Protein has always been a costly nutrient in poultry feeds (Beski et al., 2015). The quest for the reduction in feed costs has evolved through a sequential implementation of technologies that improve feed protein utilization. Major reductions in feed costs have first occurred with the use of synthetic amino acids in poultry feeds with further increases in the efficiency of protein utilization resulting from the implementation of the ideal protein concept (Baker, 1994; Lemme, 2003). Supplementation of proteases has lastly emerged as a tool to improve dietary protein utilization (Stefanello et al., 2016). Apart from animal performance, supplemental proteases have demonstrated to reduce feed costs as well as nitrogen excretion (Leinonen and Williams, 2015; McCafferty et al., 2022).

Decreasing supplies of protein feedstuffs have been pressuring the existing growing demands of protein to feed humans worldwide (OECD/FAO, 2022). Soy, regardless of its commercially available presentation as a feedstuff (whole bean heat treated, soybean meals with variable CP contents, soybean meal extracted with the use of polar solvents, etc.), remains the primary protein source in animal feeds (Erdaw et al., 2016). The soybean market price has increased 90% in average worldwide in the last 5 years (USDA, 2022). All other protein sources, either from plant or animal origin, have followed up in parallel (FAO, 2021). The increase in protein market price is expected to continue a path of increase since protein daily intake in the world has been steadily increasing (OECD/FAO, 2021). Simultaneously, increased public concerns regarding the environment and sustainability have been pressuring for the reduction of the waste generated by animal production, which includes nitrogen in excreta. Therefore, technologies that potentially improve protein retention of animals grown to feed humans are welcome.

Digestion of protein by birds is dependent on proteases endogenously released in the gastrointestinal tract, which largely degrade protein down to peptides and amino acids that are further absorbed (Sklan and Hurwitz, 1980; Recoules et al., 2017). However, it has been demonstrated that significant amounts of dietary protein present in commercial feeds are not thoroughly digested (Parsons et al., 1997; Adedokun et al., 2008; Bryan et al., 2019). Research on the use of exogenous proteases has been ongoing for years and many commercially available products are currently utilized in poultry feeds. Reports on the effects of supplemental proteases have been related to benefits in performance (Ghazi et al., 2002; Angel et al., 2011; Moss et al., 2017; McCafferty et al., 2022), nutrient digestibility (Freitas et al., 2011; Liu et al., 2013; Stefanello et al., 2016), intestinal health (Xu et al., 2017; Cowieson et al., 2018), and immune competence of broilers (Peek et al., 2009; Cowieson et al., 2017).

Exogenous proteases that can compete and succeed commercially are required to function at corresponding pH’s in the gastrointestinal tract, but they also must sustain digestive capabilities after being heat treated at temperatures regularly used in feed processing mills (Ravindran, 2013). Broad substrate specificity of some exogenous proteases, as well as their higher concentrations at the entry of digestive tracts, may produce benefits as effective catalyzers, enabling them to break peptides down faster when compared to feeds not supplemented with proteases (Ravindran, 2013; Cupi et al., 2022).

The objective of the present research was to evaluate the effects of a novel exogenous sfericase protease when added to commercial broiler feeds. Evaluations were conducted on growth performance and extended to ileal digestibility of broilers fed varying AA and energy concentrations. We hypothesized that the protease enzyme increases protein digestibility and, therefore, live performance. It was also hypothesized that protease responses are more evident in diets having reduced AA and energy contents than their counterparts formulated to maximize growth.

Two experiments were carried out at Santa Livia Research Farm, Farroupilha, Rio Grande do Sul, Brazil. All procedures conducted in the present study were approved by the local Ethics Committee on Animal Use.

Analysis of the protease added to the experimental diets showed similar activities with the declared values in both studies ( Table 3 ). Feed intake was not affected by the treatments in any of the experiments (P>0.05). Overall mortality was low and not affected by the treatments either (Grand means were of 2.87% and 2.62% in EXP1 and EXP2, respectively). Growth performance of broilers are presented in Tables 4 , 5 , respectively for EXP1 and EXP2. There were no interaction effects between diet and protease on performance variables (P > 0.05), therefore data in the tables are presented as main factor means.

Growth in both experiments was affected by diet formulation at 35 and 42 d. Significant reductions in BWG were observed for birds fed the Matrix diets (reductions ranged from as low as 1.4% at 42 d in EXP1 to as high as 1.9% in EXP2). Standard diets presented better FCR at 35 d in EXP1 and at 35 and 42 d in EXP2 (P<0.01). A trend for improvement at 42 d was observed in EXP1 (P<0.078).

The protease added to the diets showed no significant effects on BWG (P>0.05) in both experiments. A trend to increase BWG (P<0.052) was observed at 21 d in EXP1. On the other hand, the protease led to significant improvements in FCR in all cumulative age measurements in EXP1 regardless of the protease level added. A trend for improvement (P<0.067) was observed in the cumulative FCR at 42 d in EXP2.

Digestibility responses obtained in both experiments are presented in Table 6 . No interaction was observed between the diet and protease (P>0.05). Except for an increase IDCP for birds fed the Standard diet in EXP1 (P<0.038), there were no effects of diet in the digestibility variables. Protease addition led to improvements in IDM and IDCP (P < 0.05) when compared to no protease addition in EXP1; however, IDE was only increased when 30,000 units were added. No effects of protease were observed in EXP2 (P>0.05).

An impressive number of studies with poultry fed diets supplemented with exogenous proteases have been reported in the last few years. Outcomes from these studies vary from absence of effects (Kaczmarek et al., 2014; Yuan et al., 2017; Walk et al., 2019) to significant improvements in growth performance of broilers (Freitas et al., 2011; Lee et al., 2018; Jabbar et al., 2021; McCafferty et al., 2022). Synergy between proteases with other in feed supplemental enzymes, such as phytases, xylanases and amylases have also been reported (Cowieson and Adeola, 2005; Amerah et al., 2017; Cowieson et al., 2019). In the present study, phytase was added to all feeds since this is economically mandatory in presently commercial feeds.

Enzymes only interact with one type or a group of similar substrates such that the enzyme-substrate reaction products are yielded. In the case of supplemental proteases, expected yields of dietary protein degradation resulting from supplemental proteases are absorbable short peptides or amino acids (Gilbert et al., 2014). Broiler responses to increased intestinal contents of available amino acids depend on their amount and degree of essentiality. The modern broiler has demonstrated to respond to increasing dietary intake of protein from amino acids balanced feeds with improved growth, feed conversion as well as meat yields in a wide range of contents (Lemme et al., 2006; Dozier et al., 2008; Cruz et al., 2017).

Expected benefits from supplemental proteases are dependent on the presence of protein that can function as a reactive substrate to the specific enzyme added (Vieira et al., 2014). In the present study, Matrix diets were formulated with a fair reduction in protein but a small decrease in energy when compared to the Standard diets in both experiments. These differences were expected to allow for the detection in performance and digestibility when birds were fed them and, therefore, benefits due to the protease in the Matrix diets should be able to provide a compensation for their lower AA and AME contents. Performance of broilers was negatively affected by birds fed the Matrix diets at 35 and 42 d (P<0.078 for FCR at 42 d in EXP1). However, diets with the added protease provided significant improvements in FCR in EXP1 throughout all phases, regardless of the diet and the dose added. In EXP2 the protease numerical improvements in FCR at 42 d had a P<0.067. On the other hand, protease improvements were more noticeable in FCR and could be explained by the parallel increased digestibility (IDM, IDE and IDCP) in EXP1 (which did not occur in EXP2).

An important difference in the protein profile existed between diets formulated in both experiments. Meat and bone meal, included in EXP1 but not in EXP2, have reduced AA digestibility when compared to SBM (Vieira et al., 2014). It seems that, the presence of a mix of more complex proteins in EXP1 has provided a more adequate substrate for the presently used protease.

In conclusion, commercial diets supplemented with 10,000 NFP/kg or 30,000 NFP/kg lead to improvements in FCR regardless of AA density utilized.

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

The animal study was reviewed and approved by Comite de Etica no Uso de Animais Granja Santa Livia Producoes e Pesquisa Agropecuaria.

SV, main researcher; CF, analysis and text; AF, researcher; LK, researcher; JS, planning and analyses; MU-F, planning and analyses. All authors contributed to the article and approved the submitted version.