A new study published in Veterinary Medicine and Science suggests that a carefully chosen pair of feed enzymes can dismantle many of those barriers. Researchers set out to determine whether phytase, protease, or the two together could alleviate the digestive limitations imposed by canola seed in broiler chickens, with a practical eye on which single enzyme delivers the most value when budgets allow only one. The question matters commercially: with protein-rich feed ingredients fluctuating wildly in price, industry stakeholders want to know exactly where their enzyme dollars are best spent.

The experiment involved 300 one-day-old male Ross 308 chicks, distributed in a completely randomised design across five treatments with five replicates of twelve birds each. One group received a corn-soybean meal control diet with no canola seed. A second received a diet containing 10 percent canola seed without enzymes. The remaining three groups ate the canola-seed diet supplemented with protease, with phytase, or with both enzymes together. Protease was included at 0.15 grams per kilogram of feed and phytase at 0.05 grams per kilogram. Birds were raised through starter, grower and finisher phases spanning 42 days, with feed and water available freely and standard vaccination protocols against Newcastle disease and infectious bursal disease applied.

The canola seed itself was thoroughly characterised before the trial began. Analysis showed 21.25 percent crude protein, a striking 42.76 percent ether extract, 18.39 percent crude fibre, and a gross energy content of 7063 kilocalories per kilogram. The seed also contained 5264 milligrams per kilogram of phytate phosphorus, 0.36 percent tannins and 9.37 micromoles per gram of glucosinolates, the very compounds suspected of constraining bird performance. Diets were formulated to be nutritionally comparable across treatments, with the canola diets naturally higher in fat and linoleic acid owing to the oil-rich seed.

The performance results told a clear story. Birds fed the unsupplemented canola diet ate significantly less, gaining roughly 6 to 7 grams less feed per day during the starter phase than controls, and their body weight gain suffered accordingly across the starter, grower and overall periods. Feed conversion ratio was also worse in the canola group during the grower phase. But adding enzymes changed the picture. Protease alone improved weight gain and feed intake relative to the canola-only diet, though its effect on intake was modest. Phytase alone produced a more pronounced lift in feed intake. The combination of both enzymes, however, was the standout: birds on the dual-enzyme diet ate and grew at rates statistically indistinguishable from the soybean-fed controls, effectively erasing the canola penalty.

Digestibility data revealed why. Apparent metabolisable energy, which fell to around 2687 kilocalories per kilogram in the canola-only group, rebounded to control levels of roughly 2860 kilocalories per kilogram in the phytase and combination groups. Crude protein digestibility told an equally interesting tale: the protease and combination diets actually exceeded the control diet, reaching about 74 to 75 percent digestibility compared with 71.7 percent in the soybean control. Ash and crude fibre digestibility were also restored by phytase-containing treatments. In other words, the enzymes did not merely compensate for canola’s drawbacks; in the case of protein digestion, they pushed nutrient utilisation beyond what the conventional soybean diet achieved.

The physiological mechanisms behind these gains emerged from a battery of measurements on the birds’ digestive systems. At 10 days of age, when the endogenous enzyme system is still immature, activities of amylase, lipase and total protease in the jejunum were depressed in the canola-only group but elevated in all enzyme-supplemented groups, with the combination treatment producing the highest amylase and lipase values. By day 42, when the birds’ own digestive machinery had matured, these differences had disappeared, underscoring that exogenous enzymes matter most in early life. Jejunal morphology followed a similar pattern: villus height and the villus-height-to-crypt-depth ratio, key indicators of absorptive capacity, were significantly higher in phytase and combination groups, while the canola-only diet drove deeper crypts, a sign of increased epithelial turnover under intestinal stress.

The gut microbiota and the physical properties of the intestinal contents added further layers of explanation. At day 10, birds on the combination diet harboured the highest Lactobacillus populations and the lowest Escherichia coli counts in the ileum, while Clostridium perfringens was lowest in that group compared with the canola-only treatment. Intestinal digesta viscosity, a critical determinant of how easily nutrients diffuse to digestive enzymes, was significantly elevated in the duodenum and jejunum of canola-fed birds but reduced by enzyme supplementation, particularly in the ileum. The researchers attribute this to phytase disrupting viscous phytate-protein complexes and protease breaking down peptide-fibre networks, together creating a more fluid digestive environment where enzymes and substrates can interact efficiently.

Perhaps the most striking findings concerned bone health. The unsupplemented canola diet significantly impaired tibia development: bones were lighter and shorter, breaking strength dropped to about 3074 newtons compared with 4279 newtons in controls, and bone ash, calcium and phosphorus percentages all declined. These deficits trace directly back to phytate, which forms insoluble complexes with essential minerals and renders them unavailable for skeletal mineralisation. The protease-plus-phytase combination restored bone weight, length, strength and mineral chemistry to levels comparable with or exceeding the control group, with bone ash reaching 41.93 percent and calcium 15.57 percent. The authors note that improved protein digestibility from protease likely supported collagen matrix synthesis, providing the organic scaffold upon which calcium and phosphorus are deposited, while phytase liberated the minerals themselves.

The study’s conclusion carries real weight for feed formulators. Replacing soybean meal with 10 percent canola seed without enzyme support demonstrably harms growth, gut function and bone mineralisation in broilers. Yet the combined application of protease and phytase overcame these constraints, performing better than either enzyme alone. The synergy is mechanistically elegant: phytase dismantles the structural and chemical barriers that phytate erects around proteins and minerals, creating conditions in which protease can work more effectively, while protease maximises the value of the nutrients phytase releases. For producers weighing enzyme purchases against volatile protein prices, the message is that the two enzymes are genuinely complementary, and that canola seed, long treated as a second-tier ingredient, can approach soybean meal performance when the right enzymatic tools are applied.

Subject of Research: Effects of protease and phytase supplementation on growth performance, nutrient digestibility, gut health and bone mineralisation in broiler chickens fed canola seed diets

Article Title: Protease and Phytase Alleviate Digestive Limitations of Canola Seed in Broiler Chickens

Article References: Sheikh‐Nazari, R., Jafari‐Khorshidi, K., & Jafari, M. A. (2026). Protease and Phytase Alleviate Digestive Limitations of Canola Seed in Broiler Chickens. Veterinary Medicine and Science, 12(6), Article e71259. https://doi.org/10.1002/vms3.71259

Image Credits: AI Generated

DOI: 10.1002/vms3.71259

Keywords: broiler chickens, canola seed, phytase, protease, soybean meal replacement, nutrient digestibility, gut microbiota, intestinal morphology, tibia mineralisation, antinutritional factors, feed enzymes, poultry nutrition

The economic backdrop of the study is stark. Feed represents more than half of total broiler production costs, and as corn and soybean meal prices climb, producers are increasingly turning to alternative raw materials. The problem is that many of these alternatives are rich in non-starch polysaccharides, or NSP, complex plant carbohydrates that monogastric animals such as chickens cannot efficiently digest. Soluble NSP, particularly β-mannans found in soybean meal and palm kernel meal, dissolve in the gut and dramatically increase the viscosity of intestinal contents. This thickened digesta slows the diffusion of digestive enzymes toward their substrates, reduces nutrient absorption, and ultimately depresses growth and feed efficiency.

β-Mannanase has long been deployed as a countermeasure. The enzyme cleaves the β-1,4-mannosidic linkages in galactomannan backbones, breaking down viscous NSP and releasing nutrients physically trapped within plant cell walls. Previous research has also indicated that β-mannanase can reduce the impact of trypsin inhibitors in soybean meal by supporting endogenous protease activity. However, conventional commercial products typically consist of single-domain enzymes, and the Korean team hypothesized that incorporating additional glycoside hydrolase domains and accessory peptides could extend the enzyme’s functional reach, improving stability through the acidic proventriculus and gizzard and maintaining activity in the near-neutral small intestine where most nutrient absorption occurs.

To test this, the researchers allocated 300 day-old broilers to five dietary treatments: a positive control basal diet, a negative control with metabolizable energy reduced by 120 kcal/kg, and three enzyme-supplemented versions of the reduced-energy diet. The first enzyme (M1) was a conventional β-mannanase produced by fermentation with Bacillus subtilis, with an activity of approximately 800,000 U/kg. The second (M2) incorporated upgraded glycoside hydrolase functionality, and the third (M3) added accessory peptides designed to support enzyme-substrate interaction and gastrointestinal stability. The upgraded products were manufactured through optimized fermentation, with temperature modulated between 40 and 60 °C and pH between 5 and 6, to maximize enzyme secretion and consistency under feed manufacturing conditions.

In vitro characterization revealed meaningful differences among the three products. At pH 3.0, the conventional M1 enzyme retained relatively high activity, while the upgraded M3 showed a marked reduction. As pH rose to 6.0, however, the picture reversed: M3 and M2 demonstrated the highest relative activities, ranging from roughly 70 to 80 percent, while M1 declined. At pH 7.2, both upgraded enzymes significantly outperformed the conventional product. Thermal profiling showed that all three enzymes retained more than 80 percent activity at 30 °C, but at 60 °C the conventional M1 proved more heat-tolerant, a property potentially useful during feed pelleting yet apparently less decisive under living digestive conditions. Pepsin digestion assays further showed that M2 and M3 resisted proteolytic degradation better than the control diets, suggesting the upgraded enzymes could survive the hostile environment of the upper digestive tract.

The in vivo results were striking. Final body weight was significantly higher in the positive control and M3 groups compared with the unsupplemented reduced-energy negative control, and during the final phase, weight gain was higher in the positive control and M3 treatments while feed conversion ratio was lower in all enzyme-supplemented groups. Notably, feed intake did not differ among treatments, indicating that the performance benefits stemmed from improved nutrient utilization rather than simply eating more. In practical terms, the upgraded enzymes allowed birds to extract more energy and protein from a diet deliberately stripped of 120 kcal/kg, effectively recovering much of the nutritional value that NSP-rich ingredients would otherwise lock away.

Digestibility measurements reinforced this interpretation. Dry matter digestibility was higher with M2 and M3 than in the negative control, and crude protein digestibility in the M3 group exceeded both control diets. Apparent ileal digestibility of the key limiting amino acids lysine, methionine, and threonine was significantly improved by M2 and M3, while valine digestibility rose with all three enzyme products. Digesta analysis using ELISA-based assays showed elevated trypsin activity in all enzyme-supplemented groups and higher protease concentrations with M2 and M3, pointing to a secondary stimulation of the birds’ own digestive machinery. The researchers suggest that by dismantling anti-nutritional NSP barriers, the exogenous enzymes improved nutrient signaling within the gut, prompting greater endogenous enzyme secretion.

Metabolic markers told a consistent story. Blood urea nitrogen, an indirect indicator of protein metabolism and nitrogen balance, was significantly lower in the M2 and M3 groups than in both controls and the conventional enzyme group, consistent with more efficient incorporation of dietary amino acids into body tissue and reduced deamination. Jejunal digesta viscosity dropped significantly in all enzyme-supplemented groups, confirming the primary mode of action of β-mannanase in degrading soluble NSP. Plasma amino acid concentrations, however, remained unchanged across treatments, which the authors attribute to tight homeostatic regulation of circulating amino acid pools; when combined with improved growth, the pattern suggests enhanced post-absorptive utilization rather than accumulation in the bloodstream.

Carcass evaluation added a further dimension. The negative control group showed the lowest thigh muscle proportion, while the positive control and the two upgraded enzyme treatments achieved the highest values. Abdominal fat deposition was highest in the full-energy positive control group, implying that enzyme supplementation supported lean tissue deposition without the fat accretion associated with higher dietary energy. Dressing yield, breast muscle, drumstick, wing, and organ weights were unaffected, and critically, no significant differences emerged in meat quality parameters including pH, water-holding capacity, cooking loss, or shear force, indicating that the enzymatic intervention improved efficiency without compromising the physicochemical properties that consumers and processors care about.

The study’s authors are careful to note its limitations: only a single level of energy reduction and enzyme inclusion was tested, and mechanistic analyses of gut microbiota, intestinal morphology, and fermentation metabolites were not performed. Dose-response studies and microbiome sequencing could clarify how the accessory peptides in M3 enhance enzyme-substrate affinity or protect against proteolytic degradation. Nevertheless, the central conclusion carries weight for a global poultry sector under pressure to cut costs and reduce the environmental footprint of production. If structurally upgraded enzyme systems can recover 120 kcal/kg of dietary energy without sacrificing performance, carcass composition, or meat quality, feed formulators gain a powerful new lever, one that depends not on feeding more enzyme but on designing it better.

Subject of Research: Effects of structurally upgraded β-mannanase enzyme supplementation on nutrient utilization and growth performance of broilers fed energy-reduced, non-starch polysaccharide-rich diets

Article Title: Dietary β-mannanase supplementation and its effect on nutrient utilization and performance in broilers fed energy-reduced diets

Article References: Choi, S. D., Tajudeen, H., Mun, J. Y., Park, S. A., Hosseindoust, A., Neves Silvestre, P., Lee, S. S., & Kim, J. S. (2026). Dietary β-mannanase supplementation and its effect on nutrient utilization and performance in broilers fed energy-reduced diets. Food Science of Animal Resources, 46(1), Article 68. https://doi.org/10.1007/s44463-026-00068-7

Image Credits: AI Generated

DOI: 10.1007/s44463-026-00068-7

Keywords: β-mannanase, broilers, non-starch polysaccharides, feed enzymes, nutrient digestibility, energy-reduced diets, poultry nutrition, amino acid digestibility, feed conversion ratio, glycoside hydrolase, intestinal viscosity, carcass traits