microbial profile has a significant impact on overall poultry health, immune response, and growth performance. The resident microbiota is regulated by unique host–microbial and microbial–microbial interactions which have developed through evolution [
1,
2]. However, the microbiota is strongly influenced by dietary composition, environmental factors, and the health status of the animal [
3]. The gastrointestinal tract is the interface between the outside world and the internal body, making it a defensive barrier against harmful pathogens and foreign bodies. Promoting good gut health is fundamental to maintaining a high-performing flock, as healthy birds can devote the majority of their energy uptake to production rather than combating disease. Good gut health results in improved feed conversion ratio, increased weight gain, decreased mortality, and an increased performance index, thus, it is imperative for farmers to promote and maintain good gut health in their flock [
3].
Previously, livestock gut health, particularly in poultry, has been modulated by the inclusion of antibiotics in the diets, as they are able to control pathogens through direct manipulation of the gastrointestinal tract microbiota [
4]. This manipulation can reduce competition between existing microbes due to a decrease in diversity and abundance, resulting in improved digestion, absorption, and metabolism of essential nutrients [
4]. However, over time, animals have become significantly less responsive to antibiotics, and the dose rate has had to increase by 11 times since the initial implementation of antibiotics as growth promotors [
5]. The increasing dosage is synonymous with an increase in antimicrobial resistance, which has become a global emerging threat to public health. Therefore, a movement to eliminate antibiotics from livestock production has emerged. Although this is a necessary change, it presents challenges to farmers who are required to seek alternative additives that will promote gut health and production performance.
Recent literature has deemed enzymes to be an advantageous additive for gut health promotion in broilers [
6]. From hatching to day 7 of life, the broiler gastrointestinal tract undergoes significant morphological changes, which are heavily influenced by diet. In young birds, the gastrointestinal tract is short, and there is rapid passage rate and limited digestion due to the underdevelopment of villi, and a lack of endogenous digestive enzymes secreted. As the bird approaches 2 weeks of age, the secretion and activity of digestive enzymes increase, which improves nutrient utilization and gut functionality. However, the endogenous and mucosal enzymes have different developmental timetables, which has a direct influence on feed digestibility. Exogenous enzymes are used in animal feed to facilitate the breakdown of antinutritional factors, such as dietary fiber and phytic acid, lower the digesta viscosity, prevent over-fermentation and diarrhea, improve the nutritive value of the feed, and improve production performance and feed efficiency of the animal [
7]. Research has demonstrated that the addition of exogenous protease was able to modify the gut morphology, such as increasing villus length and supplementing the lack of endogenous enzyme secretion [
8]. In addition, the addition of exogenous protease allows for a reduction in the amount of diet crude protein with no adverse effect on the feed intake, weight gain, and survivability rates in broilers [
9].
Super-dosing multienzymes is an emerging practice that is thought to improve nutrient digestibility through synergistic enzyme action. As different enzymes target different compounds, multienzymes should be more effective than single-strain enzymes, as there will be more nutrients acquired from the diet. Poultry naturally produce a plethora of digestive enzymes, however, the digestive process with endogenous enzymes alone still leaves up to 25% of feed undigested due to the presence of antinutritive factors, which the animal cannot combat with endogenous enzymes [
7]. There is substantial literature surrounding the effective use of single-strain enzymes, such as phytase, however, publications on multienzyme super-dosing are lacking. Although the literature surrounding this topic is scarce for broilers, Hamdi et al. [
10] found that super-dosing phytase improved meat yield and feed conversion ratio (FCR). Furthermore, a review by Cowieson et al. [
11] concluded that there may be considerable opportunity to improve production performance by super-dosing phytase, however, the exact mechanism is still unknown. Therefore, it is important to investigate the effects of super-dosing multienzymes to fill the knowledge gap surrounding the effects and mode of action of this nutritional strategy.
It is hypothesized that super-dosing multienzymes from day 0 post-hatch can improve gut morphology and nutrient digestibility due to the increased bioavailable nutrient content, and promote a diverse and stable microbial community, which will ultimately improve flock performance.
2. Materials and Methods
All experimental procedures involving the use of animals complied with the “Australian Code of Practice for the Care and Use of Animals for Scientific Purposes” and were approved by the Animal Ethics Committee of the University of Queensland. The animal ethical certificate was obtained prior to the commencement of this trial (number: SAFS/510/18/BIOPROTON).
2.1. Birds and Experimental Design
One-hundred and ninety-two 1-day-old mixed-gender broiler chickens (Ross 308) were purchased from a commercial hatchery (Woodlands Hatchery, QLD) and transferred to the isolation shed, a closed shed with isolated cold room panels at the Queensland Animal Science Precinct (QASP) facility at Gatton Campus, University of Queensland. The birds were weighed and randomly assigned to 1 of 4 experimental groups in a randomized complete block design (RCBD). Each experimental diet was fed to 6 replicate pens (1 × 1 m
2) with 8 birds in each (
n = 48 per experimental group). The experimental diets (
Table 1) were in mash form and included a standard wheat–corn–soybean diet (control) with 4 enzyme inclusion levels (0, 350, 700, and 1000 g/ton). No antibiotics were added as growth promotors. All nutrients were supplied to meet nutrient recommendations (
Table 2). The enzyme Natuzyme (Bioproton Pty. Ltd. QLD, Acacia Ridge, Australia) is a multienzyme blend that includes phytase, xylanase, cellulase, amylase, protease, beta-glucanase, and mannanase at specific activities (
Table 3), currently recommended at 350 g/t by the manufacturer. The birds had ad libitum access to feed and water for the entire trial period. As per standard procedure, the grow-out period was divided into 3 phases (starter diet: day 1–14; grower diet: day 14–28; finisher diet: day 28–42) and nutrient levels were adjusted accordingly as per Ross 308 guidelines. On day 35, a natural indigestible and inert marker, acid insoluble ash, was added at a level of 0.2% to each experimental diet as a dietary digestibility marker. The lighting program, temperature, and humidity followed the Ross 308 guidelines. The lighting program provided 23 h of light at an 30–40 lux intensity and 1 h dark (less than 0.4 lux) for the first 7 days and a minimum of 4 h of darkness and a light period of 10 lux intensity after 7 days. The temperature was set at 32 °C and 40% relative humidity for the first 7 days and there was a 2 °C reduction per week after 7 days until the temperature reached 24 °C at 27 days and 40% relative humidity. The temperature and relative humidity were maintained until the end of the trial.
At the end of the experiment, one bird with a body weight (BW) similar to the mean BW of the pen was euthanized by cervical dislocation and eviscerated to collect gut tissue for gut morphology, ileal digesta for nutrient digestibility, cecal content for microbial profile, and bone samples for bone mineralization studies.
2.2. Gut Morphology
The gastrointestinal tracts from the base of the gizzard down to the rectum were dissected, and sections (~1 cm) were cut from the mid-regions of the duodenum, jejunum, ileum, and cecum, flushed with distilled water, and immersed in 10% formalin solution. Fixed tissues were then loaded into appropriately sized cassettes for further gut histo-morphological analysis. Each fixed intestinal tissue sample was dipped in wax and a 5 mm section was cut and embedded in paraffin (Medite TES Valida embedding station). Embedded intestinal segments were cut at a thickness of 6 μm (Leica semi-automated RM2245 rotary microtome, Leica Microsystems, VIC, Melbourne, Australia) and mounted onto slides. Then slides were stained by hematoxylin and eosin (HE), dried in the oven overnight, and cleaned to be scanned by light microscopy. The slides were scanned by an Aperio ScanScope XT (Leica Microsystems, VIC, Melbourne, Australia) and studied for the villus height, crypt depth, villus width, and the number of goblet cells. Then, the villus surface area was calculated and the villus height to crypt depth ratio measured.
Villus height was measured from the tip of the villus to the crypt between individual villi. Crypt depth was measured from the valley between the bases of the villi to the submucosa. Villus width was calculated from the mean value of villus width at one-third and villus width at two-thirds of the height of the villus. The area between 4 villi was used from 3 cuts per sample to count the number of goblet cells. The average of the 3 measurements was then reported as the number of goblet cells per surface area.
2.3. DNA Extraction and 16S rRNA Gene Amplicon Sequencing
Cecal content samples were collected from 1 bird per pen at 42 days of age, post-mortem. Digesta from the cecum was collected into a 10 mL tube. Dissecting instruments were cleaned with 70% ethanol after use on each bird. Samples were put on ice immediately and stored at −20°C prior to DNA extraction.
For each poultry digesta sample, 50 mg was transferred into a sterile 2 mL screw-cap tube containing sterile 0.1 mm and 1.0 mm zirconia beads (total weight 0.4 g; ratio 1:1). Microbial cell lysis was achieved by bead beating in a Qiagen TissueLyser II (Qiagen, Hilden, Germany; 30 Hz, 60 s, 1 repetition conditions), for total genomic DNA (gDNA) extraction. Post lysis, tubes were left for 2 min, for phase separation of the lysate, and each sample supernatant (~600 μL) was then processed for gDNA extraction and purification using the Maxwell 16 blood DNA purification kit (Promega, AS1010, Madison, WI, USA) and the automated Maxwell 16 MDx instrument (Promega, Alexandria NSW, Australia), according to the manufacturer’s instructions. The instrument was set to standard elution volume (SEV) mode and the gDNA eluted into 300 μL of elution buffer. Thereafter, the gDNA samples were quantified and purity verified with the NanoDrop 1000 spectrophotometer (Thermo Scientific, Brisbane, Australia), and subsequently submitted to the Australian Centre for Ecogenomics (ACE; the University of Queensland, Brisbane, Australia) for amplicon sequencing.
The variable region of the 16S rRNA gene was amplified between the V6 and V8 regions, using the universal forward and reverse primer pair of 926F (5′-TCG TCG GCA GCG TCA GAT GTG TAT AAG AGA CAG AAA CTY AAA KGA ATT GRC GG-3′) and 1392wR (5′-GTC TCG TGG GCT CGG AGA TGT GTA TAA GAG ACA GAC GGG CGG TGW GTR C-3′). The gDNA was fragmented and tagged with specific index adapter sequences on both ends of the gDNA fragments using the Nextera XT DNA Library Preparation kit (Illumina, San Diego, CA, USA), enabling dual-indexed sequencing of pooled libraries. The libraries were pooled and sequencing was then carried out on a 2 × 300 bp V3 MiSeq sequencer (Illumina, San Diego, CA, USA). Post-sequencing, quality control, indexing, quantification, and normalization steps were carried out by the sequence provider.
2.4. Bioinformatics Analysis
Raw data files were provided by ACE as fasta files. Analysis of these sequencing files were carried out by using Quantitative Insight into Microbial Ecology 2 (QIIME2) [
12]. Operational taxonomic units (OTUs) were assigned using the Greengenes 13_8 database [
13], using a threshold setting of 97% sequence identity. These data were then normalized to relative abundance using cumulative sum scaling (CSS), a widely used method for normalizing microbial community composition data, and the data were also log2 transformed to account for the non-normal distribution of taxonomic count data, in Calypso Version 8.84 [
14]. This software was also used to generate α-diversity parameters, hierarchical clustering and principal coordinate analysis (PCoA) plots. To examine the alpha diversity parameters of each sample, rank tests including richness, evenness, Chao1 index, Shannon index, and Simpson’s index were carried out. The richness examined the number of different OTUs present within each sample and the evenness was a measure of the relative abundance of the different OTUs making up the richness. Additionally, PCoA (beta diversity) was conducted based on Bray–Curtis distance metrics focusing on the top 20 OTUs, to explore how the overall microbiota composition differed across the 24 samples and four diets.
2.5. Nutrient Digestibility
Post-processing, the gastrointestinal tract was removed from the carcass. The ileal content was evacuated into a 10 mL O-ring tube and placed on ice and left in a −20 °C freezer until freeze drying occurred. This process was repeated for all 24 birds. The sample was weighed pre-freeze drying and post-freeze drying to calculate dry matter. To determine the ash and organic matter content, 2 g of the sample were placed in a crucible and burned in a muffle furnace for 3 h at 500 °C [
15], and it was calculated by the following equation:
To determine carbon, nitrogen, and sulfur content in the sample, 1 g of sample was placed into a ceramic boat in the Leco CNS 928 combustion analyzer (LECO Australia, NSW, Castle Hill, Australia) and analyzed.
The nutrient ileal digestibility was calculated using the following equation:
where Ni represents a concentration of the nutrient in ileal digesta; Md represents a dietary concentration of marker; Nd represents a dietary concentration of the nutrient under the study; and Mi represents the concentration of marker in ileal digesta [
16].
2.6. Bone Mineralization
The meat was removed from the right tibia bone before freezing the bone samples for mineral analyses. The right tibia bone of 24 chickens were defrosted and broken into small pieces using pliers. The entire tibia bone was collected from the right leg of each broiler chicken at 42 days and cleaned of adhering tissue. Bones were dried to a constant weight at 105 °C, then burned to ash in a muffle furnace at 600 °C. Ash was dissolved in concentrated HCl for mineral determination (5 mL of 6 M hydrochloric acid and 35 mL of distilled water), and the solution was filtered into a 250 mL glass bottle and made up to a final volume of 50 mL with distilled water. Thereafter, the Ca and P levels were measured using a Thermo iCAP 6000 series inductive coupled plasma (ICP) spectrophotometer (Thermo Electron Corporation, Str. Rivoltana, 20090 Rodana, Milan, Italy). The Ca and P composition (g/kg in ash) was calculated using iTEVA Analyst software (Cambridge, UK).
2.7. Statistical Analysis
Analysis of variance was performed on the least square means (LSM) values of the pens as an experimental unit using mixed models of SAS [
17]. All values of
p ≤ 0.05 were deemed statistically significant, and all values of 0.05 ≤
p ≤ 0.1 were considered a tendency. The reported LSM was separated using Tukey’s post hoc test.
5. Conclusions
This study aimed to ascertain whether super-dosing multienzymes would improve gut morphology, change the cecal microbial profile, and improve nutrient digestibility in broiler chickens. Results revealed that Natuzyme super-dosing tended to improve gut morphology, and significantly enhanced nutrient digestibility. The microbial profile was not significantly altered by increasing the Natuzyme dose rate, however, three bacterial species were unique to a higher level of Natuzyme in the diet. It was concluded that a dose of 700 g/t could be recommended to optimize gut morphology, nutrient digestibility, and support gut microbial diversity. Further investigations are required to study the functions and roles of Romboutsia spp., Ruminococcus gauvreauii, and Barnesiella spp. in the poultry digestive system and identify potential links between these species and improved feed utilization and efficiency.