Comparative effects of proteases on performance, carcass traits and gut structure of broilers fed diets reduced in protein and amino acids

A 32-day study was conducted at the Broiler Research Unit of Suliman Al-Amaireh & Partners Co., Jordan. All experimental procedures were conducted in accordance with commercial practices and were approved by the institutional animal care and use committee (IACUC) of the Jordan University of Science & Technology (16/04/12/206). All experimental procedures were compliant with all local animal welfare legislation. A total of one thousand one-day-old Ross 308 broilers (41 g at hatch) were sourced from the commercial hatchery of Suliman Al-Amaireh & Partners Co and were feather sexed at hatch. Birds were randomly allocated to five dietary treatments with ten replicates of 20 mixed-sex broilers (10 males and 10 females) each: a positive control (PC) diet formulated to meet Ross 308 AA requirements as per 2019 nutrient specifications [26], a negative control (NC) diet reformulated to provide 3.5% lower CP and AA compared to PC, NC supplemented with 3,000 U/kg (300 mg/kg) of a multi-protease (PR1) solution, containing 3 different coated acidic, neutral and alkaline proteases produced from Aspergillus niger, Bacillus subtilis and Bacillus licheniformis respectively (Kemzyme Protease, Kemin Europa N.V., Herentals, Belgium), NC supplemented with 15,000 U/kg (200 mg/kg) of a commercial serine protease (PR2) produced from Bacillus licheniformis, and NC supplemented with 30,000 U/kg (500 mg/kg) of a commercial alkaline protease (PR3) produced from Bacillus licheniformis. The selection of the proteases evaluated in this trial was based on their commercial relevance and their application dose match the commercial recommendation of their suppliers. Analysis of enzyme recovery was not performed in the present experiment, due to feed spoilage in transit between the trial site and experimental laboratory.

Birds were reared on a solid floor covered with clean wood shavings. The temperature and ventilation of the building were monitored daily and were managed according to the breed recommendations [26]. A regular lighting program (0–3 days 24 h/light, 4–7 days 23 h/light, and 8–final age 20 h/light) was provided by fluorescent bulbs placed above the pens.

Two dietary phases were provided: starter (d 0–14) and grower (d 15–32). All diets were produced according to commercial practices and fed as pellets (2 mm diameter and 3 mm length in starter diet; and 3 mm diameter and 5 mm length in grower diets). Pelleting conditions were acceptable for the heat-stability of all the proteases (< 60°C). All diets contained background phytase, NSP-degrading enzymes, and biosurfactants. The composition of the experimental diets is listed in Table 1. Dry matter, CP, ash, ether extract and crude fiber from experimental feeds were determined with NIRS (NIRS DS2500 F, FOSS, Hillerød, Danmark) and are shown in Table 2. Feed and water were provided ad libitum throughout the study.

Birds were weighed individually on arrival from the hatchery. Pen bird body weight (BW), body weight gain (BWG) and feed intake (FI) were recorded at 14, 28 and 32 days. Feed conversion ratio (FCR) was calculated by dividing pen FI by pen BWG. Daily mortality was recorded per pen. Final BWG, FI and FCR were calculated on day 32, and all birds were slaughtered.

At slaughter, 4 male birds from each replicate with an average BW of 2,050 g, were selected and euthanized for carcass traits evaluation. The male gender selection was decided due to higher and therefore closer BW to the average 2,050 g. Birds were slaughtered as per the Halal method according to Jordanian law. The skin along with the feathers was removed after slaughtering, carcasses were eviscerated by hand and individual carcasses were weighed. The whole breast as well as the abdominal fat were removed from the carcass and weighed individually. Carcass traits were expressed as a percentage of the carcass weight.

At 32 days 1 male bird per pen (a total of 50 birds: 10 birds per treatment) was randomly selected and euthanized and samples of the duodenum, jejunum, and ileum were collected and fixed in 10% neutralized formalin for three days and sent to the Histopathology lab of The Jordan University of Science and Technology (Irbid, Jordan). The samples were then dehydrated through ascending concentrations of alcohol starting from 60% concentration, 70%, 80%, 90%, and absolute (100%) ethanol. They were transferred to xylene for one hour and then soaked in liquid paraffin and embedded in paraffin using specialized molds. The mold was left to cool down at room temperature. The embedded samples were sectioned at 4–5 μm thickness using a rotary microtome and subsequently were stained with hematoxylin and eosin. They were then examined by a light microscope connected with a camera (Olympus BX51, Olympus, Tokyo, Japan). Each slide of the three parts of the small intestine was pictured at 40X magnification. The morphometric measurements of each sample, namely villi height (VH), villus width (VW), and crypts depth (CD) were taken using image J software (https://imagej.nih.gov/ij/). Only well-oriented sections were considered for measurements. At least six readings of each slide (total of 11 slides of each intestinal segment) of well-oriented sections were taken and considered for statistical analysis. The ratio of villus height: crypt depth (VH:CD) for each replicate was calculated from the average measurement.

Data are presented as means with overall SEM and were analyzed in the Fit Model platform of JMP 15 (SAS Institute, Cary, NC, USA) with protease supplementation as the main factor. The pen was considered the experimental unit for performance. The individual broiler sampled was considered the experimental unit for carcass traits and histology. No outlier data were identified or excluded from the dataset. In all statistical analyses, differences were considered significant at p < 0.05.

Mortality was considered low (<5%) and was not different among the different treatments. Performance results per feeding period and overall (0 to 32 days) are shown in Table 3. By day 14, broilers from PR1 and PR2 showed higher (p < 0.05) BW compared to PR3, while no difference (p > 0.05) was found compared to PC and NC. Between days 14 and 28, broiler fed NC diets supplemented or not with any of the protease enzymes (PR1, PR2, PR3, and NC) showed better (p < 0.05) FCR than broilers fed the PC diet. Across the total trial period (0 to 32 days), the best feed efficiency (p < 0.05) was realized when feeding birds with the PC diet and the NC diet supplemented with 300 mg/kg of the multi-protease solution (PR1).

The morphometric changes in the duodenal, jejunal, and ileal villi of birds from the different dietary treatments at 32 days are presented in Table 4. There were significant differences between treatments in all gut segments. Briefly:

Table 3 presents the effect of dietary supplementation of the different proteases on the selected carcass traits of male broilers at 32 days of age. Carcass weight as well as carcass yield was higher (p < 0.05) for PR1 treatment compared to all other treatments, and was higher (p < 0.05) for PR2 compared to NC and PR3. Breast weight was higher (p < 0.05) for PR1 and PR2 compared to NC and PR3. Breast % was higher (p < 0.05) for PR2 compared to NC and PR1. No effects (p > 0.05) were detected for the rest of the carcass traits evaluated.

Broiler growth performance is largely influenced by the supply of high levels of digestible AA [27–29] and several previous studies have shown growth performance impairment following their dietary reduction [30–34]. However, meeting these requirements involves formulating diets with highly digestible proteinaceous, and very often expensive, feed ingredients. Keeping the balance between broiler growth performance and production profitability is not an easy task for the modern poultry industry, and the sustained rise of in the cost of feed raw materials remains a primary challenge [35], especially in an increasingly volatile and disrupted global supply chain environment. Maximizing the utilization of CP and AA at the lowest possible feed cost can offer nutritionists a window of opportunity to improve overall broiler profitability. Reformulating diets to lower cost and reduced CP and AA levels with the use of proteases has proven to be an effective strategy in this respect [36–40]. Growth performance improvements following the application of proteases to broiler diets have been previously reported [14–18,20,21,34,37,41–43] and are in line with the magnitude of FCR improvement (5.7 points) achieved in the current study by the supplementation of the multi-protease to the diet reduced in CP and AA. However, the supplementation of the serine and the alkaline monocomponent proteases did not result in improved growth performance in our study. Many of the previous studies that showed growth performance improvements of dietary protease supplementation were performed with monocomponent proteases [14,16–18,20,21,34,41–43], which is not aligned with the lack of growth performance effect seen in the present study. On the other hand, several previous studies where the application of protease had limited effect on growth or nutrient digestibility ascribed this to the presence of other feed additives in the diet, in particular high levels of phytase and NSP-degrading enzymes [23,24,43]. Both classes of enzymes are known to improve nitrogen retention and AA digestibility through indirect mechanisms: phytase reduces the anti-nutritive and protein-binding effect of phytate [5], while NSP-degrading enzymes reduce the caging effect of fiber, allowing endogenous enzymes access to dietary CP [10,11]. The diets in the present study were formulated along with commercial guidelines and contained 1000 FTU/kg of phytase, a combination of 3 NSP-degrading enzymes, amylase, and neutral protease, as well as a biosurfactant also known to have indirect effects on nitrogen utilization [12]. It could be hypothesized that the addition of the multiprotease with a wide effective pH range could have been determinant to improve feed efficiency in the presence of multiple performance enhancing additives.

The benefits of the dietary supplementation of protease on growth performance are ascribed to increased CP digestibility [44–47], and they may be attributed to changes in intestinal morphology [38,48], reductions in the impact of antinutrients including trypsin inhibitors [49–51], and shifts in the microbiome [20,21]. The specific mode of action of dietary protease supplementation on intestinal morphology has not been well understood. Our results showed that protease supplementation had significant impact on intestinal morphology. All tested proteases provided a longer VH in the duodenum and jejunum compared to the NC. Longer VH is closely linked with greater capacity for nutrient absorption [52,53]. VH was depressed between the PC and NC, with the reductions in AA level linked to significantly shorter villi in the ileum, and near significant reductions in the duodenum and jejunum. The same pattern is seen for the reduction in VH:CD ratio in the different intestinal segments: the earlier segments showed no (duodenum) or minimal (−16.0%, jejunum) reductions, whilst a severe reduction in VH:CD was recorded in the ileum (9.47 PC vs. 5.82 NC, −38.5%), the primary site of nutrient absorption. This will provide a smaller area available for the absorption of nutrients, likely contributing to depressed feed digestibility and efficiency. It is possible that the ileum is most heavily affected by the gut health effects of undigested CP, as this is often the site where Clostridium perfringens, a pathogen which benefits from poor CP digestibility, is recovered from infected broilers [54].

Ding et al. [55] reported that CP reduction led to decreased trypsin activity in the pancreas and duodenum content with both VH and VH:CD ratio reduced in the duodenum, jejunum, and ileum. However, protease supplementation reverted these effects and increased the activity of trypsin in the pancreas and VH in the duodenum, jejunum and VH:CD ratio in the ileum. Similarly, in the present study VH in the duodenum and jejunum was significantly improved by all proteases, with the alkaline protease linked to the longest villi in all segments. This finding is also in line with other previous research studies [15,24,55,56]. This suggests that the monocomponent alkaline protease tested in the present study was the most effective at increasing gut health and absorptive capacity in the small intestine. However, this did not translate into the best growth performance. It could be hypothesized that protease supplementation may provide more AA to be utilized by certain beneficial bacterial groups that are known to stimulate the production of mucin and the proliferation of epithelial cells [57], and that this effect could differ between proteases. Further studies evaluating the effect of the different proteases in the microbiome composition and intestinal morphology would be needed to validate this hypothesis.

Growth performance of broilers from modern genetics is more responsive to CP and AA compared to energy [27], and protein deposition in the chicken carcass is increased with additional AA intake [58]. Increases in overall BWG were not significantly different between treatments in the present study, including between the nutritionally adequate PC and the reformulated NC. However, there were significant differences between treatments on carcass weight. The reformulation to lower AA content significantly reduced the carcass weight and carcass yield of the NC, compared to the PC. This difference was partially ameliorated by the addition of the alkaline monocomponent protease and totally recovered by the addition of the serine monocomponent protease, while addition of the multi-protease was able to significantly increase carcass weight and yield above the level of the PC. This is in line with the findings of Cho et al. [37], who saw increased carcass weights and breast yields following supplementation of multi-protease to both nutritionally adequate and reformulated diets. Xu et al. [15], also found that the dietary supplementation of a multi-protease increased breast muscle weight. This effect was possibly linked to higher slaughtering weight because it was not translated to higher breast muscle yield. Similarly, in the present study, higher carcass weight resulted in higher breast weight but not in higher breast meat yield following the multi-protease supplementation. These improvements in efficiency and carcass weight and yield are likely largely driven by improvements in AA digestion and protein deposition, however, a digestibility assay was not performed in the present study and this hypothesis could not be confirmed. Previous studies have demonstrated that both monocomponent and multi-proteases improve precaecal AA digestibility of all [7,38] or some AA [16,20,59]. Protease supplementation can also improve energy retention, though results are inconsistent between and even within studies [17,60]. Improved nutrient absorption following protease supplementation has been previously linked with reduced FI [14,36], though significant differences in FI were not seen in the present study.

In conclusion, the addition of a multi-protease to broiler diets reduced in CP and AA improved feed efficiency compared to all other tested proteases. Both the multi-protease and the monocomponent serine protease increased carcass weight, carcass yield, and breast weight and supported gut health and morphology. The monocomponent serine protease also improved breast %. Our findings show that a multi-protease can be supplemented to broiler diets reduced in CP and AA to enhance feed efficiency in the presence of 3 NSP-degrading enzymes, amylase, neutral protease, phytase, and biosurfactants. The addition of monocomponent proteases with a more limited pH range was not able to achieve the same performance improvements. Further studies should be performed to elucidate the effects of the different proteases in CP and AA digestibility, and microbiome composition.