Dr. Bharat L Sadarao, Dr. Sushant Labh, Dr. Partha Das, and Dr. Venket Shelke
Kemin Industries South Asia Pvt. Ltd., Chennai, INDIA
INTRODUCTION
In contemporary broiler diets, protein is the second-most essential and most costly nutrient, mainly supplied from maize and soybean meal and other sources5. Although corn and soybean meals are highly digestible, the presence of anti-nutritional factors (ANFs), such as trypsin inhibitors, lectins, and non-starch polysaccharides (NSPs), are not easily digested by birds, particularly during the starter period20,7,10. Due to this, a sizeable portion of protein (18–20%) bypasses the digestive system without being fully absorbed and digested3,4. As a result, the use of protease enzyme products in poultry diets, such as exogenous proteases, has gained momentum to improve the general performance of animals and birds10. However, the gastrointestinal tract (GIT) has a wide range of pH, from the very acidic proventriculus environment to the somewhat neutral environment of the small intestine to the alkaline environment in the large intestine. Proteases, like other enzymes, are pH-specific, and this wide pH range compromises the one-size-fits-all approach19. This warrants the use of a combination of proteases, which can act efficiently on such a wide pH range.
Several studies have been conducted on the effects of mono-component protease in broiler chickens3,11,12,14,13,9,23, but very few on multi-component protease27,6. The current study aims to compare the effects of two exogenous protease enzymes, one single protease, and the other multi-protease, on performance parameters like body weight, feed conversion ratio (FCR), mortality rate, European efficiency factor (EEF), and economic returns in commercial broilers. Kemin has developed a new multi-protease compound named KEMZYME® Protease Dry. KEMZYME® Protease Dry has an innovative stable coating that protects it from heat denaturation during the feed pelleting process and provides gastric stability in the intestinal tract of birds at different pH conditions15.
PROTEASE ENZYME
Proteases are enzymes that break down protein molecules into the amino acids and peptides needed by animals and is an important factor in protein digestion as they hydrolyze the less digestible proteins in animal feeds and break them down into more usable peptides (Figure 1). Naturally, endogenous proteases are released in the stomach/gizzard and duodenum of the animal. Exogenous proteases, however, are frequently added as a supplement to feed to improve digestion and address the less readily digestible proteinaceous components, particularly in young animals with immature digestive systems. The impact of proteases, which is pH dependent, is a crucial factor in these circumstances since not all endogenous or exogenous proteases will be active at every part of the gastrointestinal tract (GIT) (Figure 2) 17,16.
KEMZYME® PROTEASE DRY
KEMZYME® Protease is an innovative, patented (nr patent US 8,815,315 B2) combination of 3 different, coated proteases: an acidic, neutral, and alkaline one, produced respectively from Aspergillus niger, Bacillus subtilis, and Bacillus licheniformis. It provides broad enzyme activity on a variety of substrates, across the GIT, maximizing protein digestibility while significantly reducing feed cost through feed reformulation following its nutritional matrix. The acid protease has a heat-stable coating and works in the proventriculus and gizzard at a pH of 2.5-3.5, which breaks down the protein into easily digestible peptides and amino acids. The neutral protease both has a heat stable and a gastric stable coating, to ensure optimal activity to further boost up protein digestion in the duodenum and jejunum at a pH 6.5-7. The alkaline protease also has a heat-stable and gastric-stable coating, aimed to act primarily in the ileum at pH 7.2-7.8, which further breaks down protein and polypeptides into digestible amino acids. Figures 3, 4 & 5 shows the mode of action of KEMZYME® Protease at different pH along with the electron microscopic scan and schematic diagram of targeted release in GIT17,16.
MATERIALS AND METHODS
Birds, housing, and management:
The trial was conducted at a commercial broiler farm facility in the western part of India. A total number of 8,03,185 one-day-old Vencobb 430Y broiler chicks of mixed sex were used in this trial. The broiler chicks were randomly classified into 2 groups, viz. control, and treatment, with 9 branches in each group, and the trial lasted for 42 days (Table 1). The broiler birds were reared in an open-sided poultry house having a mud floor covered with a 2-inch layer of rice husk as bedding material. All birds were systematically vaccinated against Newcastle disease and Gumboro disease, and other needed prophylactic measures. Each group was provided with round bottom feeders while water supply was made available round the clock through a manual drinking system. All birds were reared under identical management conditions throughout the experiment.
| Branch | Chicks Placement | |
| Control | Treatment | |
| BRANCH – 1 | 59,671 | 61,835 |
| BRANCH – 2 | 80,140 | 88,795 |
| BRANCH – 3 | 37,262 | 49,375 |
| BRANCH – 4 | 63,962 | 52,302 |
| BRANCH – 5 | 30,553 | 24,041 |
| BRANCH – 6 | 74,737 | 15,504 |
| BRANCH – 7 | 36,215 | 38,994 |
| BRANCH – 8 | 37,580 | 21,509 |
| BRANCH – 9 | 26,795 | 3,915 |
| Total Birds | 4,46,915 | 3,56,270 |
Feeding Program:
All the birds were provided with a pellet-form diet in 4 dietary phases (Table 2): pre-starter (1 to 11 days), starter (12 to 22 days), finisher 1 (23 to 35 days), and finisher 2 (36 to 42 days). A corn-soya bean meal diet was used, to formulate the basal diet and to achieve the optimal nutrient requirements as per industry recommendation (Table 3). The control and treatment diet corresponds to the basal diet containing a single protease enzyme (Cysteine protease) and multi-protease enzyme complex (KEMZYME® Protease Dry), respectively, added on top of the feed at a dose of 300 grams per metric ton, implying that no enzyme nutritional matrix was included in the ration calculation (Table 2). However, the dose of a single protease enzyme (Cysteine protease) is 250g/MT of feed as recommended by the company. Feed cost was measured by calculating raw material costs.
| Groups | Description |
| Control | Basal diet with on-top Single Protease Enzyme@ 300g/MT of feed against the standard dose of 250g/MT as recommended by the company. |
| Treatment | Basal diet with on-top Multi-Protease Enzyme (KEMZYME® Protease Dry) @ 300g/MT of feed as recommended by the company. |
| Raw Ingredients/Medicines | Pre-Starter (Kg/MT) | Starter (Kg/MT) | Finisher 1 (Kg/MT) | Finisher 2 (Kg/MT) |
| Maize | 470 | 476 | 480 | 505 |
| Soybean Meal | 335 | 285 | 261 | 230 |
| Meat and Bone Meal (MBM) | 25 | 30 | 30 | 30 |
| Maize Gluten Meal | 30 | 40 | 40 | 40 |
| Broken Rice | 100 | 130 | 150 | 150 |
| Crude Soybean Oil | 6 | 10 | 15 | 22 |
| Di-Calcium Phosphate (DCP) | 6.20 | 3.00 | 1.50 | 1.20 |
| Calcite | 6.80 | 5.00 | 4.20 | 4.00 |
| Sodium Bicarbonate | 2.50 | 2.30 | 2.10 | 2.10 |
| L-Lysine | 3.50 | 3.70 | 3.50 | 3.40 |
| DL-Methionine (98.5 %) | 3.20 | 2.80 | 2.50 | 2.20 |
| Salt | 2.50 | 2.50 | 2.30 | 2.20 |
| L- Threonine | 1.00 | 1.00 | 1.00 | 1.00 |
| Choline Chloride (60%) | 1.00 | 1.00 | 1.00 | 1.00 |
| Acidifier 1 | 1.00 | 1.00 | 0.00 | 0.00 |
| Acidifier 2 | 0.00 | 0.00 | 1.00 | 1.00 |
| Toxin binder | 1.00 | 1.00 | 1.00 | 1.00 |
| Anti-Mycoplasma | 1.00 | 1.00 | 0.00 | 0.00 |
| Organic Trace Mineral | 0.70 | 0.70 | 0.50 | 0.50 |
| NSP Enzyme | 0.10 | 0.10 | 0.10 | 0.10 |
| Protease Enzyme | 0.30 | 0.30 | 0.30 | 0.30 |
| Betaine HCl | 0.50 | 0.50 | 0.50 | 0.50 |
| AGP 1 | 0.50 | 0.50 | 0.50 | 0.50 |
| AGP 2 | 0.13 | 0.13 | 0.13 | 0.13 |
| Vitamin Premix | 0.60 | 0.60 | 0.50 | 0.50 |
| Liver Powder | 0.60 | 0.60 | 0.50 | 0.50 |
| Anti-coccidian | 0.50 | 0.50 | 0.50 | 0.50 |
| Chromium & Vitamin C | 0.20 | 0.20 | 0.20 | 0.20 |
| Antioxidant | 0.15 | 0.15 | 0.15 | 0.15 |
| Phytase | 0.20 | 0.20 | 0.20 | 0.20 |
| Haloquinoline | 0.20 | 0.20 | 0.20 | 0.20 |
| Total Feed (Kg) | 1000 | 1000 | 1000 | 1000 |
| Cost per kg feed (INR) | 39.34 | 38.81 | 38.10 | 38.07 |
| Average Feed Cost per Kg (INR) | 38.58 | |||
| Nutrient Composition of the Diet | ||||
| Metabolizable Energy (Kcal/kg) | 2950 | 3030 | 3110 | 3170 |
| Crude Protein (%) | 23.00 | 21.90 | 20.70 | 19.70 |
| Digestible Lysine (%) | 1.35 | 1.25 | 1.17 | 1.10 |
| Chloride (%) | 0.90 | 0.83 | 0.78 | 0.74 |
| Available Phosphorous (%) | 0.48 | 0.44 | 0.42 | 0.41 |
| Sodium (%) | 0.20 | 0.20 | 0.19 | 0.18 |
| Chloride (%) | 0.27 | 0.26 | 0.25 | 0.24 |
| Crude Fiber (%) | 3.30 | 3.20 | 3.10 | 3.00 |
| Ether Extract (%) | 4.00 | 4.30 | 5.00 | 6.00 |
Parameters measured:
Performance parameters such as body weight (BW), feed intake (FI), feed conversion ratio (FCR), corrected feed conversion ratio (CFCR), European Efficiency Factor (EEF)*, Cost of Production (COP), and mortality rate were recorded and compared, the Return on Investment (ROI) from the groups were calculated after 42 days.
*EEF= (Livability ×Average weight)/ (Mean Age × FCR) ×100
Statistical analysis:
Statistical analysis of the data for performance parameters was done using Stat-graphics Centurion XVI.II software. Data were analyzed by one-way ANOVA with confidence limits set at 95%. The results were reported as the mean ± standard error (SE). A P-value of ≥0.05 was considered statistically non-significant.
RESULTS
The performance data of commercial broilers fed with control and treatment diets containing a single protease and a multi-protease, respectively, were presented in Table 4. No significant differences (P≥0.05) were observed among the groups in any of the measured performance parameters. However, it was noticed that the inclusion of multi-protease enzyme (KEMZYME® Protease Dry) in the treatment group had a positive impact on final body weight (BW), feed conversion ratio (FCR), mortality percentage, and European efficiency factor (EEF), changes comparing with control groups where it showed a numerical increase in all above performance parameters. Similarly, a lower cost of production (CoP) was observed in the treatment group than in the control group. Calculation of economic benefits revealed that the addition of KEMZYME® Protease Dry in the treatment group diet delivered a return on investment (RoI) of 4:1 over the control group (Table 5).
| Parameters | Mean ± Standard error | ||
| Control | Treatment | P-Value | |
| BW | 2.84 ± 0.045a | 2.85 ± 0.042a | 0.911 |
| FCR | 1.62 ± 0.014a | 1.61 ± 0.01a | 0.638 |
| CFCR | 1.41 ± 0.013a | 1.4 ± 0.016a | 0.689 |
| Mortality | 5.02 ± 0.229a | 4.74 ± 0.225a | 0.639 |
| EEF | 385.23 ± 4.525a | 389.33 ± 5.078a | 0.568 |
aData within a row with the same superscripts is not significantly different (P≥0.05).
Table 4: Effects of protease enzyme on broiler chicks’ growth performance (n= 18).
Body Weight and FCR:
Final body weight (BW) and FCR were not affected by the on-top supplementation of any protease mixes in the basal diet during the trial period of 42 days (Figures 6 and 7).
EEF and Mortality:
The European efficiency factor (EEF) was observed to be 4 points higher in the KEMZYME® Protease Dry treated group compared to the control group (Figure 8). The treatment group observed a lowered mortality of 0.29% than the control group, but the difference was not statistically significant (Figure 9).
Figure 8: EEF in trial groups Figure 9: Mortality % in trial groups
Cost of production (CoP) and return on investment (ROI):
In economic terms, the cost of production expressed per kilogram of live broiler weight was lower by 1 rupee 9 paise (INR 1.09) in broilers fed the treatment group diet than in the control group, which demonstrated a return on investment (ROI) of 4:1 in favor of treatment group (Figure 10 and Table 5).
Figure 10: Cost of Production (CoP)/kg live weight in trial groups
| Parameters | Control | Treatment |
| Number of birds | 356270 | 446915 |
| Mortality (Number) | 18899 | 21597 |
| Trial Period (Days) | 43.30 | 43.33 |
| Cull Birds (Number) | 1134.00 | 1399.00 |
| Livability (%) | 94.38 | 94.85 |
| Final Body Weight (Kg) | 2.844 | 2.851 |
| Feed Conversion Ratio (FCR) | 1.62 | 1.61 |
| CFCR | 1.41 | 1.40 |
| Avg. Feed Cost (INR) | 38.58 | 38.58 |
| Chick Cost (INR) | 30 | 30 |
| Total Birds Sold | 336237 | 423919 |
| Total Live Weight Sold (Kg) | 949802 | 1207083 |
| Total Feed Consumed (Kg) | 1552250 | 1943100 |
| Production Cost/Kg Live Weight (INR) | 74.30 | 73.21 |
| Live Weight Price Per Kg (INR) | 95 | 95 |
| Live Weight Per MT of Feed (Kg) | 611.89 | 621.22 |
| Gross Income Per MT of Feed (INR) | 58129.29 | 59015.43 |
| Net Income Per MT of Feed (INR) | 19552.18 | 20438.32 |
| Extra Income Per MT of Feed (INR) | 886.14 | |
| Product Inclusion Cost Per MT (INR) | 225 | |
| Return On Investment | 3.94 |
Table 5: Economic calculations and return on investment (ROI) details.
DISCUSSION
The performance parameters, including body weight, feed conversion ratio, European efficiency factor, and livability, were not significantly different (P≥0.05) between treatments. However, numerically improved body weight, FCR, EEF, and livability were recorded in the treatment group supplemented with multi-protease enzyme KEMZYME® Protease Dry. However, a single protease enzyme (cysteine protease) supplementation has been observed to have inconsistent performance results in the control group compared to the treatment group.
Many of the earlier experiments have shown benefits in growth performance from dietary protease supplementation using multi-protease complexes rather than single proteases or mono-component proteases. They noticed that the variations in broiler performance are consistent with the effect of the multi-protease complex on the bird’s capacity to better utilize ingested nutrients from the partially digestible substrates present in the feed ingredients through enhanced nutrient digestibility in terms of energy, amino acids, and nitrogen26,18,2,22,8,21. However, despite improvements in apparent ileal digestibility, a set of trials evaluating the supplementation of mono-component protease enzymes such as acidic, neutral, alkaline, and keratinase proteases, among others, failed to produce beneficial effects on poultry growth performance due to decreases in the intake of digestible amino acids (AID)28,24,25. Similarly, Zheng et al., 2023 and Wealleans et al., 2023 in their experiment observed that the addition of mono-component proteases with a more limited pH range was not able to achieve the same performance improvements26,28.
Therefore, while supplementing animal feed with protease enzymes, the manufacturers, source, type, features, dosage, activity, quality, ability, and specificity should all be carefully taken into account1,28,24. A holistic protease solution like KEMZYME® Protease Dry used adequately and appropriately, enhances protein utilization in poultry by facilitating protein digestion, followed by higher amino acid absorption. This will reduce production costs and result in a profit opportunity for broiler producers.
CONCLUSION
It was concluded that the supplementation of a multi-protease complex (KEMZYME® Protease Dry) in a standard broiler basal diet from day one till liquidation age had a positive role in improvements in overall production performance than the single protease enzyme. The current study also revealed that broilers fed with multi-protease diets showed higher European efficiency factor (EEF), better livability, and lowest broiler production costs, which could be attributed to generating a better return on investment (RoI) of 4:1 than the control group supplemented with a single protease enzyme. The usage of the multi-protease complex in the present study may create an opportunity for commercial poultry producers to optimize production costs while maintaining the performance of their birds and increasing their profitability.
References are available upon request.