3.1. Growth Performance
In this study, diet did not have a significant impact on daily feed intake (
p ≥ 0.16;
Table 2); however, birds fed the lower crude protein diet had significantly lower final body weight (
p = 0.03). Moreover, ADG from day 0 to 7, from day 7 to 14, and overall ADG (day 0 to 14) were all greater in chicks fed the adequate protein diet (
p ≤ 0.04). Similarly, FCR for all the evaluated periods was improved for chicks fed the adequate protein diet (
p ≤ 0.04). These results concerning bird performance are not surprising since amino acid deficiency is known to reduce growth rate and feed efficiency in broilers. Thus, our results are consistent with the findings from previous studies [
13,
14,
30]. Contrary to what was observed for protein levels in the diet, the presence of protease did not have a significant impact on any of the animal performance traits evaluated, except for FCR assessed in the first week of the study (i.e., days 0 to 7;
p = 0.03). Furthermore, no significant interaction between the diet and protease was observed (
p ≥ 0.19) for any of the performance traits. Collectively, our findings do not support our initial hypothesis that protease would improve growth performance in neonatal chicks.
The mechanism, kinetics, and preferred substrates of exogenous proteases are not well understood [
6]. Proteases break down proteins by hydrolyzing peptide bonds of specific amino acids. Protease selectivity depends on the accessibility of the peptide bonds within the substrate. For instance, denatured proteins are more easily degradable than compact proteins, which resist enzyme action. Moreover, proteases differ in their source (most commercial proteases are isolated from bacteria or fungi), optimal pH, mode of action, and preferred substrate [
3,
6,
31]. Measuring growth performance and nutrient digestibility are common ways to evaluate commercial enzymes; however, these approaches provide little information on how these specific enzymes actually function [
3]. Consequently, many specific questions on how commercial proteases function within the chicken’s gastrointestinal tract remain unanswered.
In previous studies, supplemental protease in the diet from 1 to 14 days had no effect on body weight gain, feed intake, and feed efficiency of chickens fed soybean-meal diets [
32,
33]. Cowieson et al. also failed to observe, on days 7–14, a protease effect on bird body weight gain and feed intake, but protease appeared to increase the gain to feed ratio [
34]. Interestingly, in all of these studies, a positive effect of protease on body weight gain and feed efficiency was observed after 14 days [
32,
33,
34]. This indicates that exogenous protease efficacy can be impacted by the age of the birds. It has been shown that age affects the secretion of endogenous trypsin. Noy and Sklan observed that the release of endogenous trypsin into the duodenum is not very efficient until day 21 of age [
35]. In addition to age, diet is known to influence pancreatic output and enzyme composition [
6]. Although more research is needed, there is evidence that the addition of exogenous protease to the diet reduces pancreatic production and secretion of endogenous proteolytic enzymes [
1,
15,
32,
36,
37,
38], an effect that could even result in a decrease in protein digestibility [
38]. Although pancreatic enzyme secretion was not quantified in the present study, a similar physiological response may have occurred in our birds, resulting in the lack of protease impact on chick performance.
3.2. Microbial Fermentation Byproducts: Ammonia and Short Chain Fatty Acids
Ammonia is a marker of microbial fermentation and a byproduct of the deamination of amino acids [
39]. As shown in
Table 3, the adequate protein diet without protease had numerically greater (
p = 0.13) excreta ammonia concentration than the low protein diet. Lowering dietary crude protein tends to decrease excreta ammonia concentration [
39]; however, in the present study, the differences in crude protein and amino acids between the adequate and low protein diets might not have been extreme enough to result in differences in the excreta ammonia concentration. Additionally, no protease effect or interaction effect between diet and protease was observed on the concentration of excreta ammonia (
p ≥ 0.17). In their meta-analysis, Lee et al. concluded that if birds are performing well, a response due to protease addition is unlikely [
40]. Therefore, the lack of response to protease on excreta ammonia concentration may be due to the fact that both diets in our study provided enough amino acids to meet the birds’ requirements for growth.
Short chain fatty acids are end products of bacterial fermentation of carbohydrates and amino acids.
Table 3 shows that, among all treatments, acetate was found at the greatest concentration (87–89%), followed by butyrate (6–8%), propionate (0.9–2%), and small amounts of the others, which were as expected at the chickens’ age [
9,
10]. The majority of short chain fatty acids were likely produced from the fermentation of carbohydrates that escaped digestion in the small intestine. Some gastrointestinal microbes ferment amino acids and can deaminate them rapidly [
41]. Therefore, the trend (
p = 0.09) for a greater concentration of short chain fatty acids in the excreta of chicks fed the diet with higher protein level seems logical.
Reduction in dietary crude protein tended to increase propionate concentrations (
p = 0.08). In addition, while the molar proportions of propionate were decreased by the addition of protease in the diet with adequate protein, adding protease to the low protein diet resulted in an increase in propionate (
p = 0.02 for the interaction diet × protease). Branched chained fatty acids, such as isovalerate, isobutyrate, and valerate, are primarily attributed to protein fermentation [
39]; however, none of the treatments impacted either valerate or isobutyrate. For isovalerate, a trend was observed for both the type of diet and for the inclusion of protease (
p = 0.09). More specifically, excreta from birds fed the adequate protein diet had a greater proportion of isovalerate than from birds fed the low protein diet (0.73% vs. 0.50%, respectively); and isovalerate proportions for no protease versus 200 g/t protease were 0.74% and 0.49%, respectively. Assuming that protease increases protein digestibility, the amino acids and polypeptides absorbed by the chicken would increase. In this scenario, protease could decrease the amount of amino acids available to bacteria, potentially decreasing the total and individual short chain fatty acid concentrations [
42,
43]. However, in the present study, protease had essentially no effect on short chain fatty acid concentrations. The lack of impact on excreta ammonia and short chain fatty acid concentrations further contradicts our original hypothesis that protease would improve protein digestibility.
3.3. Microbial Diversity of Excreta Microbiota
Diet had little influence on the alpha diversity indices (
Table 4). The two microbial richness indices shown—number of observed OTUs and Chao1—as well as Faith’s phylogenetic diversity index were not significantly changed by diet. However, another microbial diversity index (Shannon diversity index) tended to be lower (
p = 0.07) in the excreta of chicks fed the low protein diet. Additionally, the microbial population tended to be more evenly distributed in the adequate protein diet compared with the low protein diet (Evenness
p-value = 0.06). Inclusion of protease had some effects on both richness and diversity of the microbial populations: the number of observed OTUs was increased (
p = 0.04), and Chao1 tended to be increased (
p = 0.09) when protease was added to the diet. Similarly, Faith’s phylogenetic diversity was increased (
p = 0.05) by protease inclusion. No interactions between diet and protease were observed for any of the alpha diversity indices. The principal coordinate analysis describing β-diversity (
Figure 1) showed no differentiation (
p = 0.99) between the microbial populations of chickens fed adequate protein diets and those fed low protein diets. Likewise, the inclusion of protease resulted in no β-diversity changes (
p = 0.99). The first three principal components accounted for 32.18% of the variance.
3.4. Microbial Composition of Excreta Microbiota
As shown in
Table 5,
Firmicutes (40.9–53.6%) had the highest relative abundance, followed by
Proteobacteria (34.0–53.9%),
Bacteroidetes (2.8–10.8%), and
Actinobacteria (0.3–1.1%). Tong et al. [
44] and Singh et al. [
45] reported that chickens at age 6-7 weeks had the following bacterial relative abundance in their excreta:
Proteobacteria (46.4–78.8%),
Firmicutes (12.0–27.5%),
Bacteroidetes (7.1–27.2%), and
Actinobacteria (0.8–1.9%) [
46,
47]. Oakley and Kogurt found that
Firmicutes dominated the microbiota almost exclusively after week 1, and their study ended after 6 weeks [
11]. In studies involving pasture-raised chickens, Lourenco et al. also found a predominance of
Firmicutes in both the cecal contents and excreta of broilers, even at earlier ages (i.e., one-day-old chicks), regardless of their diets [
46,
47]. Therefore, it appears that the age of the bird, the surrounding environment, diet, and genetics all impact the composition of the chicken microbiota and account for differences in their microbial populations [
10,
16,
48].
Diet tended to decrease the abundance of Actinobacteria (
p = 0.09), while protease tended to decrease abundance of
Proteobacteria (
p = 0.09) and increase the presence of
Bacteroides (
p = 0.09); however, neither diet nor protease significantly changed
Firmicutes (
p ≥ 0.29). Excreta
Firmicutes and
Bacteroides have been linked to nutrient absorption. An increase in
Firmicutes could lead to greater nutrient absorption, whereas an increase in
Bacteroides could decrease nutrient absorption [
11,
49]. The ratio of
Firmicutes to
Bacteroides in our study had high variability both within and between treatment groups (SEM = 69.56); therefore, no diet or protease effect was observed. Protease could have decreased nutrient absorption, as indicated by the increased presence of
Bacteroides; however, since protease had no effect on overall body weight gain and FCR, any changes that might have occurred in nutrient absorption were likely of small magnitude.
At the genus level, an unclassified genus from the family
Enterobacteriaceae accounted for most of the relative abundance (27.1–45.3%) in the excreta of the chicks, followed by
Lactobacillus (11.6–25.8%),
Enterococcus (6.8–12.6%),
Bacteroides (1.2–10.2%), an unclassified member of the family
Plancoccaceae (1.6–7.3%),
Klebsiella (2.5–5.6%),
Ruminococcus (2.3–4.8%),
Proteus (0.6–4.8%),
Acinetobacter (1.0–3.7%), and other minor genera (
Table 6). Bacterial substrate preferences, growth requirements, and nutrient availability in the digesta determined this distribution of the bacterial population within the chick’s microbiota [
12].
An unidentified member of the family of
Enterobacteriaceae accounted for the majority of bacteria in this study. The classification of
Enterobacteriaceae includes 44 genera and 107 species. Genera
Alterococcus, Brenneria, Buttiauxella, Cedecea, Citrobacter, Edwardsiella, Erwinia, Escherichia, Leminorella, Pantoea, Pectobacterium, Photorhadus, Salmonella, Serratia, Shigella, Xenorhabdu, and
Yersisna are some of the best-known members of the
Enterobacteriaceae family. All require glucose, vitamins and amino acids for growth [
50]; however, the unclassified member of the family
Enterobacteriaceae detected in the present study was not significantly influenced by diet (
p = 0.41), protease (
p = 0.21), or their interaction (
p = 0.14).
Lactobacillus populations were not affected by protease or diet, but an interaction between diet and protease was observed (
p = 0.02): while the abundance of
Lactobacillus was decreased by the presence of protease in the adequate protein diet, the opposite effect was observed in the low protein diet.
Lactobacillus species are found throughout the digestive tract, predominantly in the small intestine. They are thought to contribute to nutrient absorption and are involved with bile salt hydrolysis [
16,
51].
Lactobacillus are gram-positive and facultatively anaerobic, and are fastidious with complex nutritional requirements, including fermentable carbohydrates, amino acids, peptides, vitamins, salts, and fatty acids; however, each
Lactobacillus species usually has characteristic nutrient requirements, and require a different profile of amino acids [
52]. Apajalahti and Vienola hypothesized that protease would decrease lactobacilli located in the small intestine [
42]; however, our data do not support this hypothesis, as protease did not consistently decrease excreta lactobacilli.
The reduction in protein in the diet decreased the relative abundances of
Proteus and
Acinetobacter (
p = 0.01), which is logical given that both
Proteus and
Acinetobacter utilize amino acids as substrates.
Proteus are gram-negative, facultatively anaerobic bacteria, known to deaminate phenylalanine and tryptophan, decompose tyrosine, hydrolyze urea, and catabolize glucose and other carbohydrates [
53].
Acinetobacter bacteria are gram-negative and aerobic. Most
Acinetobacter grow in media containing a single source of carbon and energy, and they frequently use amino acids as their sole source of nitrogen [
54].
3.5. Microbial Correlation with Feed Efficiency
Regression analysis identified strong associations between the genera
Proteus and
Bacteroides with overall FCR, and
Figure 2 summarizes those relationships. Our data revealed that while
Bacteroides had a positive relationship with FCR (
= 0.60;
p = 0.005),
Proteus had a negative relationship (
= −0.57;
p = 0.009). Since in the present study FCR was expressed as the ratio of feed consumed:body weight gain, lower values indicate a better FCR (more efficient birds). Consequently, a greater abundance of
Bacteroides was associated with poorer feed efficiency, whereas a greater abundance of
Proteus was associated with improved feed efficiency. Our results are in line with the ones reported by Singh et al. [
45], who found a greater abundance of
Bacteroides in the excreta of broilers that had poorer FCR. In humans, Jumpertz et al. [
49] found that an increase in the population of
Bacteroides led to a decrease in nutrient absorption. Regarding the genus
Proteus, Singh et al. [
55] reported that birds with better feed conversion had a lower abundance of this genus in their excreta; however, in their study, FCR was assessed during the last two weeks of broilers’ life cycle (from 35 to 49 days-old). In contrast, in our study, FCR was assessed in the first two weeks of boilers’ life, which may explain these contradictory results. Furthermore, the association of
Proteus with improved animal performance has been demonstrated: the addition of
Proteus spp. to the diets of fish at 4 g/kg resulted in improved weight gain, increased body length, and improved FCR [
56], which is in line with our results.