Feed analyses showed minor discrepancies to the calculated nutrient levels, which may have contributed to deviations in the observed results from our hypothesis. The differences between the analysed composition compared to the calculated composition may be due to the 2019 Australian drought, which resulted in a feed supply shortage, which may have led to poorer-quality cereal grains and soybean meal in the diet.
4.1. Growth Performance
The hypothesis for the trial was that multi-enzyme super-dosing would compensate for the reduced-ME content of the diets and that these energy saving effects of the multi-enzyme would restore the growth performance parameters in birds fed with reduced-ME diets. The aim was to re-define matrix values for different multi-enzyme inclusion rates to be used for cost-effective feed formulation practices in the poultry feed industry. Currently, a 70 kcal/kg ME reduction is recommended when 350 g/ton multi-enzyme is supplemented. The results revealed that reducing the dietary ME content did not affect ADG and FBW, which was at the cost of significantly increased ADFI and consequently higher FCR. Super-dosing the multi-enzyme, however, did compensate for reduced ME content and restored the growth performance of broiler chickens, including ADFI and FCR. This fact was evidently reflected in the FCR value for the STD-150 kcal diet with no multi-enzyme inclusion, which was significantly reduced from 1.60 to 1.48 with the 350 and 700 g/ton multi-enzyme inclusion rates. Adding the multi-enzyme at 1000 g/ton did not result in a further improvement to the FCR even though it was still significantly better than STD-150 kcal/kg with no multi-enzyme (FCR 1.51 vs. 1.60 for STD-150 kcal/kg plus 1000 g/ton multi-enzyme and STD-150 kcal/kg with no multi-enzyme). Only birds consuming the STD-150 kcal/kg with no enzyme included had a significantly lower final body weight and ADG. It was expected that the birds on the STD-200 kcal/kg and no enzyme diet would have a worse growth performance than birds fed the STD-150 kcal/kg diets; however, the opposite results were observed, and the birds on STD-200 kcal/kg outperformed the birds on the STD-150 kcal/kg diet. The birds on the STD-200 kcal/kg diet had the highest ADFI, which resulted in the highest daily ME intake, which might be the reason for a heavier final BW and higher ADG in this group compared to the birds on the STD-150 kcal/kg diet.
These production performance results are predominantly positive and are consistent with the current literature [
18,
19]. A lack of significant difference in FCR reveals that multi-enzyme inclusion mitigated the negative effect of reduced ME on FCR, highlighting the energy saving effects of the multi-enzyme. As presented in
Table 3, the birds on a reduced-ME diet and no multi-enzyme inclusion compensated for the energy deficiency by maintaining higher feed consumption (
p ≤ 0.001). The higher feed intake compensates for the reduced dietary ME level, as the total ME intake was increased, and neither final BW, nor ADG were therefore affected by the reduced-ME diets when the multi-enzyme cocktail was added at all levels. Therefore, although there is no difference in ADG between the standard and reduced-ME diets due to the increased feed intake, there is a significant increase in ADFI to compensate for the reduction in energy, which results in differences in FCR between the STD and reduced-ME diets.
The results of the trial demonstrated that the addition of the multi-enzyme cocktail into reduced-ME diets did not affect ADFI, which was consistent with previous reports where multi-enzymes were added to corn–soybean diets [
19]. The birds fed on a negative control diet with reduced nutrient density had poorer weight gain and feed efficiency than those given the positive control diet with the recommended nutrient density. Supplementing the multi-enzyme cocktail to both negative and positive control diets improved the weight gain and feed efficiency compared to the no multi-enzyme inclusion. In the aforementioned study, similar to our findings, there was no effect of multi-enzyme inclusion on feed intake either.
Multi-enzyme super-dosing at 1000 g/ton significantly improved FBW and ADG compared to the control diet (main effects of the multi-enzyme inclusion rate). The overall improvement in performance when the multi-enzyme was added to the diet is due to the increased nutrient bioavailability and ability of the enzymes to combat antinutritional factors present in the ingredients. Corn, soybean meal, and wheat all contain antinutritional factors that decrease performance if not remedied. Soybean meal contains trypsin inhibitors, which block the degradation of protein, thus decreasing the overall availability of protein in the diet [
20], while corn and wheat contain phytic acid, which limits phosphorus, calcium, and zinc bioavailability [
3]. The multi-enzyme cocktail that was evaluated in the present work contains protease, which alleviates the effect of the trypsin inhibitors in soybean meal, as it improves protein hydrolysis in the presence of trypsin and therefore increases the digestible protein content of the diet [
21]. Additionally, the multi-enzyme cocktail also contains phytase, which breaks the bond between phytic acid, phosphorus, calcium, and zinc to increase mineral availability [
22]. However, there was no difference in the final BW, ADG, or FCR between the different enzyme dose rates, suggesting that increasing the dose rate to “super-dosed” levels is not necessary to improve performance, which is consistent with other findings [
5,
23].
Overall, all performance traits in this experiment revealed a positive result in terms of ME level and multi-enzyme interaction. Body weight values for all of the experimental groups were similar and statistically greater than the STD-150 kcal/kg diet. This is to be expected, as the other experimental groups were either a STD diet or diets that had their energy reduction compensated for by the addition of the multi-enzyme cocktail, with the exception of the STD-200 kcal/kg diet. As ADG is a factor of final BW, the observed trend was similar for ADG. However, a point of interest is the better growth performance of the birds on the STD-200 kcal/kg diet compared to the STD-150 kcal/kg diet. This could be attributed to the fact that the STD-200 kcal/kg diet could have triggered the compensatory growth mechanism in the chickens. Compensatory growth occurs when an animal has restricted access to feed or nutrients and consequently increases feed consumption, utilization, and conversion efficiency. Although the STD-150 kcal/kg diet also had an energy reduction, this reduction may not have been sufficient to trigger the metabolic response of compensatory growth. Compensatory growth from feed, energy, and protein restriction has been documented in poultry, with Sunder et al. [
24] and Leeson [
25] observing similar findings. Although compensatory growth enables the birds consuming reduced energy diets to perform similarly to the birds fed a standard diet in terms of FCR, there was a large increase in mortality rate (
Table 3). This indicates that producers cannot improve flock performance solely through manipulating compensatory growth, as the flock mortality rate will be greater. The ROSS308 guidelines (2014) indicate that expected mortality rate in a flock is 5%, whereas reduced energy with no multi-enzyme supplementation yields a greater mortality rate—double that figure. This high incidence of mortality decreased when the multi-enzyme cocktail was added into the reduced energy diets, thus indicating that a multi-enzyme mixture is able to compensate for the reduced ME in the diets.
4.2. Organ Development
Organ development is an important factor in poultry production, and it is crucial to minimize fat depositions in the organs to prevent poor carcass and meat quality. In the present trial, the significantly higher abdominal fat deposition in the STD diet compared to the reduced-ME diets was to be expected, as the ME intake and ME:Protein intake was greater for birds fed with the STD diet. In contrast, the birds on the reduced-ME diets needed to devote and utilize the limited energy supply to maintain functions, thus less energy was left for abdominal fat deposition. Reduced abdominal fat is an advantage in poultry production, as consumers do not desire chicken carcasses with a high fat content [
26].
The ME levels and multi-enzyme dose rate interaction had no significant effects on organ weights, except for gizzard weight. There was a tendency for the gizzard to be heavier in the treatment groups with reduced-ME levels and multi-enzyme supplementation, particularly in the STD-200 kcal/kg diet (
Table 4). This interaction was mainly correlated to the effect of reduced energy diets that tended to linearly increase the gizzard weight (
p = 0.09). This may be due to the increased muscularity required by the gizzard to break down drier feed, as the energy reduction was a result of the decrease in oil quantity in the diet. Dry feed is generally retained in the gizzard–proventriculus system longer than moist feed, as previously reported [
27], thus resulting in an increase in the gizzard muscle mass.
4.3. Meat Quality
Breast pH tended to be influenced by the multi-enzyme addition to the reduced-ME diets (
p = 0.06), which was led by energy levels when tested as a main effect (
Table 5). The results revealed that as the energy level was reduced in the diet, the breast pH became more acidic. Post-mortem, rigor mortis ensues, whereby muscle metabolism changes from aerobic to anaerobic and where lactic acid is produced as a by-product, causing a decline in pH [
28]. Although rigor mortis in all biological organisms eventually leads to the production of lactic acid and thus a decline in pH, the energy source of the diet and quantity in the muscle ante mortem may influence the ultimate pH. As indicated by production performance, the feed intake increased in reduced energy diets to compensate for the lack of energy. To create an energy reduction, dietary soybean oil was replaced with corn, which is rich in carbohydrates, and the glucose is stored in the muscle as glycogen. It is a simpler process for the body to utilize glycogen and to convert this to energy via the anaerobic pathway with lactic acid as by-product rather than by utilizing the fat; thus, it can be speculated that the lower pH in the reduced energy diets may be due to the increased carbohydrate content of the diet, resulting in higher glycogen storage within the muscles.
In the present study, the values relating to the meat lightness (L*) were above 57 (
Table 5), indicating meat that was lighter than normal; however, there was no significant difference among the experimental groups. It is likely that the lighter breast might be related to the temperature of the scalding bath used for de-feathering. The scalding bath was set at 60 °C, which might have been too hot, causing the chicken breast to be slightly poached, resulting in slightly higher lightness values. It has been reported that breast colour lightness (L*) values above 49 are suggestive of a poor WHC and increased shear force and a low pH, as the lighter colour is indicative of increased reflectance caused by the increased water leakage that accumulates on the surface of the breast and reflects the light rays during the measurement of the colour [
29]. In the current trial, no differences were observed for the breast lightness and the WHC among the experimental groups; however, all of the recorded values were greater than the values reported in the literature for both normal and pale meat [
30]. The result of the current study is consistent with the data of other researchers who have reported a lighter meat colour (greater L* value) when broiler chickens were fed on low-energy diets [
31,
32]. Reports [
32] also showed that a low-ME enzyme-added diet increased the yellowness of the breast muscle (a*) in comparison with the standard ME and low-ME diet with no enzyme addition, which is slightly different to our findings. Lightness (L*) and redness (b*) were also not influenced by the dietary treatments in their study.
A higher WHC relates to an acidic pH. As the pH of poultry meat decreases to 5.3–5.4, it reaches the isoelectric point for many major proteins [
33]. Proteins with a charge that is closer to neutral imply that there is little polarity in the molecule; therefore, water is not attracted to it and will thus purge itself from the meat. A less acidic pH is associated with greater protein polarity, allowing an increased WHC, which is reflected in the present results (
Table 5).
Birds on the reduced energy diets tended to have a heavier breast weight (
Table 4; main effects of the energy levels). This might be due to a slightly greater protein intake (gram per day) for birds on the reduced energy diets because of increased ADFI in response to the lower energy content. These findings agree with the results reported [
34], where breast muscle weight (%) tended to decrease when the dietary ME levels decreased from 2805 to 2997 kcal/kg. A similar pattern could be observed in the results reported elsewhere [
33]; however, the broiler chickens on the low-ME diet with or without enzyme addition had heavier carcasses compared to the birds on the standard ME diet.
The other meat quality parameters were not affected by the experimental diets. These outcomes are in line with the results reported [
35], where with the exception of meat hardness, low-ME diets supplemented with enzyme did not affect any other meat quality parameters compared to the standard diet. Similarly, it has been reported that enzyme supplementation does not affect the physical properties of breast meat, the including pH and WHC, in broiler chickens [
36]. Our data were also in agreement with the findings from [
37], which observed no differences in meat composition or meat quality parameters when the birds were fed with commercial enzymes, including xylanase and phytase, applied individually or in combination.