The diets of laying hens are predominantly composed of cereals and soybean meal, which naturally contain various anti-nutritional factors—including non-starch polysaccharides (NSP), trypsin inhibitors, and phytate—that can impede nutrient absorption. To mitigate these effects, the poultry industry commonly incorporates exogenous enzymes into feed formulations. Xylanase is frequently included in poultry diets, especially when viscous cereals are used, to reduce intestinal viscosity by breaking down soluble non-starch polysaccharides (NSP) such as arabinoxylans and β-glucans (Melo-Durán et al., 2021);(Choct, 2015). β-Glucans are a type of non-starch polysaccharide found in the cell walls of various organisms, including yeast, bacteria, fungi, and cereal grains(Choi & Kim, 2023).

Recent nutritional studies highlight the potential of enzyme supplementation as a promising strategy to optimize the utilization of by-product feed ingredients, while simultaneously reducing formulation costs by conserving energy and amino acids. These enzymes function by hydrolyzing anti-nutritional factors, thereby enhancing nutrient digestibility and improving the metabolizable energy (ME) and amino acid availability in the diet (Cowieson et al., 2019). Xylanase is an enzyme that specifically targets non-starch polysaccharides (NSPs) by cleaving the internal β-xylosidic linkages within the xylan backbone, resulting in the production of xylo-oligosaccharides (XOS) (Morgan et al., 2022).

The use of xylanase is widely adopted in commercial laying hen diets as a strategic approach to depolymerize non-starch polysaccharides (NSPs), reduce digesta viscosity, and support intestinal health. This is particularly relevant in the current production context, which emphasizes reduced dependence on antibiotic growth promoters(Mahmood & Guo, 2020). Xylanase supplementation has been shown to reduce digesta viscosity in the jejunum and ileum, lower caecal pH, and decrease excreta moisture content. Additionally, it enhances energy retention and facilitates the breakdown of both soluble and insoluble fractions of arabinose, glucose, and xylose derived from total non-starch polysaccharides (NSPs) (Nguyen et al., 2021).

The inclusion of protease in poultry diets has been shown to enhance amino acid digestibility by hydrolyzing dietary proteins that escape complete degradation by the bird’s endogenous gastrointestinal proteolytic enzymes (Woyengo et al., 2023). The incorporation of exogenous proteases into poultry feed can enhance protein hydrolysis by complementing the activity of endogenous digestive enzymes, thereby improving the overall digestion and utilization of dietary proteins. (Liu et al., 2024). This strategy not only enhances nutrient digestibility and improves feed conversion efficiency but also reduces the moisture and nutrient content of excreta. Moreover, the use of protease contributes to the sustainability of poultry production by decreasing nitrogen excretion into the environment and supporting overall production efficiency (Stefanello et al., 2024).

The effectiveness of multi-enzyme combinations remains an area requiring further investigation, as the specific interactions and impacts of different enzyme mixtures are not yet fully understood. Currently, there is limited research assessing the effects of various exogenous enzyme combinations in laying hens fed vegetable protein-based diets supplemented with alternative sources of soybean meal. Conducting comprehensive studies on various enzyme combinations and expanding the existing knowledge is essential for advancing nutritional research and optimizing dietary strategies in laying hens (Huang et al., 2024). In such scenarios, accurately predicting the birds’ response is essential to enable the formulation of diets with a strategically balanced nutrient profile, thereby ensuring performance outcomes comparable to those achieved with nutritionally complete diets (Cowieson et al., 2006).

A thorough understanding of the intrinsic nutritional value of raw materials, along with the expected response to exogenous enzymes—such as matrix values—is highly valuable for feed formulators. A firm grasp of enzyme chemistry makes it possible to draft lower-cost rations that are still unlikely to shortchange the flock on essential nutrients. In the trial described here, 45- to 53-week-old Hy-Line layers received mash feeds spiked with xylanase, protease, or both, all built around a heavy loading of processing by-products. A range of unconventional feedstuffs-palm kernel meal, distillers’ dried grains plus solubles, soybean meal, rice bran, and wheat bran-were blended into the formulations. This study was conducted to evaluate the effects of xylanase and/or protease enzyme supplementation in laying hen diets on nutrient digestibility and overall bird performance.

Table 2 summarizes the expected and measured activities of xylanase and protease across the various dietary treatments. Xylanase activity reached 1,700 and 1,620 EPU/kg in treatments T2 and T4, respectively, while protease activity peaked at 4,564 and 4,804 U/kg in treatments T3 and T4. Measured xylanase activity was close to or slightly higher than the expected values in the supplemented groups (T2 and T4). In treatments T3 and T4, the measured protease levels were slightly lower than the expected values.

The effects of exogenous enzyme supplementation on AMEn are detailed in Table 3. Birds receiving the xylanase-supplemented diet (T2) exhibited a significant increase (p<0.05) in AMEn values compared to the negative control group (T1), reflecting improved energy utilization. Although the protease (T3) and combined xylanase+protease (T4) groups showed numerically higher AMEn values than T1, these differences did not reach statistical significance (p>0.05).

The production performance results, summarized in Table 3, indicate that both protease supplementation (T3) and the combined xylanase and protease treatment (T4) significantly enhanced hen day production (HDP) and egg mass (EM) compared to the control group (T1) (p<0.05). Digestible lysine intake (dLI) was significantly greater (p<0.05) in the combined xylanase and protease group (T4) compared to T1. However, no significant differences were observed among the treatment groups regarding egg weight (EW), feed intake (FI), feed conversion ratio (FCR), feed cost (FC), AMEn intake (AMEI), or body weight gain (Gain).

Apparent ileal amino acid digestibility (Table 4) was significantly enhanced following enzyme supplementation, with the combined treatment group (T4) exhibiting the highest digestibility values for lysine (0.87%), methionine+cystine (0.80%), threonine (0.77%), and arginine (0.93%) (p<0.05). Collectively, the combined use of xylanase and protease (T4) consistently yielded favorable outcomes in terms of energy utilization, nutrient digestibility, and production performance.

This study demonstrates that dietary supplementation with xylanase and protease, administered individually or in combination, markedly enhances nutrient utilization and production performance in laying hens consuming diets rich in by-products. The enzyme activity measurements presented in Table 2 provide valuable insights into enzyme stability and functionality under practical feeding conditions. Xylanase activity in Treatments T1 and T3 was recorded at 290 EPU/kg of feed. Meanwhile, protease activity was measured at 265 U/kg and 282 U/kg of feed in Treatments T1 and T2, respectively. This value is considered indicative of the activity of endogenous enzymes naturally present within the feed ingredients. The concentration of endogenous xylanase can vary according to the composition and source of the raw feed materials(Melo-Duran et al., 2024). Interestingly, the xylanase activity observed in treatments T2 (1,700 EPU/kg) and T4 (1,620 EPU/kg) slightly surpassed the anticipated levels, which may reflect satisfactory thermal stability of the enzyme during feed processing. Contrary to the expected trend, the measured protease activity in Treatments 3 and 4 was lower than anticipated. This reduction may be attributed to diminished thermostability at elevated temperatures, resulting in partial inactivation of the enzyme (Li et al., 2015).

The results of this study demonstrated that xylanase supplementation enhanced both apparent metabolizable energy corrected for nitrogen (AMEn) and digestible lysine (dLys) compared to the negative control (NC). Specifically, Treatment 1 (NC) exhibited an AMEn value of 2,585 kcal/kg, while Treatment 2 (NC + xylanase) showed an increased AMEn value of 2,754 kcal/kg. The observed improvement in AMEn value of 169 kcal/kg closely aligns with the metabolizable energy (ME) increase of 154 kcal/kg and exceeds the values reported by (Andrade et al., 2021) and (Whiting et al., 2023), which were 150 kcal/kg and 122 kcal/kg of ME, respectively. Primarily, xylanase-mediated hydrolysis of arabinoxylans reduces digesta viscosity, thereby improving nutrient accessibility and absorption (Cowieson & Roos, 2016). The observed increases in apparent metabolizable energy (AME) following xylanase supplementation are attributed to modifications of fiber fractions and enhanced starch digestibility(Cowieson et al., 2015).

Xylanase catalyzes the degradation of insoluble, highly branched arabinoxylans in the plant cell wall, thereby increasing the accessibility of both endogenous and exogenous enzymes to starch and protein within the endosperm cells (Hassaan et al., 2019). Supplementation with xylanase has been demonstrated to improve nutrient digestibility and reduce ileal digesta viscosity (Vargas et al., 2024). The absence of a significant improvement in apparent metabolizable energy corrected for nitrogen (AMEn) with protease supplementation alone (T3) was anticipated, given that proteins contribute relatively little to the overall energy metabolism in poultry. Nonetheless, the numerical increase observed in the combined enzyme treatment (T4) suggests potential synergistic interactions.

The production performance results revealed an interesting distinction between the effects of energy and protein utilization. The significant increases in hen-day production (4.8–5.2%) and egg mass observed in protease-supplemented treatments (T3 and T4) underscore the critical role of amino acid availability in supporting egg production. This conclusion is further reinforced by the improved digestibility of essential amino acids such as lysine, methionine, and threonine in these treatment groups. The observation that xylanase supplementation alone (Treatment 2) enhanced apparent metabolizable energy corrected for nitrogen (AMEn) without significantly impacting production parameters suggests that energy was not the primary limiting factor for egg production in this trial. Furthermore, the lack of significant effects on feed conversion ratio across all treatments may indicate that the benefits of enzyme supplementation predominantly supported productive performance rather than improvements in feed efficiency under the specific conditions of this study.

In this study, amino acid digestibility was significantly improved in Treatment 4 (NC + xylanase + protease), particularly for lysine (dLys), methionine + cysteine (dMet + Cys), threonine (dThr), and arginine (dArg). The combined supplementation of xylanase and protease demonstrated enhanced efficacy, yielding higher amino acid digestibility values compared to the negative control (Treatment 1). Previous research supports that such synergistic enzyme combinations can improve feed conversion ratios and nutrient utilization, ultimately promoting better growth performance in broiler chickens (Lin et al., 2023). The improvement in digestible amino acids (dAA) observed in this study was lower than the +3.7% increase reported by (Cowieson & Roos, 2016). Protease supplementation has been shown to enhance ileal dry matter digestibility by approximately 2.3% and ileal digestible energy by 2.7% (Zavelinski et al., 2024). Protease supplementation has been shown to enhance ileal amino acid digestibility and frequently improve the digestibility of non-protein nutrients. Proteolysis frequently alters the bulk archi tecture of the nutrient matrix in commercial diets, and that disturbance alone can largely explain the observed outcome. Beyond that primary change, a constellation of secondary influences-endogenous enzyme release, the general condition of the gastrointestinal lining, and the energy of the intestinal transporters-appears to fine-tune the beneficial response (Cowieson et al., 2015).

Enzymes are notorious for their compartmentalized specificity, yet the pairing of xylanase and protease reveals an intriguing counter-narrative. The observed synergy suggests that boosting one well-behaved enzyme with another can yield a metabolic bonus far shy of what either could promise alone. By cleaving the xylosic backbones in the plant cell wall, xylanase opens a window for protein and starch to escape into the digestive pool, an act sometimes likened to prying a stubborn lid off a jar. In that freshly exposed space, protease steps in and dismantles any lingering protein scaffolding, thereby scattering leftover fiber into fragments that dissolve more readily in gut fluids. Considered together, the two enzymes turn a recalcitrant feedstuff into a supple mash almost by brute invitation instead of surgical precision (Amerah et al., 2016).

It is important to note, however, that the impact of exogenous enzymes on amino acid digestibility has been reported to vary according to the intrinsic digestibility of the diet (Romero et al., 2014). The differences in performance quite likely trace back to the unique physical and chemical signatures of each cereal type. When enzymes are added, any lift in energy output usually hinges on how rich the starter ration already is, what substrates happen to be present, the innate digestibility of those ingredients, the overall condition of the digestive tract, and the way the enzyme blend shifts the microbial community living in the gut (Amerah et al., 2016). The measured increases in nutrient availability when xylanase is paired with protease highlight a general trend in animal nutrition: blended enzymatic treatments often outperform isolated supplements. Subsequent laboratory and on-farm studies will need to dissect the biochemical pathways responsible for that advantage and translate those insights into user-friendly algorithms that forecast enzyme performance in shifting ingredient matrices.

In conclusion, the findings of this study indicate that the supplementation of xylanase and protease significantly enhances nutrient utilization and the productive performance of laying hens fed diets with high levels of by-products. The combined use of both enzymes yielded the most pronounced improvements, highlighting the potential advantages of integrated enzyme strategies over single-enzyme applications in practical feed formulations. These results carry significant implications for sustainable poultry production, especially in contexts where feed ingredient quality varies and the use of agro-industrial by-products is critical for maintaining economic feasibility.

Nahrowi and A. Jayanegara serve as editors of the Tropical Animal Science Journal but have no role in the decision to publish this article. The authors declare that they have no conflicts of interest—financial, personal, or otherwise—that could have influenced or be perceived to influence the content of this manuscript.

The authors gratefully acknowledge funding support from Charoen Pokphand Indonesia.