The efficient conversion of feed into its basic components for optimal nutrient absorption is vital for both broiler and broiler breeder production and welfare. Gut health, an intricate and complex area combining nutrition, microbiology, immunology, and physiology, has a key role to play. When gut health is compromised, digestion and nutrient absorption are affected which, in turn, can have a detrimental effect on feed conversion leading to economic loss and a greater susceptibility to disease.

The industry has made huge efforts in recent years to develop solutions focusing on gut health. This is not only due to a direct link to improved feed efficiency and profitability, animal welfare or food safety, but also due to changes in consumer preferences and regulatory requirements.

When it comes to poultry gut health, coccidiosis and necrotic enteritis are major economic challenges, particularly when present in a subclinical form where symptoms may not be observable. Due to epithelial damage and inflammation, these subclinical infections reduce feed efficiency and result in an opportunity for potential pathogens.

Poultry trials challenged with Eimeria and Clostridium perfringens showed that NEXT ENHANCE® 150 feed additive – an encapsulated, highly concentrated blend of thymol with carvacrol – promotes healthy intestinal microbial flora, as well as supports gut barrier function, inflammation processes and immunity. NEXT ENHANCE® 150 has a positive effect in the reduction of coccidial faecal oocyst shedding and minimize damage to intestinal epithelium in infected birds . Use of NEXT ENHANCE® 150 for reduction in coccidial oocysts in excreta could lead to the development of new strategy for the prevention of avian coccidiosis.

Coated essential oil blend – protected for better performance:
Where essential oils are concerned, thymol and carvacrol are scientifically well-documented compounds. Due to their phenolic structure (having a cyclic ring with a hydroxyl group attached) they are recognized as efficient compounds showing a variety of beneficial effects in the gut. NEXT ENHANCE® 150 (NE150) is a highly concentrated blend containing thymol and carvacrol protected by a unique coating. This ensures that the active ingredients are stable during pelleting and can ultimately result in a controlled release to the lower parts of the intestinal tract.

Multi-faceted approach:

Thymol and carvacrol are highly effective against a wide range of potential pathogens. In a variety of studies, NE150 has demonstrated its potential to establish a healthy microbial composition by promoting higher lactic acid-producing bacteria and reduce pathogenic species. An example of the antimicrobial effects in broilers is shown in Figure 1.

In the study, an Eimeria challenge model was used, which typically increases the levels of C. perfringens. It is well known that C. perfringens is the causative agent for necrotic enteritis but requires other predisposing factors to become clinical or subclinical. The invasion of intestinal cells by the Eimeria parasite is seen as the major predisposing factor because it creates tissue damage and leakage of plasma proteins used by C. perfringens. Broilers, receiving NE150 showed lower levels of C. perfringens as well as lower levels of Enterobacteriaceae, a large family of pathogens including E. Coli or Salmonella. As a result, NE150 is shown to lower the risk of a bacterial overgrowth, which is key in the development of necrotic enteritis.

Biomarkers can be used to study the effect of protected thymol and carvacrol on intestinal integrity and gut barrier function. The stability of the tight junctions (a unique connection between cells), for example is linked to the amount of occludin, which increases the physical barrier function of the intestine. Under challenge conditions and during inflammation processes, occludin is known to be downregulated. A broiler study with C. perfringens challenge showed lower levels of occludin, which increased with the addition of NE150. This lowers the risk of pathogen translocation, or the uncontrolled transfer of different molecules from the lumen into the bloodstream. It has also been shown that broilers receiving NE150 under Eimeria or C. perfringens challenges showed significantly lower serum endotoxin levels, indicating improved mucosal barrier integrity. Adding NE150 to the ration has also shown increased villus height to crypt depth ratio, another well recognized marker for intestinal health. Macroscopic intestinal lesions are another relevant indicator of gut health. In Eimeria and C. perfringens challenge studies, these lesions were significantly reduced with NE150. Fewer intestinal lesions can result in a lower inflammation processes and can directly translate to performance improvements.

During a host-pathogenic infection, pro-inflammatory cytokines are released to activate the immune system. However, the inflammation needs to be controlled as a prolonged and persistent activation of pro-inflammatory cytokines can result in mucosal damage as well as impact the stability of tight junctions. The inflammation also consumes a lot of energy, which impacts performance. NE150 is shown to downregulate pro-inflammatory cytokines, which helps to protect intestinal barrier function and save energy. Conclusively, thymol and carvacrol lower the risk of performance depression and intestinal damage caused by inflammation.

Thymol and carvacrol are also known to have anti-oxidative capacities due to their chemical structure. To understand how NE150 could benefit broilers’ oxidative status, a study was done measuring various biomarkers, such as super oxide dismutase (SOD) and glutathione peroxidase (GSH-Px). These enzymes are responsible for the conversion of reactive oxygen species (ROS) to harmless substances. ROS are a result of normal metabolism but are toxic to the organism and increase significantly during an infection or stress. It was shown that both enzymes increased (Figure 2) when NE150 was added to the diet. As a result, the malondialdehyde levels, a marker for lipid peroxidation, were significantly reduced in broilers. NE150 can therefore, directly and indirectly, help to maintain a balance between ROS and the defense system, which lowers the risk of tissue and cell damage as well as performance losses.

Consistent performance improvement

With its broad impact on gut health it is not surprising that broilers receiving NE150 show a consistent improvement in feed conversion ratio with an average of 3.7% when used at the recommended dosage rate. In addition, NE150 can be used in feeding programs to support the birds under coccidiosis, necrotic enteritis or gut health challenges to help alleviate negative effects on the animals.

This array of trials shows that producers can use NE150 in their strategy to improve feed conversion ratio, thereby reducing production cost efficiently and increasing the profitability of commercial broiler productions.

References available upon request.

Source: Novus International Inc

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Evaluating Protease Enzymes

Trypsin Inhibitor, The Hidden Enemy In Soyabean Meal

Enzymes increase nutrients available in feed ingredients. Since enzymes are substrate specific, the benefit of the enzyme in the diet is dependent on the mix of raw materials and the amount of substrate available.

Assessment of added value of enzymes isn’t simple. The most accurate method is to use in vivo techniques with animals consuming semi-purified diets. Using this method, the direct effect of an enzyme can be understood for each raw material. As this method is expensive and not available to do it for everybody, the matrix approach based on nutrients contribution values given by the supplier has been widely adopted to evaluate an enzyme.

Using a set of nutrient matrix values for an enzyme is a practical approach and providing unique matrix values to a given enzyme ensures it can be applied simply to any kind of diet, regardless of the raw materials or the amount of substrate. This approach has been easy to adopt in practical formulations but has consistently demonstrated enzymes failing to meet expectations. These failures have been due to the lack of or an excess of substrate, and/or over-formulation.

When evaluating enzymes, a few concepts need to be made clear:

1. Substrate: the specific substance on which an enzyme act

2. Enzyme Effect: nutrients that a given enzyme will make available due to the direct enzymatic effect and the additional benefits accrued by the reduction of the substrate in the diet

3. Avoid over-formulation: enzymes need “nutritional space” to express and thus diets need to be near the deficiency point to make a good estimation of the enzyme’s potential.

Trials for evaluating enzymes

The test of an enzyme requires being aggressive in the formulation and pushing the limits. Under-performing chickens will help provide a better evaluation of the enzymes and understand how accurate the formulation is.

Challenge Model: In this model, the diet with significant reduction of nutrients, that the enzyme will liberate and make available to the birds, needs to be formulated. Different enzymes can then be added on top.

Objectives of the trial:

1. “AA room” is created for enzymes to show their potential.

2. Each supplier has different recommendations of how proteases affect the feed. This trial allows simplifying the comparison.

3. Proteases can’t increase the AA digestibility 10% linearly. As a result, the AA ratio will be unbalanced and subsequently the performance of negative control as well as treatments will be lower than that of the control group.

4. The most aggressive protease will have the best performance compared to T2.

5. If any of the enzyme groups shows the same performance as the control group (T1), it signals some over formulation as no protease can increase 10% linearly on all the AA.

Response Model:

The model is a variation of the ‘Challenge model’ discussed in the previous section. In this model, two control diets will be used; the current diet (this group is optional if there aren’t enough treatments) and a diet group with anywhere between 5% to 10% lower AA specifications. The control 2 specifications will be used for the treatment groups. There will be a reformulation following the matrix value recommendation of each protease supplier.

Objectives of the trial:

1. Having T1 compared to T2 will assist in acknowledging any over-formulation or amino acid imbalance.

2. Having lower specifications in AA and CP creates enough space for the enzyme to express to potential.

3. This design allows each supplier to give their ideal recommendations.

4. If the enzyme recommendation is too aggressive, the enzyme group will clearly show lower performance than T2 as long as there is a gap of performance between T1 and T2.

5. If the enzyme recommendation is conservative and the enzyme can deliver additional benefits it will be reflected in greater performance than T2 as long as there is a gap of performance between T1 and T2.

Previous article by Author: Trypsin Inhibitor, The Hidden Enemy In Soyabean Meal

As global animal production has rapidly shifted towards reduced Antibiotic free, “Gut health” has become a popular expression and all-encompassing concept in the scientific community. The gastro-intestinal tract must provide a barrier function protecting against harmful environmental elements (e.g. toxins and pathogenic microbes), while simultaneously permitting appropriate nutrient absorption. Successful animal performance depends on the interplay between the intestine, microbiota, diet, and a multitude of environmental factors.

The shift to antibiotic free production or better gut health often results in the increase of soybean meal inclusion as there are limited in the number of efficacious protein sources that successfully reduce soybean meal content. Soybean meal is the most widely used major protein source in poultry production across the world. However, SBM contains various anti-nutritional factors that may affect intestinal homeostasis and impair nutrient utilization in poultry. The main anti-nutritional factors in SBM, are trypsin inhibitors (TI), oligosaccharides, such as raffinose and stachyose, and the antigen Glycinin, β-conglycinin and Lectins. Diets that include high levels of soybean meal contain proportionally higher anti-nutritional factors and may pose the risk of impaired performance.

Chen et al. (2016) analyzed the content of TI and Urease Activity (UA) in more than 1000 samples of SBM from all over the world and observed a high degree of variability in the reported ANF values, both within the same country and amongst different origins.

Chen et. al., The Journal of the American Oil Chemists’ Society, 2020

The elevated variability and its potential negative impact on performance highlights the importance of knowing the content of anti-nutritional factors in SBM for poultry formulations.

In this article, we will review mainly the role of Trypsin Inhibitors (TI) in broilers.

Why Should we care about TI?
Trypsin and chymotrypsin are important digestive enzymes that are secreted by the pancreas as the inactive enzyme precursors trypsinogen and chymotrypsinogen. Trypsin activates itself via positive feedback and converts chymotrypsinogen and other inactive enzymes into their active forms. As Tis are protein in nature and one of the most anti nutritional components of SBM, they compete to bind to trypsin therefore affecting the digestion process. They have been correlated with rapid feed passage and decrease in digestibility of broilers with a relevant economic impact. The analysis is still more expensive, complex and time consuming for TI, for this reason, other parameters are commercially used as indirect SBM quality indicators, such as Urease activity and Protein solubility.

There are two types of TI present in Soya, Kunitz TI which is larger molecule & Bowman-Birk TI which is smaller molecule. But soyabean seed contain around 14% more Bowman Birk TI than Kunitz TI.

Consequence of TI for Soya Bean and bird performance:
Excessive quantities of TI in feed will cause pancreatic hypertrophy leading to poor growth and decreased performance (Pacheco et al. 2014; García-Rebollar et al. 2016; Rada et al. 2017). This pancreatic hypertrophy is a compensatory modulation by the body to offset the effect of ingested trypsin inhibitors (Liener 1981; Waldroup et al. 1985).

Linear increase in pancreas size with increasing TI content in SB

TI also affect the nutritive value of SBM. Because of loss of endogenous protein there is reduced digestion which affects the nitrogen balance, gut viscosity resulting into reduced live weight and negative impact on feed efficiency. Palliyeguru et al. (2011) demonstrated dietary soya TI elicited an increased severity of sub-clinical necrotic enteritis. When amino acid digestibility is compromised, the ileal ingesta will have a relatively high content of undigested amino acids that pass into the large intestine and cecal tonsils, where microbial fermentation will occur. C. perfringens, a pathogenic agent of necrotic enteritis, needs specific amino acids and peptides for its proliferation (Nakamura et al., 1968).

Using the Data from the simulation conducted by Havenstein et al.(2003) with “1957” broilers versus ‘2001” broiler it is possible to estimate the TI intake of the “1957” birds fed 1957 diets and compare it with the estimate of TI intake by the “2001” birds fed 2001 diets.

Because of improvement of modern broilers in terms of average feed intake and body weight they consume more than three times TI than 1957 birds considering the same amount of TI in SBM.

The effects of TIA are particularly strong in young animals. It has been shown that overcooking of soybean meal decreases digestibility of amino acids (Lee and Garlich, 1992; Parsons et al., 1992). The explanation for the decreased amino acid digestibility and reduced growth responses appear to be related to the Maillard reaction with cross-linking involved to a lesser extent.

Correlation of TI (AOAC) & indirect Parameters for SBM quality:
Currently, the analytical technique most commonly used to measure soybean meal quality is protein solubility, perhaps combined with the urease test. The urease test has been used for some time as a measure of soybean meal processing. Trypsin inhibitors (TI) and urease activity (UA) are the two most relevant quality measurements for soybean products as feed ingredients for animals. TI were reported to be correlated with UA, so feed processing plants use UA as an indicator of TI in soybean meal (SBM). Chen et.al (2019) conducted a study to determine the levels of TI and UA in 414 SBM samples from 19 different countries and to validate whether TI and UA are correlated. They found that TI were poorly correlated with UA in solvent extracted SBM samples, suggesting that UA should not be used as a surrogate indicator for TI content in soybean products.

How to deal with TI in SBM?
Soybean meal (SBM) is the most important source of dietary protein for poultry. Although TI is reduced by heat treatment, overheating has a negative impact on protein quality and amino acid digestibility. Exogenous Protease enzymes can improve digestibility of feedstuffs, lower feed costs and improve animal performance. Proteases improve animal performance and nutrient digestibility by decreasing digesta viscosity, improving endogenous enzyme activity and decreasing pancreas weight (Bedford and Classen, 1993; Bedford and Schulze, 1998; Erdaw et al., 2017a,b; Yan et al., 2017).

As mentioned earlier the determination in the laboratory of the TI content of SBM and its relationship with AA availability is tedious and time-consuming and provides inconsistent results. Also, the traditional processes of treating SBM can’t remove the anti-nutritional factors to a safe level. Therefore, use of exogenous protease is very effective in reducing the deleterious effect of TI in SBM. Liu et al., in 2013 conducted a study wherein they used a protease enzyme (Cibenza EP150) with different levels of TI and found that protease enzyme was able to destroy almost all trypsin inhibitors (both Bowman-Birk & Kunitz TI) present in soyabean meal (at 1:1 ratio) and destroy substancially even in higher concentración (2:1) of TI as well.

Wedekind et al., in 2020 showed that addition of exogenous protease (Cibenza EP150) in a diet containing FFS(with TI 8.15mg/g) improved the amino acid digestibility and at the same time reduce the pancreas weight also indicating a potential amelioration of the negative effect of TI from FFS.

Conclusion:
There are lot of scientific evidences on the negative effect of soybean trypsin inhibitors in chickens. They can not only adversely affect the productive performance of chickens but can also impair their intestinal health. The beneficial responses of protease are likely due to decreases in endogenous amino acid losses, but in vitro evidence also demonstrates the ability of protease to hydrolyze Bowman-Birk and Kunitz-trypsin inhibitor proteins. Thus, there might be both direct and indirect mechanisms whereby amino acid digestibility is improved with proteases and so is the bird’s performances.