The Art of Silage: A Guide to Maximizing Quality and Nutritional Value

by Vesna Jenkins, Global Product Manager, Biomin BioStabil

Silage quality directly impacts animal health and farm profitability. This guide delves into the scientific principles and practical steps necessary to produce silage of the highest caliber.

Optimal Dry Matter

The journey to exceptional silage begins with harvesting at the ideal dry matter percentage. This critical timing ensures the preservation of yield and energy content. Striking the right balance is key; harvesting too early can lead to nutrient-poor silage, while too late can compromise the forage’s structural integrity. Aim for a dry matter content of 32-38% depending on forage type for optimal results.

Wilting Wisdom

When wilting is part of the process (e.g. grass, clover or alfalfa silage), efficiency is paramount. Achieving the desired dry matter in just a few hours help to prevent spoilage and retain the forage’s nutritional value. It’s a delicate dance between removing excess moisture and maintaining the feed’s quality.

Ensiling Excellence

Post-harvest, the clock is ticking. Compacting and sealing the forage within 24 to 48 hours is vital to create an anaerobic environment. This step is crucial to ensure anaerobic conditions for optimal fermentation. Pack the silage in thin layers with heavy enough machinery such as dual wheeled heavy tractors to achieve optimal dry matter density of around 250 kg per cubic meter. Pay special attention to the edges for even compaction. Once filled, seal the clamp with high quality overlapping sheets ensuring the edges are weighted down to prevent air ingress.

Rapid Acidification

The role of silage inoculants cannot be overstated. The proven science of the silage inoculant Biomin® BioStabil accelerates the pH drop, locking in dry matter, energy, and protein. This rapid acidification is a defense mechanism against pathogenic bacteria and mycotoxin producing fungi, ensuring the silage remains safe and nutritious.

Feed-Out Finesse

Proper management of the clamp face is crucial to prevent spoilage and ensure livestock health. Cut sufficient depth from the clamp face daily to prevent newly exposed silage near the face from having time to spoil. Maintain a smooth and clean silage face to minimize spoilage. Spilled debris on the ground can easily go moldy, presenting hazards for animals if fed out. Use the proven silage inoculant Biomin® BioStabil for longer aerobic stability in the silage and TMR.

Silage making is both a science and an art. By following these guidelines and choosing the right inoculant, farmers can secure the nutritional integrity of their forage.

Consult with an EW Nutrition representative to select the perfect BioStabil inoculant tailored to your forage type and conditions, and elevate your silage from good to great.

Mycotoxins in poultry – External signs can give a hint

Part 4: Paleness

By Dr. Inge Heinzl, Editor and Marisabel Caballero, Global Technical Manager Poultry

We already showed bad feathering, mouth and beak lesions, bone issues, and foot pad lesions as signs of mycotoxin contamination in the feed, but there is another indicator: paleness. Paleness can signify a low count of red blood cells resulting from blood loss or inadequate production of these cells. Other possibilities are higher bilirubin levels in the blood due to an impaired liver, leading to jaundice or missing pigmentation.

Hen With Pale Comb And Wattles Large — Hen with pale comb and wattles (adapted from Bozzo et al., 2023)

The mycotoxins mainly causing anemia are Aflatoxins, Ochratoxin, DON, and T-2 toxin

Anemia can be diagnosed using parameters such as red blood cell count, hemoglobin levels, and hematocrit/packed cell volume (PCV). Numerous studies have examined the impact of mycotoxins on hematological parameters. They reveal their propensity to affect red blood cell production by impairing the function of the spleen and inducing hematological alterations. On the other hand, anemia can be caused by blood loss. Due to affecting coagulation factors, mycotoxins can lead to internal hemorrhages. The gut wall damage, probably due to secondary infections such as coccidiosis and necrotic enteritis, can entail bloody diarrhea in various animal species.

Impact on the production of blood cells

Low values of blood parameters such as red blood cells, hemoglobin, and hematocrit can result from inadequate production due to impacted production organs. The World Health Organization (WHO, 1990) and European Commission (European Commission, 2001) have identified hematopoietic tissues as targets for necrosis caused by T-2 toxin. Chu (2003) even stated that “the major lesion of T-2 toxin is its devastating effect on the hematopoietic system in many mammals, including humans”. Pande et al. (2006) suggested that reduced hemoglobin values result from decreased protein synthesis due to mycotoxin contamination, a notion supported by Pronk et al. (2002), who described trichothecenes as potent inhibitors of protein, DNA, and RNA synthesis, particularly affecting tissues with high cell division rates. Additionally, the European Commission (2001) highlighted the sensitivity of red blood cell progenitor cells (in this trial, the cells of mice, rats, and humans) to the toxic effects of T-2 and HT-toxins. DAS also seems to attack the hematopoietic system, as shown in humans (WHO, 1990). A further cause for anemia might be low feed intake or nutrient absorption, which inhibits adequate iron absorption and leads to iron deficiency. In their case report, Bozzo et al. (2023) assumed that renal failure and a resulting impaired excretion capacity caused by OTA might even increase the half-life of the toxins. This would enhance their effects on their target organs, such as the liver and bone marrow, and lead to anemia.

Several studies utilizing different animal species and mycotoxin dosages have been conducted to assess the effects of Aflatoxins, Ochratoxin, and T-2 Toxin on hematological parameters. The following table provides a summary of some of these studies.

Animal species	Dosage	Impact	Reference
T-2 Toxin and other Trichothecenes
Broilers	T-2 – 0, 1, 2, and 4 mg T-2 toxin/kg n=30 per group	Significant reduction in hemoglobin at 1, 2, and 4 ppm; PCV significantly reduced at 4 ppm	Pande et al., 2006
Broilers	T-2 – 0 and 4 mg/kg diet n=60 per group	Decrease in hemoglobin, mean corpuscular volume, and mean corpuscular hemoglobin concentration	Kubena et al., 1989a
Broilers	4, 16, 50, 100, 300 ppm for seven days n=5-20 chickens per group	Anemia; significant reduction of hematocrit (50 and 100 ppm); survivors had atrophied lymphoid organs and were anemic	Hoerr et al., 1982
Yangzhou goslings	0, 0.2, 0.4, 0.6, 0.8, 1.0, 2.0 mg/kg; n=6 per group	Red blood cell count decreased in the 2.0 mg/kg group along with an increase in mean corpuscular hemoglobin (p<0.05) and reduced mean platelet volume (P<0.05)	Gu et al., 2023
Broilers	2 ppm; 32 birds per group	Anemia, as indicated by significantly (P<0.05) lower total erythrocyte count (TEC) values, lower hemoglobin levels, and packed cell volume; additional thrombocytopenia could be the cause of bleeding	Yohannes et al., 2013
DON
Broilers	5 and 15 mg/kg of feed for 42 days	Decrease in erythrocytes, mean corpuscular volume (MCV), and mean corpuscular hemoglobin concentration (MCHC) at 15 mg/kg; decrease in hematocrit and hemoglobin at both levels of DON.	Riahi, 2021
Piglets	0.6 mg/kg and 2.0 mg/kg	Significant decrease in mean corpuscular volume	Modrá et al., 2013
Broilers	16 mg/kg diet n=60 per group	Significant decrease in mean corpuscular volume	Kubena et al., 1989c
Ochratoxin
Broilers	2 mg/kg diet singly or combined with DAS 6 mg/kg	Reduced mean corpuscular hemoglobin values	Kubena et al., 1994
Broilers	2 mg/kg diet	Significant decrease in hemoglobin, hematocrit, mean corpuscular volume and mean corpuscular hemoglobin concentration	Kubena et al., 1989b
Aflatoxins
Broilers	2.5 µg/g	Decrease in red blood cell count	Huff et al., 1988
Broilers	≥1.25 µg/g	Significant decrease in hemoglobin and erythrocyte count	Tung et al., 1975
AFB1 + OTA
Laying hens	Natural feed contamination OTA – 31 ± 3.08 µg/kg and AFB1 – 5.6 ± 0.33 µg/kg dry weight	Anemia signs (pale appearance of combs and wattles), evidenced by the discoloration of the content of the femoral medullary cavity.	Bozzo et al., 2023

Table 1: The effects of different mycotoxins on hematological parameters – hematopoiesis

In their meta-analysis, Andretta et al. (2012) reported that the presence of mycotoxins in broiler diets decreased the hematocrit and the hemoglobin concentration by 5% and 15%, and aflatoxin alone decreased the parameters by 6% and 20%.

It should be evident that a simultaneous occurrence of several mycotoxins even aggravates the situation. In an experiment involving Sprague Dawley rats, administering T-2, DON, NIV, ZEA, NEO, and OTB decreased hematocrit and red blood cell counts across all mycotoxins. However, for DON, NIV, ZEN, and OTB, red blood cell values showed partial recovery after 24 hours (Chattopadhyay, 2013). Perhaps the organism learns to cope with the mycotoxins.

The examples show that Trichothecenes, such as T-2 toxin, DON, and others, as well as Ochratoxins and Aflatoxins, impact blood parameters such as hematocrit, hemoglobin, red blood cell count, and mean corpuscular volume. All these changes might lead to paleness of the skin and birds’ feet and combs.

Blood loss caused by bleeding or destruction of erythrocytes

The second possibility for anemia is blood loss due to injuries or lesions. In addition to directly causing hemorrhages, mycotoxins can promote secondary infections such as coccidiosis, which damages the gut and may produce bloody feces.

Parent-Massin (2004) e.g. reports on rapidly progressing coagulation problems after the ingestion of trichothecenes leading to septicemia and massive hemorrhages. Table 2 shows more examples of mycotoxins causing paleness due to blood loss.

Animal species	Dosage	Impact	Reference
T-2 Toxin and other Trichothecenes

Cats	T-2 toxin – 0.06-0.1 mg/kg body weight/day	Bloody feces, hemorrhages	Lutsky et al., 1978
Cats	T-2 toxin – 0.08 mg/kg BW every 48 h until death	Bloody feces	Lutzky and Mor, 1981
Pigeon	DAS in oat, sifting	Emesis and bloody stools	Szathmary (1983)
Calves	0.08, 0.16, 0.32, or 0.6 mg/kg BW per day for 30 days; 1 calf per treatment	Bloody feces at doses ≥0.32 mg/kg BW per day	Pier et al., 1976
Ochratoxin
Rats	Single dosages of 0, 17, or 22 mg/kg BW in 0.1 Mol/L NaHCO₃, gavage	Multifocal hemorrhages in many organs	Albassam et al., 1987

DON
Broilers	0, 35, 70, 140, 280, 560, and 1120 mg/kg body weight	Ecchymotic hemorrhages throughout the intestinal tract, liver, and musculature; relationship to hemorrhagic anemia syndrome seems warranted	Huff et al., 1981
Sterigmatocystin (ST)
10-12-day old chicks (93-101 g)	10 and 14 mg/kg BW intraperitoneal	Hemorrhages and foci of necrosis in the liver	Sreemannarayana et al., 1987
Aflatoxins
Broiler chickens	100 µg/kg feed	Hemorrhages in the liver	Abdel-Sattar, 2019
Turkeys	500 and 1000 ppb in the diet	Bloody diarrhea, spleens with hemorrhages, petechial hemorrhages in the small intestine	Giambrone et al., 1984
Broilers	0, 0.625, 1.25, 2.5, 5.0, and 10.0 mg/kg of diet combined with Infectious Bursal Disease	Slight hemorrhages in the skeletal muscles; decreased hematocrit and hemoglobin due to hemolytic anemia.	Chang and Hamilton, 1981
Broilers	0, 1, and 2 mg AFB1/kg of diet	Downregulation of the genes involved in blood coagulation (coagulation factor IX and X) and upregulation of anticoagulant protein C precursor, an inactivator of coagulation factors Va and VIIIa, and antithrombin-III precursor with 2 mg/kg	Yarru, 2009
Pigs	1-4 mg/kg, 4 weeks 0.4-0.8 mg/kg, 10 weeks	Hemorrhages	Henry et al., 2001

Table 2: The effects of different mycotoxins on hematological parameters – blood loss

Poor pigmentation

The fourth reason for paleness can be inadequate pigmentation. According to Hy Line (2021), the so-called pale bird syndrome is characterized by poor skin and egg yolk pigmentation and is caused by reduced absorption of fat and carotenoid pigments in compromised birds. This is also the case when the diets contain pigment supplements. Tyczkowski and Hamilton (1986) observed in their experiment with chickens exposed to doses of 1-8 µg of Aflatoxins/g of diet for three weeks that aflatoxins can cause poor pigmentation in chickens, probably by impairing carotenoids absorption but also transport and deposition. Osborne et al. (1982) asserted that carotenoids were significantly (P<0.05) depressed by 2 ppm ochratoxin as well as by 2.5 ppm aflatoxin in the diet.

Another possibility is oxidative stress due to the mycotoxin challenge. As pigments also serve as antioxidants, they may be expended for this purpose and are no longer available for pigmentation.

Paleness in poultry – a reason to think about mycotoxins

Paleness can have different causes, some of which are influenced by mycotoxins. If your chickens or hens are pale, checking the feed concerning mycotoxins is always recommended. A feed analysis can give information about possible contamination (see our tool MasterRisk).

In the case of contamination, effective products binding the mycotoxins and mitigating the adverse effects of these harmful substances can help protect your birds. As paleness is usually not the only effect of mycotoxins but also a decrease in growth, toxin binders can help maintain the performance of your animals.

References:

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Albassam, M. A., S. I. Yong, R. Bhatnagar, A. K. Sharma, and M. G. Prior. “Histopathologic and Electron Microscopic Studies on the Acute Toxicity of Ochratoxin a in Rats.” Veterinary Pathology 24, no. 5 (September 1987): 427–35. https://doi.org/10.1177/030098588702400510.

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Chang, Chao-Fu, and Pat B. Hamilton. “Increased Severity and New Symptoms of Infectious Bursal Disease during Aflatoxicosis in Broiler Chickens.” Poultry Science 61, no. 6 (June 1982): 1061–68. https://doi.org/10.3382/ps.0611061.

Chattopadhyay, Pronobesh, Amit Agnihotri, Danswerang Ghoyary, Aadesh Upadhyay, Sanjeev Karmakar, and Vijay Veer. “Comparative Hematoxicity of Fusarium Mycotoxin in Experimental Sprague-Dawley Rats.” Toxicology International 20, no. 1 (2013): 25. https://doi.org/10.4103/0971-6580.111552.

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Huff, W.E., R.B. Harvey, L.F. Kubena, and G.E. Rottinghaus. “Toxic Synergism between Aflatoxin and T-2 Toxin in Broiler Chickens.” Poultry Science 67, no. 10 (October 1988): 1418–23. https://doi.org/10.3382/ps.0671418.

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Kubena, L.F., R.B. Harvey, T.S. Edrington, and G.E. Rottinghaus. “Influence of Ochratoxin A and Diacetoxyscirpenol Singly and in Combination on Broiler Chickens.” Poultry Science 73, no. 3 (March 1994): 408–15. https://doi.org/10.3382/ps.0730408.

Kubena, L.F., R.B. Harvey, W.E. Huff, D.E. Corrier, T.D. Philipps, and G.E. Rottinghaus. “Influence of Ochratoxin A and T-2 Toxin Singly and in Combination on Broiler Chickens.” Poultry Science 68, no. 7 (July 1989): 867–72. https://doi.org/10.3382/ps.0680867.

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Kubena, L.F., W.E. Huff, R.B. Harvey, T.D. Phillips, and G.E. Rottinghaus. “Individual and Combined Toxicity of Deoxynivalenol and T-2 Toxin in Broiler Chicks.” Poultry Science 68, no. 5 (May 1989): 622–26. https://doi.org/10.3382/ps.0680622.

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Riahi, Insaf, Virginie Marquis, Anna Maria Pérez-Vendrell, Joaquim Brufau, Enric Esteve-Garcia, and Antonio J. Ramos. “Effects of Deoxynivalenol-Contaminated Diets on Metabolic and Immunological Parameters in Broiler Chickens.” Animals 11, no. 1 (January 11, 2021): 147. https://doi.org/10.3390/ani11010147.

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Tyczkowski, Juliusz K., and Pat B. Hamilton. “Altered Metabolism of Carotenoids during Aflatoxicosis in Young Chickens,” Poultry Science 66, no. 7 (July 1987): 1184–88. https://doi.org/10.3382/ps.0661184.

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Yohannes, T., A. K. Sharma, S. D. Singh, and V. Sumi. “Experimental Haematobiochemical Alterations in Broiler Chickens Fed with T-2 Toxin and Co-Infected with IBV.” Open Journal of Veterinary Medicine 03, no. 05 (2013): 252–58. https://doi.org/10.4236/ojvm.2013.35040.

Mycotoxins in poultry – External signs can give a hint

Part 3: Bone disorders and foot pad lesions

By Dr. Inge Heinzl, Editor, and Marisabel Caballero, Global Technical Manager Poultry

Bone health is essential for animals and humans. Besides giving structural support, allowing movement, and protecting vital organs, the bones release hormones that are crucial for mineral homeostasis and acid balance and serve as reservoirs of energy and minerals (Guntur & Rosen, 2012; Rath, N.C. & Durairaj, 2022; Suchacki et al., 2017).

Bone disorders and foot pad lesions are considerable challenges in poultry production, especially for fast-growing birds with high final weights. Due to pain, the animals do not move, and dominant, healthy birds may restrict lame birds’ access to feed and water. In consequence, these birds are often culled. Moreover, processing these birds is problematic, and often, they must be discarded or downgraded.

Foot pad lesions, another common issue in poultry production, can also have significant economic implications. On the one hand, pain restricts birds from eating and drinking and reduces weight gain. On the other hand, for many producers, chicken feet constitute a substantial part of the economic value of the bird; therefore, discarding them represents a significant financial loss. Additionally, to push poultry production in the right direction concerning animal health and welfare, a foot pad scoring system at the processing plant is in place in European countries.

Mycotoxins affect bones in different ways

Mycotoxins, depending on their target organs, can have diverse effects on the skeleton of birds. For example, mycotoxins that target the liver can disrupt calcium metabolism, which in turn affects the mineralization of the bones (rickets) and the impairment of chondrocytes can slow down bone growth (e.g., tibial dyschondroplasia). When the kidneys are impacted, urate clearance decreases, plasma uric acid consequently increases, and urate crystals form in the synovial fluid and tendon sheaths of various joints, particularly the hock joints. These examples highlight the complex and varied ways mycotoxins can impact poultry bone health.

Inadequate bone mineralization and strength – Rickets and layer cage fatigue

Sufficient bone mineralization is essential for the stability of the skeleton. Calcium (Ca), Vitamin D, and Phosphorous (P) deficiency leads to inadequate mineralization, weakens the bone, and can cause soft and bent bones or, in the case of layers, cage fatigue – a collapse of the spinal bone- and paralysis. Inadequate bone mineralization can be caused in different ways, among them:

Decrease in the availability of the nutrients necessary for mineralization. This can occur if the digestibility of these nutrients deteriorates
Impact on the Ca/P ratio—A ratio of 1 – 2:1 is vital for adequate bone development (Loughrill et al., 2016). Mycotoxins can alter absorption and transporters for one or both elements, altering their ratio.
Impact on the Vitamin D receptor, affecting its expression or the transporters for Ca and P.

Aflatoxins can impair bone mineralization by different modes of action. An important one is the impairment of the digestibility of Ca and P: Kermanshahi et al. (2007) fed broilers diets with high levels of aflatoxins (0.8 to 1.2 mg AFB1/kg feed) for three weeks, which resulted in a significant reduction of Ca and P digestibility. Other researchers, however, did not find an effect on Ca and P digestibility with lower aflatoxin levels: Bai et al. (2014) feeding diets contaminated with 96 (starter) and 157 µg Aflatoxins (grower) per kg of feed to broilers and Han et al. (2008) saw no impact on cherry valley ducks with levels of 20 and 40 µg AFB1/kg diet.

Indirectly, a decrease in the availability of Ca and P due to aflatoxin-contaminated feed can be shown by blood or tibia levels of these minerals, as demonstrated by Zhao et al. (2010): They conducted a trial with broilers, resulting in blood serum levels of Ca and P levels significantly (P<0.05) dropped with feed contaminated with 2 mg/kg of AFB1. Another trial conducted by Bai et al. (2014) showed decreased Ca in the tibia and reduced tibial break strength.

To get more information about the effect of mycotoxins on bone mineralization and the utilization of Ca, P, and Vit. D in animal organisms, Costanzo et al. (2015) challenged osteosarcoma cells with 5 and 50 ppb of aflatoxin B1. They asserted a significant down-modulation of the expression of the Vitamin D receptor. Furthermore, they assumed an interference of AFB1 with the actions of vitamin D on calcium-binding gene expression in the kidney and intestine. Paneru et al. (2024) could confirm this downregulation of the Vit D receptor and additionally of the Ca and P transporters in broilers with levels of ≥75 ppb AFB1. They also saw a significant reduction in tibial bone ash content at AFB1 levels >230 ppb, a decreased trabecular bone mineral content and density at AFB1 520 ppb, and a reduced bone volume and tissue volume of the cortical bone of the femur at the level of 230 ppb (see Figure 1). They concluded that AFB1 levels of already 230 ppb contribute to bone health issues in broilers.

Figure 1: Increasing doses of AFB1 (<2 ppb – 560 ppb) deteriorate bone quality (Paneru, 2024): Cross-sectional images of femoral metaphysis with increasing AFB1 levels (left to right). The outer cortical bone is shown in light grey, and the inner trabecular bone in blue. Higher levels of AFB1 (T4 and T5) show a disruption of the trabecular bone pattern (less dense blue pattern with thinner and more fragmented bone strands and with wide spaces between the trabecular bone) (shown in white).

All experiments strongly suggest that aflatoxins harm bone homeostasis. Additional liver damage, oxidative stress, and impaired cellular processes can exacerbate bone health issues.

Trichothecenes also negatively impact bone mineralization. Depending on the mycotoxin, they may affect the gut, decreasing the absorption of Ca and P and probably provoking an imbalance in the Ca/P ratio.

For instance, when T-2 toxin was fed to Yangzhou goslings at 0.4, 0.6, and 0.8 mg/kg of diet, it decreased the Ca levels (halved at 0.8 mg/kg) and increased the P levels in the blood serum, so the Ca/P ratio decreased from the adequate ratio of 1 – 2 to 0.85, 0.66, and 0.59 (P<0.05) (Gu et al., 2023). The alterations of the Ca and P levels, the resulting decreasing Ca/P ratio, and an additional increase in alkaline phosphatase (ALP) suggest that T-2 toxin negatively impacts Ca absorption, increases ALP, and, therefore, disturbs calcification and bone development.

Other studies show that serum P levels decreased in broilers fed DON-contaminated feed with levels of only 2.5 mg/kg (Keçi et al., 2019). One reason for the lower P level is probably the lower dry matter intake, affecting Ca and P intake. Ca serum level is not typically reduced, which can be explained by the fact that Ca plays many critical physiological roles (e.g., nerve communication, blood coagulation, hormonal regulation), so the body keeps the blood levels by reducing bone mineralization. Another explanation is delivered by Li et al. (2020): After their trial with broilers, they stated that dietary P deficiency is more critical for bone development than Ca deficiency or Ca & P deficiency. The results of the trial conducted by Keçi et al. with DON (see above) were reduced bone mineralization, affected bone density, ash content, and ash density in the femur and tibiotarsus with a stronger impact on the tibiotarsus than on the femur.

In line with trichothecenes effects in Ca and P absorption, Ledoux et al. (1992) suppose that diarrhea caused by intake of fumonisins leads to malabsorption or maldigestion of vitamin D, calcium and phosphorus, having birds with rickets as a secondary effect.

Ochratoxin A (OTA) impairs kidney function, negatively affects vitamin D metabolism, reduces Ca absorption, and contributes to deteriorated bone strength (Devegowda and Ravikiran, 2009). Indications from Huff et al. (1980) show decreased tibia strength after feeding chickens OTA levels of 2, 4, and 8 µ/g, and Duff et al. (1987) report similar results also in turkey poults.

A further mycotoxin possibly contributing to leg weakness is cyclopiazonic acid produced by Aspergillus and Penicillium. This mycotoxin is known for leading to eggs with thin or visibly racked shells, indicating an impairment of calcium metabolism (Devegowda and Ravikiran, 2009). Tran et al. (2023) also showed this fact with multiple mycotoxins.

The co-occurrence of different mycotoxins in the feed – the standard in praxis – increases the risk of leg issues. A trial with broiler chickens conducted by Raju and Devegowda (2000) showed a bone ash-decreasing effect of AFB1 (300 µg/kg), OTA (2 mg/kg), and T-2 toxin (3 mg/kg), fed individually but an incomparable higher effect when fed in combination.

Impairment of bone growth – tibial dyschondroplasia (TD)

In TD, the development of long bones is impaired, and abnormal cartilage development occurs. It is frequent in broilers, with a higher incidence in males than females. It happens when the bone grows, as the soft cartilage tissue is not adequately replaced by hard bone tissue. Some mycotoxins have been related to this condition: According to Sokolović et al. (2008), actively dividing cells such as bone marrow are susceptible to T-2 toxin, including the tibial growth plates, which regulate chondrocyte formation, maturation, and turnover.

T-2 toxin: In a study with primary cultures of chicken tibial growth plate chondrocytes (GPCs) and three different concentrations of T-2 toxin (5, 50, and 500 nM), He et al. (2011) found that T-2 toxin decreased cell viability, alkaline phosphatase activity, and glutathione content (P < 0.05). Additionally, it increased the level of reactive oxygen species and malondialdehyde in a dose-dependent way, which could be partly recompensated by adding an antioxidant (N-acetyl-cysteine). They concluded that T-2 toxin inhibits the proliferation and differentiation of GPCs and contributes, therefore, to the development of TD, altering cellular homeostasis. Antioxidants may help to reduce these effects.

Gu et al. (2023) investigated the closely bodyweight-related shank length and the tibia development in Yangzhou goslings fed feed with six different levels (0 to 2.0 mg/kg) of T-2 toxin for 21 days. They determined a clear dose-dependent slowed tibial length and weight growth (p<0.05), as well as abnormal morphological structures in the tibial growth plate. As tibial growth and shank length are closely related to weight gain (Gu et al., 2023; Gao et al., 2010; Ukwu et al., 2014; Yu et al., 2022), their slowdown indicates lower growth performance.

Fumonisin B1 is also a potential cause of this kind of leg issue. Feeding 100 and 200 mg/kg to day-old turkey poults for 21 days led to the development of TD (Weibking et al., 1993). Possible explanations are the reduced viability of chondrocytes, as found by Chu et al. (1995) after 48 h of exposure, or the toxicity of FB1 to splenocytes and chondrocytes, which was shown in different primary cell cultures from chicken (Wu et al., 1995).

Bacterial chondronecrosis with osteomyelitis lameness (BCO) can be triggered by DON and FUM

BCO presents a highly critical health and welfare issue in broiler production worldwide, and it is estimated that 1-2 % of condemnations in birds at the marketing age result from this disease. What is the reason? Today’s fast-growing broilers are susceptible to stress. This enables pathogenic bacteria to compromise epithelial barriers, translocate from the gastrointestinal tract or the pulmonary system into the bloodstream, and colonize osteochondrotic microfractures in the growth plate of the long bone. This can lead to bone necrosis and subsequent lameness.

In their experiment with DON and FUM in broilers, Alharbi et al. (2024) showed that these mycotoxins reduce the gut’s barrier strength and trigger immunosuppressive effects. They used contaminations of 0.76, 1.04, 0.94, and 0.93 mg DON/kg of feed and 2.40, 3.40, 3.20, and 3.50 mg FUM/kg diet in the starter, grower, finisher, and withdrawal phases, respectively. The team observed lameness on day 35; the mycotoxin groups always showed a significantly (P<0.05) higher incidence of cumulative lameness.

The increase in uric acid leads to gout

In general, mycotoxins, which damage the kidneys and, therefore, impact the renal excretion of uric acid, are potentially a factor for gout appearance.

One of these mycotoxins is T-2 toxin. With the trial mentioned before (Yangzhou goslings, 21 days of exposure), Gu et al. (2023) showed that the highest dosage of the toxin (2.0 mg/kg) significantly increased uric acid in the blood (P<0.05), possibly leading to the deposit of uric acid crystals in the joints and to gout.

Huff et al. (1975) applied Ochratoxin to chicks at 0, 0.5, 1.0, 2.0, 4.0, and 8.0 µg/g of feed during the first three weeks of life. They found ochratoxin A as a severe nephrotoxin in young broilers as it caused damage to the kidneys with doses of 1.0 µg/g and higher. At 4.0 and 8.0 µg/g doses, uric acid increased by 38 and 48%, respectively (see Figure 2). Page et al. (1980) also reported increased uric acid after feeding 0.5 or 1.0 mg/kg of Ochratoxin A to adult white Leghorn chickens.

Figure 2: Effect of Ochratoxin A on plasma uric acid (mg/100 ml) (according to Huff et al., 1975)

Foot pad lesions – a further hint of mycotoxicosis

Foot pad lesions often result from wet litter, originating from diarrhea due to harmed gut integrity. Frequently, mycotoxins impact the intestinal tract and create ideal conditions for the proliferation of diarrhea-causing microorganisms and, therefore, secondary infections. Some also negatively impact the immune defense system, allowing pathogens to settle down or aggravate existing bacterial or viral parasitic diseases. In general, mycotoxins affect the physical (intestinal cell proliferation, cell viability, cell apoptosis), chemical (mucins, AMPs), immunological, and microbial barriers of the gut, as reported by Gao et al. (2020). Here are some examples of the adverse effects of mycotoxins leading to intestinal disorders and diarrhea:

Mycotoxins can modulate intestinal epithelial integrity and the renewal and repair of epithelial cells, negatively impacting the intestinal barrier’s intrinsic components; for instance, DON can significantly reduce the transepithelial electrical resistance (TEER)(Grenier and Applegate, 2013). A higher permeability of the epithelium and a decreased absorption of dietary proteins can lead to higher protein in the digesta in the small intestine, which serves as a nutrient for pathogens including perfringens (Antonissen et al., 2014; Antonissen et al., 2015).
The application of Ochratoxin A (3 mg/kg) increased the number of S. typhimurium in the duodenum and ceca of White Leghorn chickens (Fukata et al., 1996). Another trial with broiler chicks at a concentration of 2 mg/kg aggravated the symptoms due to an infection by S. gallinarum (Gupta et al., 2005).
In a trial by Grenier et al., 2016, feed contaminated with DON (1.5 mg/kg), Fumonisin B (20 mg/kg), or both mycotoxins aggravated lesions caused by coccidia.
DON impacts the mucus layer composition by downregulating the expression of the gene coding for MUC2, as shown in a trial with human goblet cells (Pinton et al., 2015). The mucus layer prevents pathogenic bacteria in the intestinal lumen from contacting the intestinal epithelium (McGuckin et al., 2011).
Furthermore, DON and other mycotoxins decrease the populations of lactic acid-producing bacteria, indicating a shift in the microbial balance (Antonissen et al., 2016).
FB1 causes intestinal disturbances such as diarrhea, although it is poorly absorbed in the intestine. According to Bouhet and Oswald (2007), the main toxicological effect ascertained in vivo and in vitro is the accumulation of sphingoid bases associated with the depletion of complex sphingolipids. This negative impact on the sphingolipid biosynthesis pathway could explain other adverse effects, such as reduced intestinal epithelial cell viability and proliferation, modification of cytokine production, and impairment of intestinal physical barrier function.
T-2 toxin can disrupt the immune response, enhance the proliferation of coli in the gut, and increase its efflux (Zhang et al., 2022).

All these mycotoxins can cause foot pad lesions by impacting gut integrity or damaging the gut mucosa. They promote pathogenic organisms and, thus, provoke diarrhea and wet litter.

Mitigating the negative impact of mycotoxins on bones and feet is crucial for performance

Healthy bones and feet are essential for animal welfare and performance. Mycotoxins can be obstructive. Consequently, the first step to protecting your animals is to monitor their feed. If the analyses show the occurrence of mycotoxins at risky levels, proactive measures must be taken to mitigate the issues and ensure the health and productivity of your poultry.

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Mycotoxins pose a threat to the horse’s digestive system

Author: Judith Schmidt, Product Manager On Farm Solutions

Alarm in the gut! Horses have a susceptible digestive system that can quickly become unbalanced. Intestinal disorders in horses are usually associated with colic. Many factors can be responsible for intestinal issues. Have you ever thought about mycotoxins? What can horse owners do to support their horse´s gut health?
The equine stomach is not robust at all. Depending on their age and use, more than half of all horses suffer from stomach pain. Their digestive system is very sensitive and very different from that of other mammals: Horses cannot vomit and often suffer from severe abdominal pain, diarrhea, or cramps if they overeat or ingest spoiled feed.

The horse´s digestive system is complex and sensitive

The horse´s stomach has a relatively small capacity of around twelve to fifteen liters. Depending on the feed’s consistency and composition, it remains in the stomach for around one to five hours before it is pressed through the stomach outlet (pylorus) into the small intestine. The horse´s entire intestine is about ten times its body length.

Figure Digestive Tract Figure 1: The horse’s digestive tract

The horse´s gastrointestinal tract is a complex network, reacting extremely sensitively to changes and, therefore, highly susceptible to disorders. It essentially consists of the head intestine (lips, oral cavity, teeth, and esophagus), stomach (blind pouch, fundus, and stomach outlet), small intestine (duodenum, jejunum, and ileum), and large intestine (caecum, colon and rectum). Each section plays a crucial role in the digestive process; any disruption can lead to health issues. Understanding this structure is key to maintaining a horse’s digestive health.

Digestive disorders can have various reasons

Intestinal problems in horses can stem from diverse causes, often a complex interplay of multiple factors. By understanding these causes more deeply, horse owners can be better equipped to prevent and manage these issues. In the following, we delve into several of these causes.

1. Too long time between the feedings

Usually, a feeding break should be at most four to six hours, as, in nature, a horse is busy eating for at least 18 hours a day. In contrast to humans, who produce stomach acid only after food intake, the horse’s stomach produces gastric acid around the clock. The continuous intake of roughage, intensive chewing, and high saliva production (a horse produces 5 to 10 L of saliva per day) is, therefore, essential to protect the stomach mucosa by neutralizing excess gastric acid.

A too-long time between feedings and, therefore, no saliva production leads to an accumulation of gastric acid in the stomach. Four hours without roughage can already cause inflammation of the mucosa and probably ulcers.

2. Excessive amounts of concentrated feed

Excessive amounts of concentrates such as wheat or rye, conditioned by less chewing activity, increase gastric acid and histamine production, and the stomach lining can be attacked. Also, in this case, the development of stomach ulcers is possible.

Furthermore, the possibly resulting hyperacidity of the organism can lead to malfunctions of the organs, the skin, and the hooves.

3. Stress

Stress can also lead to a higher production of gastric acid and, therefore, to gastric ulcers. The horse is a flight animal. When it is under stress, it prepares for the impending escape, and the muscles are preferably supplied with blood, resulting in a lower blood flow to the mucous membranes. Furthermore, the rising cortisone level reduces the hydrochloric acid-suppressing prostaglandin E. As a result, more stomach acid is produced, irritating the gastric mucosa.

Stress can be triggered, e.g., by transportation, competitions, training, a change of house, a new rider, unsuitable equipment, or poor posture.

4. Dental diseases

The teeth are essential for digestion. When feed is chewed, it is broken down and mixed with saliva. Chipped teeth cannot chew well, and the feed is not sufficiently salivated or crushed, which has a detrimental effect on digestion.

For this reason, an expert vet should check the horse´s teeth at least once a year.

5. Administration of painkillers/medication

As with humans, long-term medication administration can promote the formation of stomach ulcers. For this reason, it is essential to ensure that horses are fed a gentle diet on the stomach, especially when using oral pain therapy, and to add stomach protection if necessary.

6. Endotoxins

If pathogens such as E. coli or clostridia proliferate extremely or are killed by an antibiotic, endotoxins can be released. These toxins can cause transformation or inflammation of the gut mucosa. In drastic cases, whole areas of the mucosa can die off.

7. Mycotoxins – the hidden danger in horse feed

Mycotoxins in plants and horse feed are a common but often unnoticed danger to horses’ health. Mycotoxins are natural, secondary metabolites of molds that have a toxic effect on humans and animals and can trigger mycotoxicosis. Contaminated feed can severely affect the horse’s health and, in the worst case, lead to death.

Over 90 % of the world´s feed production is estimated to be contaminated with at least one mycotoxin (see also Global Mycotoxin Report 2023, EW Nutrition. The intake of mycotoxins via hay, grain, silage, or compound feed can hardly be avoided. Mycotoxins are an increasing problem for all horse owners. Scientific studies show that the mycotoxins DON and ZEA are most frequently found in horse feed and, therefore, are also frequently detected in sports horses’ urine and blood samples.

Due to the highly toxic metabolic products, feed contaminated with molds can lead to severe liver and kidney diseases in horses, affect fertility, trigger colic, and promote digestive issues (diarrhea and watery stools).

Figure 2: Mycotoxins and their impact on horses

How to protect the horse from mycotoxins?

The first measure against the ingestion of mycotoxins is prevention. Correct pasture management and adequate barn and feed hygiene can contribute to preventing the ingestion of toxins.

However, despite the best prophylactic measures, it is impossible to prevent mycotoxin contamination of feed completely. As mycotoxins are not visible, analyzing the feed regarding mycotoxin contamination is recommended.

To protect your horse from mycotoxins, EW Nutrition developed MasterRisk, a tool for evaluating the risk of mycotoxins. Additionally, EW Nutrition has developed a complementary feed specifically for your horse´s needs in the form of granules. The sophisticated formulation of “Toxi-Pearls” is designed to bind mycotoxins and mitigate the adverse effects of mycotoxin contamination.

The pearls contain a mixture of mycotoxin binder, brewer’s yeast, and herbs:

The contained mycotoxin binder effectively controls the most important feed myco- and endotoxins. It additionally supports the liver and immune system and strengthens the intestinal barrier.
Brewer´s yeast supports the natural strength of the gastrointestinal tract. Due to its high natural content of beta-glucans and mannan-oligosaccharides (MOS), unique surface structure, and the associated high adsorption power, brewer´s yeast has a prebiotic effect on the intestinal microbiome.
The additional unique herbal mixture consists of the typical gastrointestinal herbs oregano, rosemary, aniseed, fennel, and cinnamon. The processed beetroot is a true all-rounder. Literature shows that it has an antioxidant effect and strengthens the immune system. It also promotes bile secretion and, therefore, supports fat digestion.

Conclusion

The horse’s digestive tract is highly sensitive and must be supported by all means. Besides failures in management, such as too long breaks between feedings or too high amounts of feed concentrate, mycotoxins present a high risk in horse nutrition. To prevent horses from intestinal issues, feed and stress management, dental care, and medication in the case of disease must be optimized. Particular attention should be paid to possible mycotoxin contamination. Effective toxin risk management, which consists of analysis, risk evaluation, and adequate toxin risk-managing products, should be implemented.

Mycotoxins in poultry – External signs can give a hint

Part 2: Beak/mouth lesions

by Marisabel Caballero and Inge Heinzl, EW Nutrition

The second part of this series will focus on oral lesions as signs of mycotoxin exposure. In this segment, we will delve into the appearance and development of oral lesions, their specific locations based on the type of mycotoxin, and how toxin levels and duration of exposure impact these lesions.

A bit of history: oral lesions in poultry and their association with mycotoxin exposure

Exposure to trichothecenes, a specific group of mycotoxins that includes T-2 toxin and scirpenols- such as monoacetoxyscirpenol (MAS), diacetoxyscirpenol (DAS), and triacetoxyscirpenol, has been associated with oral lesions since the early studies related with mycotoxins:

After reports of toxicosis in farm animals, Bamburg’s group (1968) aimed to isolate the toxins produced by Fusarium tricintum, then considered the most toxic fungus found in moldy corn in Wisconsin (USA). Their experiments led to the discovery of the T-2 toxin, named after the strain of F. tricintum from which it was isolated. Today, we know that this fungus was wrongly identified; it was F. sporotrichioides (Marasas et al., 1984). However, the toxin remained known as T-2.
Wyatt’s group (1972) already described yellowish-white lesions in the oral cavity of commercial broilers in a case report from 1972. The birds also presented lesions on the feet, shanks, and heads, which raised the possibility of contact with the toxin from the litter.
In some of the earliest experimental works regarding T-2 toxin in poultry, Christensen (1972) noted the development of oral necrosis in turkey poults consuming increasing levels of feed invaded by tricintum; also Wyatt (1972) found a linear increase in lesion size and severity with increasing toxin concentrations of T-2 in broilers, starting with 1 ppm. He noted that oral lesions occurred without exception in all birds receiving T-2 toxin.
Later, Chi and co-workers (1977) tested what later were considered sub-acute levels of T-2 in broiler chickens, finding oral lesions from 0.4 ppm after 5 to 6 weeks of exposure. At higher levels, the lesions appeared after two weeks. In the same year, Speers’ group (1977) concluded that adult laying hens are more tolerant to T-2 than young chicks and also found that another mycotoxin can produce oral lesions in poultry: monoacetoxyscirpenol (MAS).
Fast forward, scientific research continued and the effects of T-2 and scirpenols, either alone or in combinations, on performance and oral lesions in poultry are today well known, as studied by Kubena et al. (1989), Ademoyero & Hamilton (1991), Kubena et al. (1994), Diaz et al. (1994), Brake et al. (2000), Schuhmacher-Wolz et al. (2010), Verma & Swamy (2015), Vaccari (2017), and reviewed by Sokolovic et al. (2008), Minafra et al. (2018), Puvača & Ljubojević Pelić (2023), and Vörösházi et al. (2024).

What are oral lesions and how do they develop?

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Oral lesions caused by feed contaminated by T-2 toxin or scirpenols first occur as yellow plaques that develop into raised yellowish-gray crusts with covered ulcers (Hoerr et al., 1982). They also have been described as white in color and sometimes caseous in nature, as well as round and small, pin-point-sized, or large sheets covering a wider part of the mouth (Wyatt et al., 1972; Ademoyero and Hamilton, 1991).

Under the microscope, the lesions show a fibrinous surface layer and intermediate layers with invaginations full of rods and cocci, suggesting that the surrounding microbiota quickly colonizes the lesion. Inflammation immediately ensues as Wyatt’s team (1972) found the underlying tissues filled with granular leukocytes.

Why do T-2 toxins and other trichothecenes cause such lesions?

T-2 toxin and other trichothecenes are known for their caustic nature (evidenced by studies of Chi and Mirocha, 1978; Marasas et al., 1969), and for incidents involving accidental exposure by laboratory personnel (Bamburg et al., 1968, cited in Wyatt et al., 1972).

Induction of necrosis has been proposed as the main toxicity effect based on in vitro experiments on human skin fibroblast models. The findings were a reduction of ATP production in the cell line together with disruption of mitochondrial DNA (mtDNA) but without an increase in reactive oxygen species (ROS) or activity of caspase-3 and caspase-7, which would be the case for apoptosis (Janik-Karpinsa et al., 2022). A further study (Janik-Karpinsa et al., 2023) found that T-2, on the same cell line, reduced the number of mtDNA copies, damaging several genes and hindering its function; consequently, ATP production is inhibited, and cell necrosis ensues.

Meanwhile, an inflammatory response is triggered, and the lesions are colonized by the surrounding microbial flora (Wyatt et al., 1972). Supporting this notion, Hoerr et al. (1981) observed no mouth lesions after directly administering toxins via crop gavage. Enterohepatic recirculation, facilitating the return of toxins to the oral cavity through saliva, can amplify their toxic effects (Leeson et al., 1995).

Oral lesions depend on…

…the toxin

Oral lesions vary depending on the type of toxin involved. The location of lesions is influenced by the specific mycotoxin in the feed. For instance, research by Wyatt et al. (1972) revealed that with T-2 toxin, lesions initially manifest on the hard palate and along the tongue’s margins. Over two weeks, these lesions progress to affect the lingual papillae at the tongue’s root, the underside of the tongue, and the inner side of the lower beak near the midline.

In contrast, Ademoyero and Hamilton (1991) found that scirpenols present a different pattern. A study including 4 mycotoxins at 5 different levels found, after three weeks of exposure, that the lesions caused by triacetoxyscirpenol (TAS) predominantly occurred in the angles of the mouth (53% of the birds in the study), sparing the tongue. On the other hand, diacetoxyscirpenol (DAS) primarily induces lesions inside the upper beak (shown 47% of the broilers), followed by the inside of the lower beak (in 32% of the birds). The lesion distribution for scirpentriol mirrors that of TAS, while monoacetoxyscirpenol (MAS) resembles DAS in its impact.

Chi and Mirocha (1978) conducted a comparative analysis of lesions caused by T-2 toxin and DAS (both 5 ppm). They observed that the severity of DAS-induced lesions was higher, leading to difficulties in mouth closure for some chicks due to encrustations in the mouth angles.

…the contamination level

Different findings regarding the dose dependency of the lesions are available. Wyatt et al. (1972) (Figure 1) showed a relationship between the lesion size and the toxin level. A clear relationship between the severity and incidence of lesions and the amount of T-2 toxin was also demonstrated by Chi et al. (1977) and Speers et al. (1976). This linear relationship in the case of T-2 toxin could be confirmed for the scirpenols TAS, STO, MAS, and DAS by Ademoyero and Hamilton (1991). They demonstrated a distinct dose-response relationship in a trial with the scirpenols STO, TAS (at 5 levels between 0-8 µg/g), MAS, and DAS (at 5 levels between 0-4 µg/g).

Figure 1: Effect of the inclusion rate of T-2 on the lesion size (Wyatt et al., 1972)

Sklan et al. (2001) tested T-2 toxin at more likely levels (0, 110, 530, and 1,050 ppb) in male chickens and found lesions in 90% of the chickens fed 500 ppb T-2 and in 100% of the ones fed 1,000 ppb of T-2 after 10 to 15 days; the higher dosage provoked the lesions of higher severity. When feeding 100 ppb of T-2, mild lesions appeared in 40% of the chickens after 25 and 35 days. Another group led by Sklan (2003) studied four groups of 12 one-day-old male turkey poults fed mash diets with 0 (control), 241, 485, or 982 ppb T-2 toxin for 32/33 days. Feed intake and feed efficiency were not affected, but oral lesions were apparent on day 7. The severity of the lesions plateaued after 7–15 days, and the lesion score was dose-related (see Figure 2). In the same trial, they also tested DAS (0, 223, 429, or 860 ppb) and found a similar dose relationship.

Figure 2: Lesion scores in poults fed T-2 toxin at different inclusion rates and lengths of exposure (Sklan et al., 2003)

A different result is found in the trial conducted by Hoerr et al. (1982), who observed lesions 2-4 days after initiating toxin exposure (T-2 toxin and DAS; 4 and 16 ppm for 21 days) and comparable lesions when feeding 50, 100, or 300 ppm of the same toxins for 7 days. They asserted that the toxin concentration did not influence the time to onset of lesions nor their severity. Most research, however, shows a clear dose-response relation.

…the duration of exposure

On one hand, chronic exposure to low levels of toxins often requires a specific duration before noticeable effects emerge. And on the other hand, symptoms may also diminish due to hormesis, an adaptive response of the organism to moderate, intermittent stress.

With high toxin levels, lesions appear very soon after exposure. For example, Diaz et al. (1994) exposed hens to a diet containing 2 mg DAS/kg feed, finding lesions in 40% of the birds after only 48 h of exposure. Chi and Mirocha (1978) noted lesions after five days with a T-2 level of 5 ppm. At a comparable level (4 ppm), Chi et al. (1977) reported lesions emerging in the second week of exposure, with nearly 75% of chicks experiencing oral lesions by the third week. Sklan et al. (2003) saw lesions already on day 7 when feeding T-2 toxin or DAS at 1 ppm.

When testing lower levels (200 ppb), Sklan et al. (2001) found lesions after 10 days. They became more severe after 15 to 20 days and then, their severity decreased. Hoerr et al. (1982) also confirmed this by reporting that the number and size of the lesions increased until day 14 but decreased thereafter. Both studies confirm the phenomenon of hormesis.

… animal factors

In general, lesions appear with lower levels of toxins in broilers compared with layers and in layers compared with breeders. Turkeys are also less sensitive than broilers (Puvača & Ljubojević Pelić (2023).

Age also has an influence: young birds usually still have a maturing immune system, and the detoxification processes might not be entirely in place. However, their feed intake is lower and for this reason, in studies like Wang and Hogan (2019), higher impact of mycotoxins is found in older chicks.

Furthermore, additional stress factors influence the impact of mycotoxins in animals. Stress factors are cumulative and, when different factors concur, the severity of mycotoxin effects can increase.

Are oral lesions key indicators for implementing effective toxin risk management?

Oral lesions are painful for the animals, distract them from eating, and deteriorate growth performance. Often they are related with mycotoxins; however, when they appear, an investigation of different factors should take place, including mycotoxin analysis, as oral lesions may have other causes. Some of the known causes of oral lesions in poultry are also very fine feed particle size, deficiency of Vitamins A, E, B6 and Biotin, excessive levels of copper sulphate, and some parasite infections.

This article aimed to help with the differential diagnosis by providing a summary of the knowledge we have about the type and shape of the lesions related to mycotoxin contamination, which can help on a differential diagnosis. Checking the feed for mycotoxins and implementing effective toxin management helps prevent their negative effects, keeps the animals healthy, and contributes to animal welfare and, consequently, performance.

References

Ademoyero, Adedamola A., and Pat B. Hamilton. “Mouth Lesions in Broiler Chickens Caused by Scirpenol Mycotoxins.” Poultry Science 70, no. 10 (October 1991): 2082–89. https://doi.org/10.3382/ps.0702082.

Bamburg, J.R., N.V. Riggs, and F.M. Strong. “The Structures of Toxins from Two Strains of Fusarium Tricinctum.” Tetrahedron 24, no. 8 (January 1968): 3329–36. https://doi.org/10.1016/s0040-4020(01)92631-6.

Brake, J., P.B. Hamilton, and R.S. Kittrell. “Effects of the Trichothecene Mycotoxin Diacetoxyscirpenol on Feed Consumption, Body Weight, and Oral Lesions of Broiler Breeders.” Poultry Science 79, no. 6 (June 2000): 856–63. https://doi.org/10.1093/ps/79.6.856.

Chi, M.S., and C.J. Mirocha. “Necrotic Oral Lesions in Chickens Fed Diacetoxyscirpenol, T—2 Toxin, and Crotocin.” Poultry Science 57, no. 3 (May 1978): 807–8. https://doi.org/10.3382/ps.0570807.

Chi, M.S., C.J. Mirocha, H.J. Kurtz, G. Weaver, F. Bates, and W. Shimoda. “Subacute Toxicity of T-2 Toxin in Broiler Chicks ,.” Poultry Science 56, no. 1 (January 1977): 306–13. https://doi.org/10.3382/ps.0560306.

Christensen, C. M., R. A. Meronuck, G. H. Nelson, and J. C. Behrens. “Effects on Turkey Poults of Rations Containing Corn Invaded by Fusarium Tricinctum (CDA.) Sny. & Hans.” Applied Microbiology 23, no. 1 (January 1972): 177–79. https://doi.org/10.1128/am.23.1.177-179.1972.

Diaz, G. J., E. J. Squires, R. J. Julian, and H. J. Boermans. “Individual and Combined Effects of T‐2 Toxin and Das in Laying Hens.” British Poultry Science 35, no. 3 (July 1994): 393–405. https://doi.org/10.1080/00071669408417704.

European Food Safety Authority. “Scientific Opinion on the Risks for Animal and Public Health Related to the Presence of T-2 and HT-2 Toxin in Food and feed1EFSA Panel on Contaminants in the Food Chain (CONTAM).” European Food Safety Authority, 2011. https://www.efsa.europa.eu/en/efsajournal/pub/2481.

Hoerr, F, W Carlton, B Yagen, and A Joffe. “Mycotoxicosis Caused by Either T-2 Toxin or Diacetoxyscirpenol in the Diet of Broiler Chickens.” Fundamental and Applied Toxicology 2, no. 3 (May 1982): 121–24. https://doi.org/10.1016/s0272-0590(82)80092-4.

Hoerr, F. J., W. W. Carlton, and B. Yagen. “Mycotoxicosis Caused by a Single Dose of T-2 Toxin or Diacetoxyscirpenol in Broiler Chickens.” Veterinary Pathology 18, no. 5 (September 1981): 652–64. https://doi.org/10.1177/030098588101800510.

Janik-Karpinska, Edyta, Michal Ceremuga, Magdalena Wieckowska, Monika Szyposzynska, Marcin Niemcewicz, Ewelina Synowiec, Tomasz Sliwinski, and Michal Bijak. “Direct T-2 Toxicity on Human Skin—Fibroblast HS68 Cell Line—in Vitro Study.” International Journal of Molecular Sciences 23, no. 9 (April 29, 2022): 4929. https://doi.org/10.3390/ijms23094929.

Janik-Karpinska, Edyta, Michal Ceremuga, Marcin Niemcewicz, Ewelina Synowiec, Tomasz Sliwiński, and Michal Bijak. “Mitochondrial Damage Induced by T-2 Mycotoxin on Human Skin—Fibroblast HS68 Cell Line.” Molecules 28, no. 5 (March 6, 2023): 2408. https://doi.org/10.3390/molecules28052408.

Leeson, Steven, Gonzalo J. Diaz, and John D. Summers. Poultry metabolic disorders and Mycotoxins. University Books, 1995.

Marasas, W.F.O., J.R. Bamburg, E.B. Smalley, F.M. Strong, W.L. Ragland, and P.E. Degurse. “Toxic Effects on Trout, Rats, and Mice of T-2 Toxin Produced by the Fungus Fusarium Tricinctum (Cd.) Snyd. Et Hans.” Toxicology and Applied Pharmacology 15, no. 2 (September 1969): 471–82. https://doi.org/10.1016/0041-008x(69)90045-3.

Minafra, Cibele, Denise Russi Rodrigues, Isabel Cristina Mores Vaccari, Vinícius Duarte, Fabiana Ramos dos Santos, Weslane Justina da Silva, Alison Batista Vieira Silva Gouveia, Lorrayne Moraes de Paulo, Janaina Borges dos Santos, and Júlia Marixara Souza Silva. “Oral Lesions in Broilers Caused by Corn Mycotoxins: Review – Original: Lesões Orais Em Frangos de Corte Provocadas Por Micotoxinas Do Milho: Revisão.” Pubvet 12, no. 07 (July 17, 2018). https://doi.org/10.31533/pubvet.v12n7a134.1-11.

O., Marasas W F, Paul E. Nelson, and T. A. Toussoun. Toxigenic fusarium species, identity and Mycotoxicology. University Park: Pennsylvania State University Press, 1984.

Puvača, Nikola, and Dragana Ljubojević Pelić. “Problems and Mitigation Strategies of Trichothecenes Mycotoxins in Laying Hens Production.” Journal of Agronomy, Technology and Engineering Management (JATEM) 7, no. 2 (April 1, 2024): 1074–87. https://doi.org/10.55817/isad5453.

Schuhmacher-Wolz, Ulrike, Karin Heine, and Klaus Schneider. “Toxicity of HT-2 and T-2 Toxins.” European Food Safety Authority, 2010. https://www.efsa.europa.eu/en/supporting/pub/en-65.

Sklan, D., E. Klipper, A. Friedman, M. Shelly, and B. Makovsky. “The Effect of Chronic Feeding of Diacetoxyscirpenol, T-2 Toxin, and Aflatoxin on Performance, Health, and Antibody Production in Chicks.” Journal of Applied Poultry Research 10, no. 1 (March 2001): 79–85. https://doi.org/10.1093/japr/10.1.79.

Sklan, D., M. Shelly, B. Makovsky, A. Geyra, E. Klipper, and A. Friedman. “The Effect of Chronic Feeding of Diacetoxyscirpenol and T-2 Toxin on Performance, Health, Small Intestinal Physiology and Antibody Production in Turkey Poults.” British Poultry Science 44, no. 1 (March 2003): 46–52. https://doi.org/10.1080/0007166031000085373.

Sokolović, Marijana, Verica Garaj-Vrhovac, and Borka ŠImpraga. “T-2 Toxin: Incidence and Toxicity in Poultry.” Archives of Industrial Hygiene and Toxicology 59, no. 1 (March 1, 2008): 43–52. https://doi.org/10.2478/10004-1254-59-2008-1843.

Speers, G.M., C.J. Mirocha, C.M. Christensen, and J.C. Behrens. “Effects on Laying Hens of Feeding Corn Invaded by Two Species of Fusarium and Pure T-2 Mycotoxin ,.” Poultry Science 56, no. 1 (January 1977): 98–102. https://doi.org/10.3382/ps.0560098.

Verma, Yamini, and Madhu Swamy. “Clinico-Pathological Effect of FeedingFusarium Sporotrichioidesand t-2 Toxin Contaminated Diet in Broiler Chicken.” Indian Journal of Veterinary Pathology 39, no. 1 (2015): 58. https://doi.org/10.5958/0973-970x.2015.00013.9.

Vörösházi, Júlia, Zsuzsanna Neogrády, Gábor Mátis, and Máté Mackei. “Pathological Consequences, Metabolism and Toxic Effects of Trichothecene T-2 Toxin in Poultry.” Poultry Science 103, no. 3 (March 2024): 103471. https://doi.org/10.1016/j.psj.2024.103471.

Wyatt, R. D., B. A. Weeks, P. B. Hamilton, and H. R. Burmeister. “Severe Oral Lesions in Chickens Caused by Ingestion of Dietary Fusariotoxin T-21.” Applied Microbiology 24, no. 2 (1972): 251–57. https://doi.org/10.1128/aem.24.2.251-257.1972.

Wyatt, R. D., J. R. Harris, P. B. Hamilton, and H. R. Burmeister. “Possible Outbreaks of Fusariotoxicosis in Avians.” Avian Diseases 16, no. 5 (October 1972): 1123. https://doi.org/10.2307/1588839.

Unlocking Optimum Poultry Performance: Harnessing the Power of GH10 Xylanase

Author: Ajay Bhoyar, Global Technical Manager, EW Nutrition

Exogenous feed enzymes are increasingly utilized in poultry diets to manage feed costs, mitigate the adverse effects of anti-nutritional factors, and enhance nutrient digestion and bird performance. These enzymes are primarily employed to bolster the availability of nutrients within feed ingredients. Among the various enzymes utilized, those capable of breaking down crude fiber, starch, proteins, and phytates are commonly integrated into animal production systems.

In monogastric animals such as poultry and swine, a notable deficiency exists in the endogenous synthesis of enzymes necessary for the hydrolysis of non-starch polysaccharides (NSPs) like xylan (McLoughlin et al., 2017). This deficiency often manifests in poultry production as a decline in growth performance, attributed to increased digesta viscosity arising from the prevalence of NSPs in commonly utilized poultry feed ingredients. Without sufficient endogenous enzymes to degrade xylan, NSPs can increase digesta viscosity, encase essential nutrients, and create a barrier to their effective digestion. In response to this issue, monogastric animal producers have implemented exogenous enzymes such as xylanases into the feeds for swine and poultry to degrade xylan to short-chain sugars, thus reducing intestinal viscosity and improving the digestive utilization of nutrients (Sakata et al., 1995; Aragon et al., 2018)

Understanding Xylanase Enzymes

Xylanase enzymes belong to the class of carbohydrases that specifically target complex polysaccharides, such as xylan, a backbone nonstarch polysaccharide (NSP) prevalent in plant cell walls. These enzymes catalyze the hydrolysis of xylan into smaller, more digestible fragments, such as arabino–xylo-oligosaccharides (AXOs) and xylo-oligosaccharides (XOs), thereby facilitating the breakdown of dietary fiber in poultry diets.

Mechanism of action

It is generally agreed that the beneficial effects of feed xylanase are primarily due to the reduction in viscosity. Studies have shown that supplementing xylanases to animal feeds reduces digesta viscosity and releases encapsulated nutrients, thus improving the overall feed digestibility and nutrient availability (Matthiesen et al., 2021). The reduction in digesta viscosity by adding xylanase is achieved by the partial hydrolysis of NSPs in the upper digestive tract, leading to a decrease in digesta viscosity in the small intestine (Choct & Annison, 1992).

GH10 vs. GH11 Xylanases

Well-characterized xylanases are mostly grouped into glycoside hydrolase families 10 (GH10) and 11 (GH11) based on their structural characteristics (amino acid composition), mode of xylan degradation, the similarity of catalytic domains, substrate specificities, optimal conditions, thermostability, and practical applications.

Why are GH10 xylanases more efficient in animal production?

While both GH10 and GH11 xylanases act on the xylan main chain, these two enzyme types have different folds, substrate specificities, and mechanisms of action (Biely et al., 2016). The GH10 xylanases are more beneficial in animal feed production due to their efficient mechanism of action, broader substrate specificity, and better thermostability, as discussed below.

GH10 xylanase exhibits broader substrate specificity

Generally, the GH10 xylanases exhibit broader substrate specificity and can hydrolyze various forms of xylan, including soluble and insoluble substrates. On the other hand, GH11 xylanases have a narrower substrate specificity and are primarily active on soluble xylan substrates. GH10 xylanases exhibit higher catalytic versatility and can catalyze the cleavage of the xylan backbone at the nonreducing side of substituted xylose residues, whereas GH11 enzymes require unsubstituted regions of the xylan backbone (Collins et al., 2005; Chakdar et al., 2016).

As a result, GH10 xylanases generally produce shorter xylo-oligosaccharides than members of the GH11 family (Collins et al., 2005). Moreover, as shown in Fig.1, the GH10 xylanase can rapidly and effectively break down xylan molecules.

Fig.1.: Activity of a bacterial GH10 xylanase against soluble and insoluble arabinoxylans

Higher thermostability

Enzymes are proteins, and the protein’s primary structure determines their thermostability. The enzyme protein tends to denature at higher than tolerable temperatures, rendering it inactive. An enzyme’s high-temperature tolerance ensures its efficacy throughout the pelleted feed manufacturing. This results in consistent enzyme activity in the finished feed, subsequent gut health, and predictable performance benefits.

Xylanases with higher thermostability are more suitable for applications requiring high-temperature processes. An intrinsically heat-stable bacterial xylanase maintains its activity even under high-temperature feed processing conditions, such as pelleting.

A study conducted at the University of Novi Sad, Serbia (Fig. 2), with three pelleting temperatures (85 °C, 90 °C, and 95 °C) and conditioning times of 4 and 6 mins, showed that Axxess XY, an intrinsically thermostable GH10 xylanase, demonstrated more than 85% recovery even at 4 to 6 mins conditioning time and 95 °C temperature.

Fig.2: Optimum recovery of Axxess XY at elevated conditioning time and temperatures

Maintaining consistently optimum enzyme activity is crucial for realizing the benefits of enzyme inclusion in feed under challenging feed processing conditions.

Conclusion

In conclusion, exogenous feed enzymes, including xylanase, have gained widespread recognition for their pivotal role in poultry nutrition. The increasing use of xylanase is attributed to its ability to effectively manage feed costs while incorporating high-fiber ingredients without compromising poultry performance. However, the efficacy of xylanase is based on several factors, including its mode of action, substrate specificity, catalytic efficacy, and thermostability. Selecting the appropriate xylanase enzyme tailored for specific needs is crucial to harnessing its full benefits.

A GH10 xylanase, such as Axxess XY, designed explicitly as a feed enzyme, offers distinct advantages in poultry production. Its efficient mechanism of action, broader substrate specificity, and superior thermostability make it a preferred choice for optimizing animal performance. Notably, Axxess XY exhibits exceptional activity against soluble and insoluble arabinoxylans, thereby enhancing nutrient utilization, promoting gut health, and ultimately elevating overall performance levels in poultry.

Incorporating specialized GH10 Xylanase enzymes like Axxess XY represents a strategic approach to unlocking the nutrients in feedstuffs, ensuring optimal performance, and maximizing profitability in the poultry business.

References

Aragon, Caio C., Ana I. Ruiz-Matute, Nieves Corzo, Rubens Monti, Jose M. Guisán, and Cesar Mateo. “Production of Xylo-Oligosaccharides (XOS) by Controlled Hydrolysis of Xylan Using Immobilized Xylanase from Aspergillus Niger with Improved Properties.” Integrative Food, Nutrition and Metabolism 5, no. 4 (2018). https://doi.org/10.15761/ifnm.1000225.

Bedford, Michael R., and Henry L. Classen. “Reduction of Intestinal Viscosity through Manipulation of Dietary Rye and Pentosanase Concentration Is Effected through Changes in the Carbohydrate Composition of the Intestinal Aqueous Phase and Results in Improved Growth Rate and Food Conversion Efficiency of Broiler Chicks.” The Journal of Nutrition 122, no. 3 (March 1992): 560–69. https://doi.org/10.1093/jn/122.3.560.

Biely, Peter, Suren Singh, and Vladimír Puchart. “Towards Enzymatic Breakdown of Complex Plant Xylan Structures: State of the Art.” Biotechnology Advances 34, no. 7 (November 2016): 1260–74. https://doi.org/10.1016/j.biotechadv.2016.09.001.

Chakdar, Hillol, Murugan Kumar, Kuppusamy Pandiyan, Arjun Singh, Karthikeyan Nanjappan, Prem Lal Kashyap, and Alok Kumar Srivastava. “Bacterial Xylanases: Biology to Biotechnology.” 3 Biotech 6, no. 2 (June 30, 2016). https://doi.org/10.1007/s13205-016-0457-z.

Choct, M., and G. Annison. “Anti‐nutritive Effect of Wheat Pentosans in Broiler Chickens: Roles of Viscosity and Gut Microflora.” British Poultry Science 33, no. 4 (September 1992): 821–34. https://doi.org/10.1080/00071669208417524.

Collins, Tony, Charles Gerday, and Georges Feller. “Xylanases, Xylanase Families and Extremophilic Xylanases.” FEMS Microbiology Reviews 29, no. 1 (January 2005): 3–23. https://doi.org/10.1016/j.femsre.2004.06.005.

Matthiesen, Connie F., Dan Pettersson, Adam Smith, Ninfa R. Pedersen, and Adam. C. Storm. “Exogenous Xylanase Improves Broiler Production Efficiency by Increasing Proximal Small Intestine Digestion of Crude Protein and Starch in Wheat-Based Diets of Various Viscosities.” Animal Feed Science and Technology 272 (February 2021): 114739. https://doi.org/10.1016/j.anifeedsci.2020.114739.

McLoughlin, Rebecca F, Bronwyn S Berthon, Megan E Jensen, Katherine J Baines, and Lisa G Wood. “Short-Chain Fatty Acids, Prebiotics, Synbiotics, and Systemic Inflammation: A Systematic Review and Meta-Analysis.” The American Journal of Clinical Nutrition 106, no. 3 (March 2017): 930–45. https://doi.org/10.3945/ajcn.117.156265.

Sakata, T., M. Adachi, M. Hashida, N. Sato, and T. Kojima. “Effect of N-Butyric Acid on Epithelial Cell Proliferation of Pig Colonic Mucosa in Short-Term Culture.” DTW – Deutsche Tierärztliche Wochenschau 102, no. 4 (1995): 163–64.

Mycotoxins in poultry – External signs can give a hint

Part 1: Impact on Feathering

By Dr. Inge Heinzl, Editor, and Marisabel Caballero, Global Technical Manager, EW Nutrition

Mycotoxins are known to decrease health and performance in poultry production. Their modes of action, such as reducing protein synthesis and promoting oxidative stress and apoptosis, lead to cell destruction and lower cell replacement, affecting several organs and tissues.

When different stress factors collude, such as high temperatures and humidity, poor ventilation, high stocking density, and management events, the effects of in-feed mycotoxins can reach a higher level, which may include external signs.

The most common and recognized external sign of mycotoxicosis is mouth lesions caused by trichothecenes, which are highly associated with the presence of T-2 in the feed. However, other signs may appear, such as paleness of combs, shanks, and feet, as well as leg problems, ruffled feathers and poor feather coverage, feed passage, and abnormal feces.

In a series of articles, we want to report on external signs facilitating a differential diagnosis of mycotoxin contamination. This is necessarily followed by feed or raw material mycotoxin analysis and strategies to avoid or mitigate the effects of mycotoxin contamination in poultry production. In the first article, we will cover feathers.

A healthy plumage is crucial for growth and reproduction

Feathering is a crucial aspect of poultry health and productivity. Feathers are essential for thermoregulation, locomotion, adequate skin protection, and reproductive success, protecting hens from injury during mating. Inadequate feathering can lead to lower feed efficiency (Leeson and Walsh, 2004) as well as loss in fertility and chick production (Fisher, 2016). Mycotoxins in poultry feed can compromise feather quality in poultry production animals. This first article delves into the relationship between mycotoxins and poor feathering, exploring different mycotoxins and their mechanisms of action.

In which way do mycotoxins compromise feathering?

On the one hand, chronic mycotoxin exposure impairs the digestive process, hindering the absorption and utilization of vital nutrients essential for feather growth. This disruption can lead to malnutrition, directly impacting the quality and health of feathers. On the other hand, mycotoxins also interfere with metabolic processes critical for feather development, such as keratin synthesis (Wyatt et al., 1975; Nguansangiam, 2004). Enzymatic pathways involved in synthesizing keratin, the protein building block of feathers, are particularly vulnerable to mycotoxin-induced disruptions. The presence of mycotoxins in feed has been associated with the manifestation of sparse feathering and the sticking out of feathers at an unnatural angle (Emous and Krimpen, 2019). In the case of multiple mycotoxins occurring in the feed, even at singularly unimportant concentrations, a negative impact on feathering is possible. Different mycotoxins have different target organs and consequences for the animal, so their ways of compromising feathering also vary. As feathering needs protein availability, all mycotoxins affecting the protein metabolism or the absorption of nutrients also impact the feathering process. Let us look at the most prominent mycotoxins.

1. T-2 toxin

Due to climate change, T-2 toxins are on the rise. In the US, more than 50% of the tested samples contained T-2 toxin; in Europe, we found it in 31%, and in China, in 82% of the samples (EW Nutrition, 2024). The highest level was found in Europe, with 850 ppb.

Adverse effects of T-2 toxin in goslings were shown by Gu et al. (2023), who exposed the animals to 6 different levels of T-2 toxin, from 0.2 to 2.0 mg T-2 toxin/kg of feed. The goslings showed a sparse covering with short, dry, rough, curly, and gloss-free feathers on their back with dosages ≥0.8 mg/kg. When zooming on, T-2 can cause necroses of the layer of regenerative cells in the feather base, implying malformation or absence of new feathers, as well as structural damage to existing feathers on the base of the ramus and barb ridges (Hoerr et al. (1982), Leeson et al. (1995)).

The effects in feather regenerative cells are dose-dependent, as confirmed by Hoerr et al. (1982), who applied different doses of T-2 toxin (1.5, 2, 2.5, and 3 mg/kg body weight/day) to 7-day-old broilers for 14 days. Delayed feather development, especially at high dosages, was noticed, as well as malformations and opaque bands in the feathers, the latter probably caused by a segmental reduction in diameter.

Manafi et al. (2015) noticed feather malformations when broiler chickens were challenged with 0.5 ppm T-2 toxin in the feed in combination with an inoculation of 2.4×10⁸ cfu Mycoplasma gallisepticum. When the chickens were challenged only with T-2 toxin, the feathers were ruffled, showing that a coincidence of stress factors even aggravates the symptoms.

2. Aflatoxins

Aflatoxins, produced by certain Aspergillus species, are among the most notorious mycotoxins. Looking at test results of the last year, Aflatoxin shows incidences between 25 (USA) over 40-65% (Europe, LATAM, MEA, and SEAP) up to 84-88% (China and South Asia) with average levels up to 42 ppb in South Asia (EW Nutrition, 2023). However, more information about the concrete impact of aflatoxins on feathering is needed. They may indirectly affect feathering because they impact digestion and the utilization of nutrients or trace minerals such as zinc, which is essential for the feather construction process. Damage to the liver impacts protein metabolism, and keratin is also necessary for feather production.

In other studies, Muhammad et al. (2017) fed 5 mg AFB1/kg to Arbor Acres broilers, and the birds showed ruffled feathers. A significantly lower feather shine was noticed by Saleemi et al. (2020) when they gave the animals 300 μg AFB1/kg of feed, and the birds of Zafar et al. (2017) showed ruffled, broken, dull, and dirty feathers after six weeks of feeding an aflatoxin-contaminated diet.

3. Ochratoxin

Ochratoxins, commonly produced by Aspergillus and Penicillium fungi, also pose a significant threat to poultry. When looking at the mycotoxin report, this mycotoxin was found in 16% (Europe) to 70% (SEAP) of the samples (EW Nutrition, 2023). Ochratoxins primarily affect feathering by compromising the structural integrity of feathers and causing delayed feathering in broilers (Leeson, 2021).

Several trials have shown the negative impact of ochratoxin on feather quality. Hassan et al. (2010) fed OTA to laying hens and saw a dose-dependent (dosages from 0 to 10 mg/kg feed) occurrence of ruffled and broken feathers in the OTA group, whereas the plumage of the control group was shiny and well-formed. Hameed et al. (2012) also realized dull feathers when feeding 0.4 and 0.8 mg OTA per kg of feed. A further dose-dependent decrease in feather quality was described by Khan et al. (2023) in broiler chicks. He injected them with dosages from 0.1 to 1.7 mg/kg body weight on day 5 of age and saw a deterioration of feather appearance (rippled feathers) in the groups with the higher dosages of 1.3 and 1.7 mg/kg. Abidin et al. (2016) observed a similar dose-dependent deterioration of the feather quality in white Leghorn cockerels when feeding 1 or 2mg OTA/kg feed.

Combinations of aflatoxins and ochratoxins were also tested. Khan et al. (2017) fed moldy feed naturally containing 56 µg OTA and 136 µg AFB1 per kg to layer hens and saw a deterioration of feather quality with increasing feeding time. Qubih (2017) noticed ruffled feathers when feeding a diet naturally contaminated with 800 ppb of OTA and 100 ppb of AFB1.

4. Scirpenol mycotoxins

Parkhurst et al. (1992) examined the effects of different scirpenol mycotoxins. After feeding graded levels of fusarium mycotoxins to broiler chicks until three weeks of age, they discovered that the impact of scirpenols stretched across the entire feathered body parts and that the degree of feather alteration is dose-dependent. The main alteration was a frayed or even missing web on the medial side of the outer end of the feather due to poor development of the barbs, barbules, and barbicels, and the tip of the feathers became square instead of rounded—the thinner and weaker shafts of the feathers inclined to show an accentuated medial curve.

Figure Feathering Affected By Scirpenol Mycotoxins Parkhurst et al. (1992)

Figure 1: Feathering affected by scirpenol mycotoxins

In their trial, Parkhurst and Hamilton realized that 15-monoacetoxyscirpenol (15-MAS) caused the most severe alterations of feathers, and they determined a minimum effective dose (MED) of 0.5 µg/g diet. The MEDs for 4,15-diacetoxyscirpenol (4,15-DAS) and 3,4,15-triacetoxyscirpenol (TAS) were higher, 2 µg/g and > 8 µg/g, respectively.

How can we enable adequate feathering in poultry?

Adequate feathering of poultry is necessary for the animal’s health and welfare and to ensure fertility and productivity. The occurrence of mycotoxins in the feed – and the probability is high! – can cause poor feathering or the development of malformed feathers.

To best equip broilers, layers, and breeders, their feed must contain all nutrients essential for healthy growth and appropriate feathering. As the risk of contamination of the feed materials is very high (see EW Nutrition’s mycotoxin report 2023), it is of crucial importance to have an efficient mycotoxin risk management in place, which includes sampling, analysis of samples, and the use of mycotoxin binders. EW Nutrition offers MasterRisk, an online tool where farmers and feed millers can feed the results of their feed analysis concerning mycotoxins and get a risk management recommendation.

In the next part of the series, we will report on beak lesions and skin paleness, two other external signs of mycotoxin contamination.

References:

Abidin, Zain ul, Muhammad Zargham Khan, Aisha Khatoon, Muhammad Kashif Saleemi, and Ahrar Khan. “Protective Effects Ofl-Carnitine upon Toxicopathological Alterations Induced by Ochratoxin A in White Leghorn Cockerels.” Toxin Reviews 35, no. 3–4 (August 22, 2016): 157–64. https://doi.org/10.1080/15569543.2016.1219374.

Emous, R. A., and M. M. Krimpen. “Effects of Nutritional Interventions on Feathering of Poultry – a Review.” Poultry Feathers and Skin: The Poultry Integument in Health and Welfare, 2019, 133–50. https://doi.org/10.1079/9781786395115.0133.

Fisher, Colin. “Feathering in Broiler Breeder Females – Aviagen.” https://aviagen.com/, 2016. http://en.aviagen.com/assets/Tech_Center/Broiler_Breeder_Tech_Articles/English/Feathering-in-Broiler-Breeeder-Females-EN-2016.pdf.

Hameed, Muhammad Raza, Muhammad Khan, Ahrar Khan, and Ijaz Javed. “Ochratoxin Induced Pathological Alterations in Broiler Chicks: Effect of Dose and Duration.” Pakistan Veterinary Journal Pakistan Veterinary Journal 8318, no. 2 (December 2012): 2074–7764.

Hassan, Zahoor-Ul, M. Zargham Khan, Ahrar Khan, and Ijaz Javed. “Pathological Responses of White Leghorn Breeder Hens Kept on Ochratoxin A Contaminated Feed.” Pakistan Veterinary Journal 30, no. 2 (2010): 118–23.

Hoerr, F.J., W.W. Carlton, B. Yagen, and A.Z. Joffe. “Mycotoxicosis Produced in Broiler Chickens by Multiple Doses of Either T‐2 Toxin or Diacetoxyscirpenol.” Avian Pathology 11, no. 3 (January 1982): 369–83. https://doi.org/10.1080/03079458208436112.

Khan, Ahrar, Muhammad Mustjab Aalim, M. Zargham Khan, M. Kashif Saleemi, Cheng He, M. Noman Naseem, and Aisha Khatoon. “Does Distillery Yeast Sludge Ameliorate Moldy Feed Toxic Effects in White Leghorn Hens?” Toxin Reviews, January 25, 2017, 1–8. https://doi.org/10.1080/15569543.2017.1278707.

Khan, Shahzad Akbar, Eiko N. Itano, Anum Urooj, and Kashif Awan. “Ochratoxin-a Induced Pathological Changes in Broiler Chicks.” Pure and Applied Biology 12, no. 4 (December 10, 2023): 1608–16. https://doi.org/10.19045/bspab.2023.120162.

Leeson, S., and T. Walsh. “Feathering in Commercial Poultry II. Factors Influencing Feather Growth and Feather Loss.” World’s Poultry Science Journal 60, no. 1 (March 1, 2004): 52–63. https://doi.org/10.1079/wps20045.

Leeson, Steve. “Effects of Nutrition on Feathering.” Poultry World, May 22, 2021. https://www.poultryworld.net/specials/effects-of-nutrition-on-feathering/.

Leeson, Steven, Gonzalo J. Diaz Gonzalez, and John D. Summers. Poultry metabolic disorders and Mycotoxins. Guelph, Ontario, Canada: University Books, 1995.

Manafi, M., N. Pirany, M. Noor Ali, M. Hedayati, S. Khalaji, and M. Yari. “Experimental Pathology of T-2 Toxicosis and Mycoplasma Infection on Performance and Hepatic Functions of Broiler Chickens.” Poultry Science 94, no. 7 (July 2015): 1483–92. https://doi.org/10.3382/ps/pev115.

Muhammad, Ishfaq, Xiaoqi Sun, He Wang, Wei Li, Xinghe Wang, Ping Cheng, Sihong Li, Xiuying Zhang, and Sattar Hamid. “Curcumin Successfully Inhibited the Computationally Identified CYP2A6 Enzyme-Mediated Bioactivation of Aflatoxin B1 in Arbor Acres Broiler.” Frontiers in Pharmacology 8 (March 21, 2017). https://doi.org/10.3389/fphar.2017.00143.

Nguansangiam, Sudarat, Subhkij Angsubhakorn, Sutatip Bhamarapravati, and Apichart Suksamrarn. The Southeast Asian J of Tropical Medicine 34, no. 4 (2004): 899–905.

Parkhurst, Carmen R., Pat B. HamiltonON, and Adedamola A. AdemoyeroERO. “Abnormal Feathering of Chicks Caused by Scirpenol Mycotoxins Differing in Degree of Acetylation.” Poultry Science 71, no. 5 (May 1992): 833–37. https://doi.org/10.3382/ps.0710833.

Qubih, T. S. “Relationship between Mycotoxicosis and Calcium during Preproduction Period in Layers.” Iraqi Journal of Veterinary Sciences 26, no. 1 (June 28, 2012): 11–14. https://doi.org/10.33899/ijvs.2012.46888.

Saleemi, M. Kashif, Kamran Ashraf, S. Tehseen Gul, M. Noman Naseem, M. Sohail Sajid, Mashkoor Mohsin, Cheng He, Muhammad Zubair, and Ahrar Khan. “Toxicopathological Effects of Feeding Aflatoxins B1 in Broilers and Its Amelioration with Indigenous Mycotoxin Binder.” Ecotoxicology and Environmental Safety 187 (January 2020): 109712. https://doi.org/10.1016/j.ecoenv.2019.109712.

Wyatt, R.D., P.B. Hamilton, and H.R. Burmeister. “Altered Feathering of Chicks Caused by T-2 Toxin.” Poultry Science 54, no. 4 (July 1975): 1042–45. https://doi.org/10.3382/ps.0541042.

Zafar, Roheena, Farhat Ali Khan, and Muhammad Zahoor. “In Vivo Amelioration of Aflatoxin B1 in Broiler Chicks by Magnetic Carbon Nanocomposite.” Pesquisa Veterinária Brasileira 37, no. 11 (November 2017): 1213–19. https://doi.org/10.1590/s0100-736×2017001100005.

How xylanase can enhance swine productivity

By Dr. Ajay Awati, Director of Enzymes, EW Nutrition

The use of by-products and high-fiber ingredients in feed formulations has increased in swine operations. Driven by both economic and sustainability goals, this shift has emphasized the importance of understanding the role of dietary fibers and carbohydrases in swine nutrition and health (Petry & Patience, 2020). These feeds rich in fiber are generally considered to have low nutritional value due to the lower digestive energy or amino acid levels when compared to concentrated feeds with high starch or proteins (Woyengo et al., 2014).

Dietary fiber is vital in pig nutrition, necessitating a baseline inclusion to support regular digestive tract functions (Wenk, 2001). Incorporating fiber into the diets of monogastric animals raises concerns due to its correlation with reduced nutrient utilization and diminished net energy levels (Noblet; Le Goof, 2001). High-fiber diets can present challenges for inclusion in monogastric animals’ feeds, especially young animals, due to their bulky nature and restricted ability to ferment fiber, impacting nutrient uptake based on fiber type, the age of the pig, and diet composition (Bach Knudsen et al., 2012).

Moreover, the apparent ileal digestibility (AID) of nutrients is adversely affected by dietary fiber, attributed to the small intestine’s deficiency in endogenous enzymes necessary for breaking down these bonds (Bach Knudsen et al., 2012).

This article aims to demonstrate how enzymatic degradation of arabinoxylans, particularly through xylanase enzymes, can mitigate anti-nutritional effects and enhance the nutritional value of high-fiber swine diets, thereby improving animal health and performance.

Into the World of Arabinoxylans

In plants classified as monocotyledonous, such as cereals, the main non-starch polysaccharides (NSP’s) components of the cell wall are arabinoxylans, cellulose and β-glucan (Bach Knudsen, 1997). Arabinoxylans represent a complex group of dietary fibers with significant implications for swine nutrition and health. Their structural heterogeneity can influence physicochemical properties, biological activities, and affect pigs’ gut microbiota and immune system. Present in both soluble and insoluble forms, it consists of a backbone of xylose residues with arabinose side chains, playing a crucial role in the nutritional dynamics within swine diets (Mudgil & Barak, 2013).

The fermentability of corn-based dietary fiber is limited by its insoluble fraction and complex branched structure; impacting the digesta transit rate and reducing the digestibility of nutrients (Gutierrez et al., 2013). Supplementing exogenous carbohydrases offers a viable approach to enhance the utilization of fiber that is otherwise difficult to ferment, potentially amplifying its positive effects.

Xylanase’s Impact on Fiber and Gut Health

Non-digestible carbohydrates may be fermented by microbial populations along the gastrointestinal tract to synthesize short-chain fatty acids that may be absorbed and metabolized by the pig. Such indigestible carbohydrates consist of specific disaccharides, oligosaccharides, resistant starches, and non-starch polysaccharides. The presence and composition of these indigestible carbohydrates in pig diets vary based on the types of feed ingredients incorporated into their meals (Navarro et al., 2019). Xylanase works on the hydrolysis of the arabinoxylan fraction of NSPs. The NSPs present in the walls of plant cells encapsulate nutrients, making them unavailable for the action of the animal’s own digestive enzymes. Moreover, NSPs exhibit a high affinity for water within the gastrointestinal lumen, leading to elevated digesta viscosity. This increased viscosity reduces gastrointestinal motility, facilitating an environment conducive to the proliferation of pathogenic microflora (Choct, 1998). The advantageous outcomes of enzyme supplementation arise from the enzymatic disruption of intact cellular membranes, leading to the release of sequestered nutrients, or are a consequence of modifying the physicochemical properties of non-starch polysaccharides, due to changes in viscosity and water-holding capacity and/or changes in the composition and content of bacteria in the intestine (Bedford, M. R., & Classen, 1992).

Arabinoxylans in Cereal Grains and Their By-products

Factors such as genetics, climate, maturity stage, fertilizer use, and post-harvest storage time influence the proportion of total cell wall polysaccharides in cereal grains. These factors vary across production systems and countries, depending on the availability of feed resources (Paloheimo et al., 2010).

Cereal grains and their by-products, including wheat bran, corn distillers dried grains with solubles (DDGS), and rice husks, serve as significant sources of arabinoxylans. Their incorporation into swine diets is growing due to economic advantages.

The ethanol industry’s growth has increased the availability of distillery by-products. Brazil alone generates an estimated 366 million tons of DDGS annually (USDA, 2017). Among these by-products, distiller-dried grains are prevalent, especially in the U.S. pork industry as feed ingredients.

Corn, wheat, and barley, as staple ingredients in swine feed, exhibit significant variations in their NSP and arabinoxylans content. In grain form, corn contains 4.7% AX with a soluble component of 0.5%, while wheat has a higher arabinoxylans content at 7.3% with 1.8% being soluble. Barley stands out with the highest arabinoxylans content at 8.4%, of which 1.2% is soluble, reflecting its rich fiber composition. The processing into flour results in a reduction of arabinoxylans content across all three cereals, highlighting the impact of processing on dietary fiber availability (Knudsen, 2014).

Rice distillers’ by-product is recognized as a valuable protein source, boasting high crude protein levels. Nonetheless, its high fiber content can restrict usage (Huang et al., 2017). Wheat bran is particularly rich in arabinoxylans, enhancing its dietary fiber content. DDGS also contain significant amounts of both soluble and insoluble arabinoxylans, resulting from the corn kernel’s residual non-starch polysaccharides (Agyekum & Nyachoti, 2017).

It is essential to understand the specific levels of arabinoxylans in these components to create balanced diets that optimize nutritional benefits while minimizing potential anti-nutritional effects.

Addressing Arabinoxylan Degradation

Xylanases target specific substrates, necessitating the presence of arabinoxylans for their effective action. The complex structure of arabinoxylans makes them resistant to degradation by the swine’s endogenous enzymes, presenting a dual challenge: how to harness the beneficial effects of soluble arabinoxylans while mitigating the negative impacts of their insoluble counterparts.

These enzymes specifically cleave the 1,4-β-D-xylosidic bonds in arabinoxylans, randomly targeting xylose linkages within the xylan structure. Each enzyme type has a unique pattern of degradation (Collins et al., 2005) and GH 10 xylanases specialize in breaking down arabinoxylans with high arabinose substitution into smaller oligosaccharides. These oligosaccharides are valuable for fermentation, serving as energy sources or prebiotics.

Also, this group of enzymes action not only reduces gut viscosity but can lead to enhanced feed efficiency, growth performance, and overall health of swine by improving the digestibility of fibrous components in feed (Lærke et al., 2015). GH 10 xylanases often have optimal activity at pH levels found in the animal gut, and their thermal stability ensures they retain activity under feed processing temperatures. Lei et al. (2016) highlighted the efficacy of xylanase in improving nutrient digestibility and overall feed efficiency. By targeting the arabinoxylans present in swine diets, xylanase enzymes facilitate a more efficient conversion of feed into energy, contributing to improved growth rates and performance metrics.

As detailed by Tiwari, Singh, & Jha (2019), arabinoxylans undergo fermentation in the gut, leading to the production of short-chain fatty acids (SCFAs) that beneficially alter the gut microbial ecology. The application of GH 10 xylanases has been highlighted for its potential to significantly enhance the degradation of arabinoxylans, thereby improving the fermentation process and increasing the yield of SCFAs. This enzymatic breakdown facilitates more efficient nutrient absorption and overall better gastrointestinal health, directly influencing swine growth and performance positively.

Swine Intestine

A study reveals that xylanase supplementation significantly reduces mortality rates in pigs in a dose-dependent manner. With mortality rates dropping from 4.16% in the control group to as low as 2.25% with the highest xylanase dosage, the results highlight xylanase’s potential to improve gut health and increase survival rates. This suggests a promising approach for boosting pig well-being and reducing the reliance on enteric antibiotics, marking a significant stride in sustainable animal nutrition practices.(Zier-Rush et al., 2016).

The research conducted by Petry et al. (2020) demonstrated that xylanase increased the digestibility of non-starch polysaccharides, particularly arabinoxylan, in diets high in insoluble corn fiber. This improvement in nutrient absorption highlights xylanase’s role in optimizing the use of high-fiber ingredients in swine diets, thereby enhancing animal health and performance. Due to its cost-effectiveness and nutrient profile, xylanase supplementation enhances the nutritional value of DDG in swine diets.

The strategic implementation of xylanase in swine diets represents a promising approach to the challenges posed by high-fiber feed ingredients. By improving the digestibility of arabinoxylans and other complex carbohydrates, xylanase supplementation can mitigate the anti-nutritional effects of insoluble fibers, enhance feed efficiency, and support optimal growth and health outcomes in swine.

Soluble And Insoluble AX

Enhancing Swine Productivity with Enzyme Solutions

With the growing incorporation of co-products in swine feed, there arises a crucial need to transform the high fiber content into a beneficial asset for the animals. The strategic incorporation of enzyme solutions, particularly xylanase enzymes, into swine feed formulations emerges as a scientifically supported approach to significantly enhance the digestibility of high-fiber diets. This method effectively addresses the nutritional intricacies posed by arabinoxylans, facilitating improved feed utilization. Moreover, the action of xylanase enzymes extends beyond enhancement of nutrient absorption; it plays a pivotal role in promoting the health and performance of swine. Such targeted nutritional strategies are vital in the context of swine production systems, highlighting the necessity of integrating these enzymatic solutions to achieve optimal animal health, growth, and productivity.

References:

Agyekum, K. A., & Nyachoti, C. M. (2017). Nutritional and metabolic consequences of feeding high-fiber diets to swine: A review. Engineering, 3(5), 716-725. https://doi.org/10.1016/J.ENG.2017.03.010
Bach Knudsen, K. E. (1997). Carbohydrate and lignin contents of plant materials used in animal feeding. Animal Feed Science and Technology, 67, 319-338.
Bach Knudsen, K. E., Hedemann, M. S., & Laerke, H. N. (2012). The role of carbohydrates in intestinal health of pigs. Animal Feed Science and Technology, 173, 41–53.
Bedford, M. R., & Classen, H. L. (1992). Reduction of intestinal viscosity through manipulation of dietary rye and pentosanase concentration is effected through changes in the carbohydrate composition of the intestinal aqueous phase and results in improved growth rate and food conversion efficiency of broiler chicks. The Journal of Nutrition, 122, 560-569.
Choct, M. (1998). The effect of different xylanases on carbohydrate digestion and viscosity along the intestinal tract in broilers. Australian Poultry Science Symposium, 10.
Collins, T., Gerday, C., & Feller, G. (2005). Xylanases, xylanase families and extremophilic xylanases. FEMS Microbiology Reviews, 29(1), 3–23. https://doi.org/10.1016/j.femsre.2004.06.005
Gutierrez, N. A., Kerr, B. J., & Patience, J. F. (2013). Effect of insoluble-low fermentable fiber from corn-ethanol distillation origin on energy, fiber, and amino acid digestibility, hindgut degradability of fiber, and growth performance of pigs. Journal of Animal Science, 91, 5314–5325. https://doi.org/10.2527/jas.2013-6328
Huang, Y. F., Gao, X. L., Nan, Z. B., & Zhang, Z. X. (2017). Potential value of the common vetch (Vicia sativa L.) as an animal feedstuff: A review. Journal of Animal Physiology and Animal Nutrition, 101, 807-823. https://doi.org/10.1111/jpn.12617
Lærke, H. N., Arent, S., Dalsgaard, S., & Bach Knudsen, K. E. (2015). Effect of xylanases on ileal viscosity, intestinal fiber modification, and apparent ileal fiber and nutrient digestibility of rye and wheat in growing pigs. Journal of Animal Science, 93(9), 4323-4335.
Lei, Z., Shao, Y., Yin, X., Yin, D., Guo, Y., & Yuan, J. (2016). Combination of xylanase and debranching enzymes specific to wheat arabinoxylan improve the growth performance and gut health of broilers. Journal of Agricultural and Food Chemistry, 64(24), 4932-4942.
Mudgil, D., & Barak, S. (2013). Composition, properties and health benefits of indigestible carbohydrate polymers as dietary fiber: a review. International Journal of Biological Macromolecules, 61, 1-6. https://doi.org/10.1016/j.ijbiomac.2013.06.044
Navarro, D. M. D. L., Abelilla, J. J., & Stein, H. H. (2019). Structures and characteristics of carbohydrates in diets fed to pigs: a review. Journal of Animal Science and Biotechnology, 10, 39. https://doi.org/10.1186/s40104-019-0345-6
Noblet, J., & Le Goff, G. I. (2001). Effect of dietary fibre on the energy value of feeds for pigs. Animal Feed Science and Technology, 90, 35-52.
Paloheimo, M., Piironen, J., & Vehmaanperä, J. (2010). Xylanases and cellulases as feed additives. In M. Bedford & G. Partridge (Eds.), Enzymes in farm animal nutrition (2nd ed., pp. 12-53). CABI Publishing. https://doi.org/10.1079/9781845936747.0012
Petry, A., Huntley, N., Bedford, M., Zijlstra, R. T., & Patience, J. (2020). Supplementing xylanase increased the digestibility of non-starch polysaccharides, particularly arabinoxylan, in diets high in insoluble corn fiber fed to swine with a 36-d dietary adaptation period. Journal of Animal Science, 98(52-52).
Petry, A. L., & Patience, J. F. (2020). Xylanase supplementation in corn-based swine diets: a review with emphasis on potential mechanisms of action. Journal of Animal Science, 98(11), skaa318. https://doi.org/10.1093/jas/skaa318
Tiwari, U., Singh, A., & Jha, R. (2019). Fermentation characteristics of resistant starch, arabinoxylan, and β-glucan and their effects on the gut microbial ecology of pigs: A review. Animal Nutrition, 5, 217-226.
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Woyengo, T. A., Beltranena, E., & Zijlstra, R. T. (2014). Nonruminant nutrition symposium: controlling feed cost by including alternative ingredients into pig diets: a review. Journal of Animal Science, 92(4), 1293-1305.
Zier-Rush, C. E., Groom, C., Tillman, M., Remus, J., & Boyd, R. D. (2016). The feed enzyme xylanase improves finish pig viability and carcass feed efficiency. Journal of Animal Science, 94(suppl_2), 115. https://doi.org/10.2527/msasas2016-244

Overcoming Challenges of Xylanase Inhibitors in Animal Feeds

By Dr. Ajay Awati, Global Director Enzymes, EW Nutrition

In recent years, the scientific understanding of xylanase inhibitors (XIs) and their impact on animal nutrition has grown significantly. Xylanase, a crucial enzyme used to enhance nutrient availability in feed, can face challenges from XIs present in cereal grains. This article explores the evolution of plant protection mechanisms, the economic impact of XIs, and the development of a novel xylanase, Axxess XY, resistant to these inhibitors.

Xylanase inhibitors – an evolutionary protection mechanism of plants

Xylanase inhibitors (XI) are a classic example of the evolutionary development of protection mechanisms by cereal plants against pathogens. Microorganisms, such as fungal pathogens, involve the degradation of xylan as one of the mechanisms in pathogenesis (Choquer et al., 2007). There are also other mechanisms by which microorganism-produced xylanases affect plants.

To protect themselves, plants evolved xylanase inhibitors to prevent the activities of xylanases. XIs are plant cell wall proteins broadly distributed in monocots. There are three classes of XIs with different structures and inhibition specificities (Tundo et al., 2022):
1. Triticum aestivum xylanase inhibitors (TAXI)
2. Xylanase inhibitor proteins (XIP), and
3. Thaumatin-like xylanase inhibitors (TLXI).

Xylanase inhibitors have an economic impact

In animal nutrition, xylanases are widely used in diets containing cereal grains and other plant materials to achieve a higher availability of nutrients. The inhibitory activity of XIs prevents this positive effect of the enzymes and, therefore, makes them economically relevant. Studies have reported that higher levels of XIs negatively impact broiler performance. For example, in one of the studies, broilers fed with grains of a cultivar with high inhibitory activity showed a 7% lower weight on day 14 than broilers fed with grains of a cultivar with less inhibitory activity (Madesen et al., 2018). Another study by Ponte et al. (2004) also concluded that durum wheat xylanase inhibitors reduced the activity of exogenous xylanase added to the broiler diets.

Xylanase inhibitors can withstand high temperatures

Even though XIs can impact the performance of exogenous xylanase in different ways, only minor attention was paid to the reduction of xylanase’s susceptibility to xylanase inhibitors during the xylanase development in the last decades. Firstly, the issue was ignored mainly through the assumption that XIs are denatured or destroyed during pelleting processes. However, Smeets et al. (2014) showed that XIs could sustain significant temperature challenges. They demonstrated that after exposing wheat to pelleting temperatures of 80°C, 85°C, 92°C, and 95°C, the recovery of inhibitory activity was still 99%, 100%, 75%, and 54%, respectively. Furthermore, other studies also confirmed that conditioning feed at 70-90°C for 30 sec followed by pelleting had little effect on the XI activity in the tested feed, showing that xylanase inhibitors are very likely present in most xylanase-supplemented feeds fed to animals.

Do we only have the problem of xylanase inhibitors in wheat?

No. After first reports of the presence of xylanase inhibitors in wheat by Debyser et al. (1997, 1999), XIs were also found in other cereal grains (corn, rice, and sorghum, etc.), and their involvement in xylanase inhibition and plant defense has been established by several reports (Tundo et al., 2022).

In most of the countries outside Europe, exogenous xylanase is used not only in wheat but also in corn-based diets. Besides broiler feeds, also other animal feeds, such as layer or swine feed being part of more mixed-grain diets, are susceptible to the inhibitory activity of XIs. Nowadays, the situation is getting worse with all the raw material prices increasing and nutritionists tending to use other feed ingredients and locally produced cereals. They need a xylanase which is resistant to xylanase inhibitors.

Xylanases’ resistance to XIs is crucial – Axxess XY shows it

To prevent xylanases from losing their effect due to the presence of xylanase inhibitors, the resistance of new-generation xylanases to these substances is paramount in the development process, including enzyme discovery and engineering.

In the past 25 years, scientists have learned much about XI-encoding genes and discovered how xylanase inhibitors can block microbial xylanases. Additionally, there has been a significant increase in understanding the structural aspects of the interaction between xylanases and XIs, mainly how xylanase inhibitors interact with specific xylanases from fungi or bacteria and those in the GH10 or GH11 family. With such understanding, a new generation xylanase, Axxess XY, was developed. Besides showing the essential characteristics of intrinsic thermostability and versatile activity on both soluble and insoluble arabinoxylan, it is resistant to xylanase inhibitors.

Axxess XY takes xylanase application in animal feeds to the next level.

Axxess XY outperforms other xylanases on the market

Recent scientific developments (Fierens, 2007; Flatman et al., 2002; Debyser, 1999; Tundo et al., 2022; Chmelova, 2019) and internal research can be summarized as follows:

High Inhibitory Figure 1: Schematic summary of the susceptibility of different xylanase to xylanase inhibitors from three main groups.

The high resistance to xylanase inhibitors is one of the reasons that a novel xylanase with bacterial origin and from the GH-10 family was chosen to be Axxess XY. EWN innovation, together with research partners, made an interesting benchmark comparison between xylanases that are commercially sold by different global suppliers and Axxess XY. For these trials, all xylanase inhibitors from wheat were extracted. The inhibitors, together with the respective xylanase, were incubated at 40⁰C (to mimic birds’ body temperature) for 30 mins. Then, the loss of xylanase activity was calculated by analyzing remaining activity after incubation. Results are shown below in Figure 2. There were varying levels of activity loss observed in the different commercially sold xylanases. In some xylanases, the losses were alarmingly high. However, Axxess XY was not inhibited at all.

Graph Fig. 2: Extracted total xylanase inhibitors from wheat incubated with the respective xylanase at 40°C for 30 mins. – Loss of activity after incubation with xylanase inhibitors

Conclusion:

Xylanase inhibitors are present in all cereal grains and, unfortunately, heat tolerant (up to 90⁰C, still 75% of inhibition activity was retained). Regardless of the diets used, there is a possibility that the xylanase used may come across xylanase inhibitors, resulting in a loss of activity. More importantly, this can lead to inconsistent performance.

For effective, consistent, and higher performance of NSP enzyme application, it is a must to use xylanase that is resistant to xylanase inhibitors.

Literature:

Chmelová, Daniela, Dominika Škulcová, and Miroslav Ondrejovic. “Microbial Xylanases and Their Inhibition by Specific Proteins in Cereals.” KVASNY PRUMYSL 65, no. 4 (2019). https://doi.org/10.18832/kp2019.65.127. LINK

Choquer, Mathias, Elisabeth Fournier, Caroline Kunz, Caroline Levis, Jean-Marc Pradier, Adeline Simon, and Muriel Viaud. “Botrytis CinereaVirulence Factors: New Insights into a Necrotrophic and Polyphageous Pathogen.” FEMS Microbiology Letters 277, no. 1 (2007): 1–10. https://doi.org/10.1111/j.1574-6968.2007.00930.x. LINK

Debyser, W, WJ Peumans, EJM Van Damme, and JA Delcour. “Triticum Aestivum Xylanase Inhibitor (Taxi), a New Class of Enzyme Inhibitor Affecting Breadmaking Performance.” Journal of Cereal Science 30, no. 1 (1999): 39–43. https://doi.org/10.1006/jcrs.1999.0272. LINK

Mycotoxins in layer and breeder feed impact hens, eggs, hatchery, and chicks

By Marisabel Caballero, Global Technical Manager Poultry

As the planet’s climate experiences changes, new patterns affect the microbial communities colonizing crops. Recently, several areas of the planet have experienced extreme temperatures, drought, changes in the humid/dry cycles, and an increase in atmospheric carbon dioxide (1,2). As a response, the fungi affecting the crops have shifted their geographical distribution, and with this, the pattern of mycotoxin occurrence also changed. For instance, in Europe, we are looking at higher frequencies and levels of Aflatoxins (AF), Ochratoxins (OT), and Fumonisins (FUM) than ten or even five years ago (2-4).

This affects animal production, as mycotoxin challenges show increased frequency, quantity, and variety. Mainly long-living animals, such as laying hens and breeders, can have a higher risk. Moreover, mycotoxins can also be carried over to the eggs, potentially risking human health in the case of layers (table eggs) and in the case of breeder hens, hatchery performance and day-old chick (DOC) quality.

Laying hens and breeders: carryover of mycotoxins into eggs

Most mycotoxins are absorbed in the proximal part of the gastrointestinal tract (Table 1). This absorption can be high, as in the case of aflatoxins (~90%), but also very limited, as in the case of fumonisins (<1%), with a significant portion of unabsorbed toxins remaining within the lumen of the gastrointestinal tract (5).

Once mycotoxins are ingested, detoxification and excretion processes are started by the body, and at the same time, organ damage ensues. The detoxification of mycotoxins is mainly carried out by the liver (6), and their accumulation happens primarily in the liver and kidneys. However, accumulation in other tissues, such as the reproductive organs and muscles, has also been found (7-9). The detoxification process’ objective is the final excretion of the toxins, which occurs through urine, feces, and bile; often, the toxins can also reach the eggs (7-20).

Table 1: mycotoxin absorption rates for poultry and their carry-over rate into eggs

Mycotoxin	Main absorption sites	Absorption rate in poultry	Carry-over rate into eggs
Aflatoxins	Duodenum, jejunum	≈90%	≈0.55%
DON	Duodenum, jejunum	≈20%	≈0.001%
Fumonisins	Duodenum, jejunum	≈1%	≈0.001%
Ochratoxin	Jejunum	≈40%	≈0.15%
T-2	Duodenum, jejunum	≈20%	≈0.10%
Zearalenone	Small & large intestine	≈10%	≈0.30%

(Adapted from 5, 7-17, 19-21)

Table 1 shows carry-over rates of mycotoxins into eggs, resulting from diverse studies (7-10, 14, 16, 19). However, the same studies indicate that results can vary broadly due to different factors, as reviewed by Völkel and collaborators (26). This variability is related to the amount and source of contamination, way of application, period, and the possible co-occurrence of various mycotoxins or several metabolites. Other factors to consider are animal-related, such as species, breed, sex, age group, production level, and health status. Environmental and management factors can play a role in carry-over rates, and finally, detection limits and analytical procedures also influence these results. In summary, highly varying carry-over has been demonstrated, and the risk needs to be considered when animals are exposed.

Mycotoxins in breeder’s feed impact hatchery performance and day-old chick quality

When hens are exposed to mycotoxins, their effects on the intestine, liver, and kidney decrease egg production and quality (10, 14, 27), and, in the case of breeders, consequently, affect hatchery performance, DOC production, and DOC quality (28-30). The main effects of mycotoxins, when we speak about DOC production, are exerted in the gastrointestinal tract, the liver, and the kidneys, affecting embryos and young chicks:

Intestine and kidneys: Mycotoxins harm the intestinal epithelium and have nephrotoxic effects, affecting calcium and vitamin D3 absorption and metabolism, necessary for eggshell quality (31). Thin and fragile shells can increase embryonic mortality, lower embryonic weight gain, and hinder hatchability (32).
Liver: The liver plays a central role in egg production as it is responsible for vitamin D3 metabolism, the production of nutrient transporters, and the synthesis of the lipids that make up the yolk. Thus, when liver function is impaired, the internal and external quality of the egg declines, which affects DOC production (31-34).
Embryo and young chicks: Studies (33-38) have found how mycotoxins affect the embryos. In general, there are two possibilities: the direct one, when the mycotoxin is transferred into the egg, and the indirect one, when the mycotoxin impacts egg quality and, therefore, leads to disease or death of the embryo. The result is a higher embryonic mortality or lower DOC quality. These, among others, result from the lower transfer of antioxidants and antibodies from the hen, low viability of the chick’s immune cells, and higher bacterial contamination. A lower relative weight of the bursa of Fabricio and the thymus is often found.

Qreshi’s team (29) studied the effects on the progeny of broiler breeders consuming feed highly contaminated with AFB1, finding suppression in antibody production and macrophage function in chicks after ten days. Similar results were found by other researchers (36, 37) evaluating the effects of AF and OTA as single and combined contamination. When both mycotoxins are present in the feed, the effect on hatchability and DOC quality are synergistic.

Due to mycotoxin contamination, the reproduction and immune response are impaired, resulting in decreased DOC production and increased early chick mortality, as they are more susceptible to bacterial and viral infections.

Mycotoxins impair table egg production and quality

Studies (22-24) have found mycotoxin contamination in commercial table eggs. A meta-analysis of mycotoxins’ concentration based on 11 published papers was completed recently (22): counting with data from 9509 samples, the meta-analysis reveals an overall presence of mycotoxins in 30% of the samples, being Beauvericin in the first place, followed by DON as well as AF and OTA in third and fourth place, respectively. The risk for humans depends on the intake of contaminated foods in terms of amount and frequency (25), and so far, it has not been estimated in most parts of the world.

Natural contamination in laying hens: a case report

Giancarlo Bozzo’s team (39) reported and published a veterinary case regarding natural mycotoxin contamination in commercial egg production: up to week 47 of age, production parameters were on top of the genetic standards. However, a drop in egg production started at around week 47, and at week 50, egg production was only 68% (figure 1).

Figure 1: production of laying hens fed naturally contaminated feed with AFB1 and OTA

The house with the reduced performance received feed with linseed. In other houses of the same complex, which did not include linseed in the feed, production was unaffected. Therefore, this raw material was considered a possible cause of the issue. Linseed was removed from the formula, and three weeks after (53 weeks of age), egg production was at 84%. Afterward, linseed got back into the formulation, and the laying rate dropped again to 70% (week 56), this time accompanied by a significant increase in mortality.

Samples were collected at week 56, and AFB1 and OTA were detected in feed and the kidneys and livers of the hens consuming it (table 2). While the levels in the feed were not considered high risk, evidence from necropsy and histopathology suggested either a higher or a prolonged exposure; a synergistic effect of both mycotoxins on hen’s health and productivity can be inferred.

Table 2: mycotoxin analysis results for feed and organs

HPLC analysis results in samples of:
toxin	Feed 1 (n=5)	Feed 2 (n=5)	Kidney (n=10)	Liver (n=10)
OTA	1.1 ± 0.1 ppb	31 ± 3 ppb	47 ± 3 ppb	24 ± 2 ppb
AFB1	ND	5.6 ± 0.3 ppb	1.4 ± 0.3 ppb	3.6 ± 0.4 ppb

The liver and kidneys were enlarged and showed signs of damage. Furthermore, urate crystals in the peritoneum and the abdominal air sac were observed, indicating renal failure. This limited the excretion of both toxins in the urine, increasing their half-life in the organism and enhancing the effects in target organs, contributing to the synergistic effect observed.

After using mycotoxin-free certified linseed, the problem receded. Though this is the best option to keep animals healthy and productive, it may not be practical in the long term due to the ubiquitous nature of the toxins and the cost and availability constraints of feed raw materials. Moreover, the mycotoxin levels present in the feed were relatively low and fell under recommended guidelines. For these reasons, in-feed toxin mitigation solutions must also be considered to reduce exposure for production animals.

In-feed intervention mitigates the effects of intermittent exposure to multiple mycotoxins

EW Nutrition conducted a study with Hy-Line W-36 layer-breeders intercalating three 10-day cycles of feed with 100ppb AFB1 + 100ppb OTA, with two 21-day cycles of non-challenged feed. An in-feed intervention (Solis Max 2.0, displayed as IFI) containing bentonite, yeast cell wall components, and a mixture of phytogenic components mitigated all effects.

Table 3: experimental groups and mycotoxin challenge

Treatment	Group	100 ppb AFB1+ 100 ppb OTA	IFI (2 kg/ton)
T-1	Control (C)
T-2	C+IFI		X
T-3	Challenge (Ch)	X
T-4	Ch+IFI	X	X

Trial design:

A total of 576 hens (18 replicates per diet, 8 hens each) and 58 roosters were randomly assigned to four diets at 28 weeks of age, as shown in Table 3. The 72-day experimental period included alternating 10-day challenge and 21-day non-challenge intervals (Figure 2). During the challenge intervals, the breeders in T-3 and T-4 were fed the mycotoxin-contaminated feed with and without the IFI.

Figure 2: trial timeline showing challenge and non-challenge intervals and days of data collection and sampling.

Mitigated effects on egg production and egg quality

The challenge decreased overall egg production (Figure 3), egg mass, and shell thickness (Table 4). The first challenge interval did not affect production, but days later, from the first non-challenge period, all parameters were lower for the challenged group.

Different letters indicate significant differences (p<0.05). Statistical tendencies (p<0.1) are indicated by (*).

Figure 3: Egg production of hens intermittently challenged with AFB1 and OTA, with and without in-feed Solis Max

The adverse effects on productivity and egg quality started after the first challenged feed was withdrawn and persisted through the following intervals until the end of the experiment. Similar effects in chronic mycotoxin challenges have been previously found (37, 39).

Table 4: Average egg quality parameters of hens intermittently challenged with AFB1+OTA, with and without an in-feed intervention (IFI)

Group	Eggshell strength (N)	Eggshell thickness (mm)	Haugh Units
Control	21,02^a	0,3661^ab	70,88
IFI	21,16^a	0,3702^a	71,68
Challenge	20,05^b	0,3630^b	70,07*
Ch+IFI	21,06^a	0,3698^a	71,06

Different letters indicate significant differences (p<0.05). Statistical tendencies (p<0.1) are indicated by (*).

Mitigated effects on the progeny in incubation trials

Three incubation trials were performed: after the first challenge and non-challenge interval and at the end of the trial period after the third challenge interval. A significant decrease in fertility and hatchability was observed for the challenged group in all incubation trials. As mycotoxins affect egg quality (22-24) and can be transferred to the eggs (10, 14, 27), the effects were also shown in the case of hatchability and offspring performance. Fertility was affected from the first challenge interval onwards, continuing to be low for the challenge group until the end of the trial. However, the hatchability of fertile eggs dropped after the withdrawal of the contaminated feed and showed the lowest value during the third challenge interval.

The in-feed supplementation of Solis Max 2.0 (IFI) resulted in the consistent recovery of egg production and egg quality throughout the whole experimental period, achieving the same levels of productivity as the non-challenged control.

Letters indicate significant differences (p<0.05). Statistical tendencies (p<0.1), indicated by (*).

Figure 4: Hatchery parameters of eggs from breeders intermittently challenged with AFB1 and OTA, with and without an in-feed intervention (IFI).

Results in hatch of fertile can be related to egg quality, as the thickness of the eggshell influences the egg’s moisture loss and exchange with the environment during the incubation period. Thinner eggshells lead to higher embryo mortality (31, 32). The group having the challenge with Solis Max showed the same performance as the non-challenged control regarding hatchery performance.

Day-old chick weight was not affected. However, weight gain and mortality after ten days were hindered for the chicks from breeders taking the mycotoxin-contaminated feed (Table 5).

Table 5: Average day- and 10-day-old chick parameters from hens intermittently challenged with AFB1+OTA, with and without an in-feed intervention (IFI)

Parameter	Control	Challenge	Ch + IFI
DOC body weight (g)	36,67	36,24	36,80
10-day body weight (g)	76,30^a	75,94^b	79,50^a
10-day mortality (%)	0,94	1,26	0,97

Letters indicate significant differences (p<0.05). Statistical tendencies (p<0.1) indicated by (*)

At the end of the experiment, oxidative stress biomarkers were measured in the blood serum of 15 hens per treatment, showing significantly lower GPx, and SOD (figure 5) in the challenged group, which indicates a depletion of the mechanisms to fight oxidative stress (40), the hens taking the in-feed product did not show this depletion.

Figure 5: Antioxidants in blood serum, glutathione peroxidase (GPx), and superoxide dismutase (SOD) from breeders intermittently challenged with AFB1 and OTA, with and without an in-feed intervention (IFI).

Intermittent exposure to AFB1 and OTA negatively affected layer breeder productivity, egg quality, and hatchability and promoted oxidative stress in the birds. Intermittent mycotoxin challenges may affect animals even after the contamination is withdrawn. In-feed interventions showed effectiveness in mitigating these effects.

Climate changes bring new mycotoxin challenges – the right in-feed solutions can help

Today’s mycotoxin scenario shows increased frequency, quantity, and variety. Mainly long-living animals, such as laying hens and breeders, can be at more risk. Additionally, the contamination can be carried over to the eggs, potentially risking human health in the case of table eggs and hatchery performance and DOC quality in the case of breeders.

From case reports, we learn the consequences of real challenges and struggles in commercial production; from scientific trials based on possible commercial situations, we realize the advantages of interventions designed to tackle those challenges.

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