By Dr. Inge Heinzl, Editor, EW Nutrition

Nowadays, intensive livestock farming with high stocking densities causes stress in the animals and affects the immune system^{9, 13}. The increase in respiratory diseases with associated losses and costs is only one of the consequences. Due to antimicrobial resistance, antibiotics should only be used in critical cases, so effective alternatives are requested to support the animals.

Respiratory problems are a conjunction of several factors

It already has a name: PRDC or the Porcine Respiratory Disease Complex describes the cooperation of viruses, bacteria, and non-infectious factors such as environmental conditions (e.g., insufficient ventilation), stocking density, management (e.g., all-in-all-out only by pens and not for the whole house) and pig-specific factors such as age and genetics, altogether causing respiratory issues in pigs. Non-infectious factors such as high ammonia levels weaken the immune system and lay the foundation for, e.g., mycoplasmas which damage the ciliated epithelial cells in the upper respiratory tract, the first line of defense, and pave the way for PRRS viruses. They, on their part, enter the respiratory tract embedded in inhaled dust. There, they harm the macrophages and breach a further barrier of defense. Another pathfinder is the Porcine Circovirus 2 (PCV2), which destroys specific immune cells and leads to a generally higher susceptibility to infectious agents. Bacteria such as Pasteurella multocida or Streptococcus suis further on can cause secondary infections^{7, 20, 22}. Also, the combination of mycoplasma hyopneumoniae and porcine circovirus, both typically low pathogenic organisms, leads to severe respiratory disease¹⁵.

Restricted respiratory function impacts growth

The main tasks of the respiratory tract are to take in oxygen from the air and to pump out the CO₂ entailed by the catabolism of the tissue. In pigs, however, the respiratory tract is also responsible for thermoregulation, as pigs don’t have perspiration glands. The animals must get rid of excessive heat by rapid breathing. If the respiratory function is affected due to disease, thermoregulatory capacity is reduced. The resulting lower feed intake leads to decreased growth performance and less economic profit¹⁷. One of the first studies concerning this topic was conducted by Straw et al. (1989)²¹. They asserted that, with every 10 % more affected lung tissue, daily gain decreased by about 37g. This negative correlation between affected lung tissue and weight gain could be confirmed by Paz-Sánchez et al. (2021)¹⁸. They saw that animals with >10% lung parenchyma impacted by cranioventral bronchopneumonia needed a longer time to market (208.8 days vs. 200.8 days in the control), showed a lower carcass weight (74.1 kg vs. 77.7 kg in the control group) and, therefore, also a lower daily gain (500.8 g/day compared to 567.2 g/d). In another study, Pagot and co-workers (2007)¹⁶ observed 7000 pigs from 14 French farms. They saw a significant negative correlation (p<0.001) between the prevalence of pneumonia and growth and a weight gain loss of about 0.7 for each point of pneumonia increase.

Plant extracts support pigs with different modes of action

People have always used herbal substances to cure illnesses, be it willow bark for pain, chamomile for anti-inflammation or an upset stomach. Ribwort and thyme are used as cough suppressants, and eucalyptus and menthol help you breathe better. What is good for humans can also be used for pigs. To use plant extracts efficiently, it is crucial to know their specific modes of action. Due to their volatile nature, essential oils can directly reach the target site, the respiratory tract, via inhalation¹.

1.   Plant extracts can act as an antimicrobial

Many essential oils show some degree of antimicrobial activity. So, the oils of, e. g., oregano, tea tree, lemongrass, lemon myrtle, and clove are effective against a wide range of gram-positive and gram-negative bacteria. LeBel et al. (2019)¹² tested nine different oils against microorganisms causing respiratory issues in pigs. They found the oils of cinnamon, thyme, and winter savory the most effective against Streptococcus suis, Actinobacillus pleuropneumoniae, Actinobacillus suis, Bordetella bronchiseptica, Haemophilus parasuis, and Pasteurella multocida, with MICs and MBCs from 0.01 to 0.156%.

Not only the direct bactericidal effect is important. 1,8 cineol, e.g., although often considered to have only marginal or no antimicrobial activity¹⁰, effectively causes leakage of bacterial membranes² and allows other harmful substances to enter the bacterial cell. However, cineol possesses noted antiviral properties.

2.  Plant extracts can have mucolytic, spasmolytic, and antitussive effects

In the case of respiratory disease, mucolytic and spasmolytic characteristics of phytomolecules are decisive in allowing efficient respiration. Mucolytic substances dissolve the mucus, make it more liquid and facilitate the removal from the respiratory tract by the ciliated epithelium. As liquifying the mucus with essential oils or phytomolecules is related to local irritation, dosage and application form are of the highest importance⁵.

The “cleanup” is called mucociliary clearance. There are also substances that do not dissolve the mucus but stimulate the mucociliary apparatus itself and increase mucociliary transport velocity¹.

Spasmolytic activity on airway smooth muscle is shown, for example, by menthol⁸ or the essential oil of eucalyptus tereticornis⁴. Menthol showed antitussive effects¹¹.

3.   Plant extracts can have immune-modulatory and anti-inflammatory effects

If animals are suffering from a respiratory disease or are in danger of catching one, a supportive influence on the immune system is helpful. One thing is to make vaccination more effective. Mieres-Castro et al. (2021)¹⁴ figured out that the combined application of influenza vaccine and cineol to mice resulted in a longer survival time, less inflammation, less weight loss, a lower mortality rate, less pulmonary edema, and lower viral titers after a challenge with the virus seven days after the vaccination than the mice without cineol.

On the other hand, if the animals are already ill, strengthening their immune defense is essential. Li et al. (2012)¹³ showed that interleukin-6 concentration was lower (p<0.05) and the tumor necrosis factor-α level was higher (p<0.05) in the plasma of pigs fed a diet with 0.18% thymol and cinnamaldehyde than in the negative control group. Also, the lymphocyte proliferation for pigs fed the diet with thymol and cinnamaldehyde increased significantly compared with the negative control (p<0.05).

4.   Plant extracts can act as an antioxidant

There are respiratory diseases in which reactive oxygen species (ROS) play an important role. In these cases, the antioxidant activity of phytomolecules is of interest. Here again, Li et al. (2012)¹³ asserted that a diet with 0.18% thymol and cinnamaldehyde increased the total antioxidant capacity level (p<0.05) in pigs compared to a negative control group.

Can Baser & Buchbauer (2010) described eucalyptus oil containing 1,8-cineole, the monoterpene hydrocarbons α-pinene (10–12%), p-cymene, and α-terpinene, and the monoterpene alcohol linalool, is used to treat diseases of the respiratory tract in which ROS play an important role.

5.   Plant extracts reduce the production of ammonia

High concentration of ammonia in the pig house stresses the pigs’ respiratory tract and makes them susceptible to disease. Ammonia develops when feces and urine merge and the enzyme urease degrades them. Yucca extract, containing a high percentage of saponins, can reduce ammonia emissions in animal houses. Ehrlinger (2007)⁵ supposes that the glyco-components of the saponins bind ammonia and other harmful gases. Another explanation can be the decreased activity of urease shown in a trial with rats¹⁹ or the reduction of total nitrogen, urea nitrogen, and ammonia nitrogen in sow manure³.

6.   Plant extracts often show diverse modes of useful action against respiratory issues

Due to their natural task – protecting the plant – essential oils typically do not show only one beneficial activity for us. Camphene, for example, in Thymus vulgaris, shows expectorant, spasmolytic, and antimicrobial properties and is used in treating respiratory tract infections. Menthol can be effectively used in cases of asthma due to its bronchodilatory activity on smooth muscle, its interaction with cold receptors, and the respiratory drive. Menthol acts antitussive in low concentration, gives the impression of decongestion and reduces respiratory discomfort and sensations of dyspnea.

Cineol, on its part, acts antimicrobial, antitussive, bronchodilatory, mucolytic, and anti-inflammatory. It promotes ciliary transport and improves lung function^{1, 6}. Mucolytic, antioxidant, antiviral, and antibacterial activity is ascribed to thymol⁵.

Trial shows: phytomolecules help to keep respiratory diseases in check

A field study was conducted on a Philippine piglet farm with a history of chronic respiratory issues during the growing phase, with a morbidity of about 10-15%. In this study, a supplement for water containing phytomolecules that support animals against respiratory diseases (Grippozon) was tested. For the trial, 360 randomly selected 28-day-old pigs (average weight: 6.64±0.44 kg) were divided into two groups with 6 replications per group and 30 piglets per replication. All piglets came from sows raised antibiotic-free, and the piglets received antibiotics neither upon weaning except in case of symptoms (scouring: Baytril-1 mL/pig;  respiratory disease: Excede – 1mL/pig). All piglets received the same feed and a regular water therapy regimen:

Control group: no additional supplements
Grippozon group:  Addition of 250 mL of Grippozon per 1000 L of water

As parameters, the incidence of respiratory disease, final weight, daily gain, FCR, and antibiotic cost, were recorded.

The phytomolecules-containing product reduced the incidence of respiratory diseases by 52 %, leading to a 53% lower cost for antibiotic treatment. The animals showed better growth performance (600 g higher average weight and 13 g higher average daily gain), altogether resulting in an extra cost-benefit of 1.76 US$ per pig.

Reduction in disease and medication ensures healthier pigs in the Grippozon-supplemented group, reflected by better performance.

We have means at hand to reduce the use of antibiotics

Respiratory disease is a big problem in pigs. Due to the still high occurrence of antimicrobial resistance, it is essential to reduce antibiotic use as much as possible. Phytomolecules offer the possibility to strengthen the animals’ health so that they are less susceptible to disease or support them when they are already infected. With the help of phytomolecules, we can reduce antibiotic treatments and help keep antibiotics effective when their use is indispensable.

References

1. Can Baser , K. Hüsnü, and Gerhard Buchbauer. Handbook of Essential Oils: Science, Technology, and Applications. Boca Raton, FL: Taylor & Francis distributor, 2010.
2. Carson, Christine F., Brian J. Mee, and Thomas V. Riley. “Mechanism of Action of Melaleuca Alternifolia (Tea Tree) Oil on Staphylococcus Aureus Determined by Time-Kill, Lysis, Leakage, and Salt Tolerance Assays and Electron Microscopy.” Antimicrobial Agents and Chemotherapy 46, no. 6 (2002): 1914–20. https://doi.org/10.1128/aac.46.6.1914-1920.2002.
3. Chen, Fang, Yantao Lv, Pengwei Zhu, Chang Cui, Caichi Wu, Jun Chen, Shihai Zhang, and Wutai Guan. “Dietary Yucca Schidigera Extract Supplementation during Late Gestating and Lactating Sows Improves Animal Performance, Nutrient Digestibility, and Manure Ammonia Emission.” Frontiers in Veterinary Science 8 (2021). https://doi.org/10.3389/fvets.2021.676324.
4. Coelho-de-Souza, Lívia Noronha, José Henrique Leal-Cardoso, Francisco José de Abreu Matos, Saad Lahlou, and Pedro Jorge Magalhães. “Relaxant Effects of the Essential Oil of Eucalyptus Tereticornisand Its Main Constituent 1,8-Cineole on Guinea-Pig Tracheal Smooth Muscle.” Planta Medica 71, no. 12 (2005): 1173–75. https://doi.org/10.1055/s-2005-873173.
5. Ehrlinger, Miriam. “Phytogene Zusatzstoffe in der Tierernährung.” Dissertation, Tierärztliche Fakultät LMU, 2007.
6. Gelbe Liste Online. “Gelbe Liste Pharmindex Online.” Gelbe Liste. Accessed January 20, 2023. https://www.gelbe-liste.de/.
7. Hennig-Pauka, Isabell. “Atemwegserkrankungen: Schutz fängt schon bei Ferkeln an.” Der Hoftierarzt, January 13, 2021. https://derhoftierarzt.de/2021/01/atemwegserkrankungen-schutz-faengt-schon-bei-ferkeln-an/.
8. Ito, Satoru, Hiroaki Kume, Akira Shiraki, Masashi Kondo, Yasushi Makino, Kaichiro Kamiya, and Yoshinori Hasegawa. “Inhibition by the Cold Receptor Agonists Menthol and ICILIN of Airway Smooth Muscle Contraction.” Pulmonary Pharmacology & Therapeutics 21, no. 5 (2008): 812–17. https://doi.org/10.1016/j.pupt.2008.07.001.
9. Kim, K.H., E.S. Cho, K.S. Kim, J.E. Kim, K.H. Seol, S.J. Sa, Y.M. Kim, and Y.H. Kim. “Effects of Stocking Density on Growth Performance, Carcass Grade and Immunity of Pigs Housed in Sawdust Fermentative Pigsties.” South African Journal of Animal Science 46, no. 3 (2016): 294–301. https://doi.org/10.4314/sajas.v46i3.9.
10. Kotan, Recep, Saban Kordali, and Ahmet Cakir. “Screening of Antibacterial Activities of Twenty-One Oxygenated Monoterpenes.” Zeitschrift für Naturforschung C 62, no. 7-8 (2007): 507–13. https://doi.org/10.1515/znc-2007-7-808.
11. Laude, E.A., A.H. Morice, and T.J. Grattan. “The Antitussive Effects of Menthol, Camphor, and Cineole in Conscious Guinea-Pigs.” Pulmonary Pharmacology 7, no. 3 (1994): 179–84. https://doi.org/10.1006/pulp.1994.1021.
12. LeBel, Geneviève, Katy Vaillancourt, Philippe Bercier, and Daniel Grenier. “Antibacterial Activity against Porcine Respiratory Bacterial Pathogens and in Vitro Biocompatibility of Essential Oils.” Archives of Microbiology 201, no. 6 (2019): 833–40. https://doi.org/10.1007/s00203-019-01655-7.
13. Li, Xue, Xia Xiong, Xin Wu, Gang Liu, Kai Zhou, and Yulong Yin. “Effects of Stocking Density on Growth Performance, Blood Parameters and Immunity of Growing Pigs.” Animal Nutrition 6, no. 4 (2020): 529–34. https://doi.org/10.1016/j.aninu.2020.04.001.
14. Mieres-Castro, Daniel, Sunny Ahmar, Rubab Shabbir, and Freddy Mora-Poblete. “Antiviral Activities of Eucalyptus Essential Oils: Their Effectiveness as Therapeutic Targets against Human Viruses.” Pharmaceuticals 14, no. 12 (2021): 1210. https://doi.org/10.3390/ph14121210.
15. Opriessnig, T., L. G. Giménez-Lirola, and P. G. Halbur. “Polymicrobial Respiratory Disease in Pigs.” Animal Health Research Reviews 12, no. 2 (2011): 133–48. https://doi.org/10.1017/s1466252311000120.
16. Pagot, E., P. Keita, and A. Pommier. “Relationship between Growth during the Fattening Period and Lung Lesions at Slaughter in Swine.” Revue Méd. Vét., , , 5, 253-259 158, no. 5 (2007): 253–59.
17. Pallarés Martínez, Francisco José, Jaime Gómez Laguna, Inés Ruedas Torres, José María Sánchez Carvajal, Fernanda Isabel Larenas Muñoz, Irene Magdalena Rodríguez-Gómez, and Librado Carrasco Otero. “The Economic Impact of Pneumonia Processes in Pigs.” https://www.pig333.com. Pig333.com Professional Pig Community, December 14, 2020. https://www.pig333.com/articles/the-economic-impact-of-pneumonia-processes-in-pigs_16470/.
18. Paz-Sánchez, Yania, Pedro Herráez, Óscar Quesada-Canales, Carlos G. Poveda, Josué Díaz-Delgado, María del Quintana-Montesdeoca, Elena Plamenova Stefanova, and Marisa Andrada. “Assessment of Lung Disease in Finishing Pigs at Slaughter: Pulmonary Lesions and Implications on Productivity Parameters.” Animals 11, no. 12 (2021): 3604. https://doi.org/10.3390/ani11123604.
19. Preston, R. L., S. J. Bartle, T. May, and S. R. Goodall. “Influence of Sarsaponin on Growth, Feed and Nitrogen Utilization in Growing Male Rats Fed Diets with Added Urea or Protein.” Journal of Animal Science 65, no. 2 (1987): 481–87. https://doi.org/10.2527/jas1987.652481x.
20. Ruggeri, Jessica, Cristian Salogni, Stefano Giovannini, Nicoletta Vitale, Maria Beatrice Boniotti, Attilio Corradi, Paolo Pozzi, Paolo Pasquali, and Giovanni Loris Alborali. “Association between Infectious Agents and Lesions in Post-Weaned Piglets and Fattening Heavy Pigs with Porcine Respiratory Disease Complex (PRDC).” Frontiers in Veterinary Science 7 (2020). https://doi.org/10.3389/fvets.2020.00636.
21. Straw , B. E., V. K. Tuovinen, and M. Bigras-Poulin. “Estimation of the Cost of Pneumonia in Swine Herds.” J Am Vet Med Assoc. 1989 Dec 15;195(12):1702-6. 195, no. 12 (December 15, 1989): 1702–6.
22. White, Mark. “Porcine Respiratory Disease Complex (PRDC).” Livestock 16, no. 2 (2011): 40–42. https://doi.org/10.1111/j.2044-3870.2010.00025.x.

By Dr. Ajay Bhoyar, Global Technical Manager – Poultry, EW Nutrition

Rancidity testing is essential in the feed industry, as a key indicator of product quality and shelf life. It is conducted to determine the level of oxidation in samples of feed or feed ingredients and it can be performed through a number of analytical methods.

Rancidity is the process by which fats and oils in food become degraded, resulting into off-odor/flavor, taste, and texture. This process is caused by the oxidation of unsaturated fatty acids and can be accelerated by factors such as exposure to light, heat, and air. Rancidity can occur naturally over time, but it can also be accelerated by improper storage or processing of animal products. Fats are highly susceptible to degradation due to their chemical nature.

How does oxidative rancidity occur?

Oxidation occurs when an oxygen ion replaces a hydrogen ion within a fatty acid molecule and higher numbers of double bonds within the fatty acid increase the possibility of autoxidation. Oxidative rancidity results from the breakdown of unsaturated fatty acids in the presence of oxygen. Light and heat promote this reaction, which results in the generation of aldehydes and ketones – compounds which impart off-odors and flavors to food products. Pork and chicken fat demonstrate a higher degree of unsaturated fatty acids compared with beef fat and are therefore more prone for rancidity.

Oxidation: a three-step process

Fat/oil oxidation is a three-step process (Initiation, Propagation and Termination). Therefore, the oxidation products depend on the time. In the first phase, called Initiation, the formation of free radicals begins and accelerates.

Once the initial radicals have formed, the formation of other radicals proceeds rapidly in this second phase called Propagation. In this part of the process, a chain reaction of high energy molecules, which are variations of free radicals and oxygen, are formed and can react with other fatty acids. These reactions can proceed exponentially, if not controlled. Also in this phase, the rate of peroxide radical formation will reach equilibrium with the rate of decomposition to form a bell-shaped curve.

In the final phase, called Termination, the starting material has been consumed, and the peroxide radicals, as well as other radicals decompose into secondary oxidation by-products such as esters, short chain fatty acids, polymers, alcohols, ketones and aldehydes. It is these secondary oxidation by-products, which can negatively affect the growth and performance of animals.

Fig. 1: Oxidation: a three-phase series of reactions

Antioxidants preserve the quality of rendered products

Chemical antioxidants are used in the rendering industry to help preserve the quality of animal by-products. Synthetic antioxidants, such as BHA, BHT, and ethoxyquin, can help prevent the oxidation of these by-products, which can cause them to become rancid. These chemical antioxidants are added in small amounts to the raw materials prior to rendering or can be incorporated into the finished products to help extend their shelf life and maintain their nutritional value. It is important to note that the use of antioxidants in the rendering industry must be done in compliance with regulations and guidelines set forth by the FDA and other governing bodies.

Natural antioxidants like tocopherols, rosemary extract, ascorbyl palmitate, etc. are also used to prevent oxidation and maintain the freshness of rendered products, if the chemical antioxidants cannot be used.

Rancidity testing

Rancidity testing is the process of determining the level of rancidity in a product. Testing for level of rancidity is used widely as an indication of product quality and stability.

There are several methods used for rancidity testing, including:

Organoleptic rancidity testing

Oxidation of fats and oils leads to a change in taste, smell, and appearance. Organoleptic testing involves using the senses (sight, smell, taste) to determine the level of rancidity. Trained testers will examine the product for visual signs of spoilage, such as discoloration or the presence of crystals, and will also smell and taste the product to detect any off-flavors or odors.

Chemical & instrumental rancidity testing

Chemical testing involves using chemical methods to measure the level of rancidity. One common method is the peroxide value test, which measures the amount of peroxides (indicators of rancidity) in the product. Another method is the p-anisidine test, which measures the level of aldehydes (another indicator of rancidity) in the product.

Peroxide value

Peroxide Value (PV) testing determines the amount of peroxides in the lipid portion of a sample through an iodine titration reaction targeting peroxide formations. Peroxides are the initial indicators of lipid oxidation and react further to produce secondary products such as aldehydes. Because peroxide formation increases rapidly during the early stages of rancidification but subsequently diminishes over time, it is best to pair PV testing with p-Anisidine Value to obtain a fuller picture of product quality.

Fig.2: Oxidation products changes with time

p-Anisidine (p-AV)

p-AV is a determination of the amount of reactive aldehydes and ketones in the lipid portion of a sample. Both compounds can produce strong objectionable flavors and odors at relatively low levels. The compound used for this analysis (p-Anisidine) reacts readily with aldehydes and ketones and the reaction product can be measured using a colorimeter. Samples that are particularly dark may not be the most applicable for this analysis as the colorimeter may not be able to adequately measure the wavelength required.

TBARS

Thiobarbituric acid reactive substances (TBARS) are a byproduct of lipid peroxidation (i.e. as degradation products of fats). This can be detected by the TBARS assay using thiobarbituric acid as a reagent. TBA Rancidity (TBAR) also measures aldehydes (primarily malondialdehyde) created during the oxidation of lipids. This analysis is primarily useful for low-fat samples, as the whole sample can be analyzed rather than just the extracted lipids.

The Instrumental testing involves using instruments to measure the level of rancidity.

Gas chromatography

One common method is the use of a gas chromatograph, which can detect the presence of volatile compounds that indicate rancidity.

Fourier-transform infrared spectrophotometer (FTIR)

FTIR method can detect changes in the chemical makeup of the product that indicate rancidity.

Free Fatty Acids (FFA)

FFA testing determines the fatty acids that have been liberated from their triglyceride structure. A titration is performed on the extracted fat from a specific sample. The FFA content is then determined through a calculation of the amount of titrant used to reach the final result. Knowing what type of fat or fat containing product is being tested is important for this analysis to ensure that the appropriate calculation is applied. As the test does not differentiate between fatty acid types, samples with high palmitic or lauric fatty acid composition should have a different calculation factor applied so as to accurately represent the free fatty acid result.

Oxidative Stability Index (OSI)

OSI indicates how resistant a sample is to oxidation. Samples are subjected to heat while air is injected – a process which accelerates oxidation reactions. The samples are monitored, and the time required for the sample to reach an inflection point is determined. This test is useful when testing the efficacy of an antioxidant added to a product. Antioxidants should inhibit free radical propagation and thus increase a samples ability to hold up under the stressing conditions imposed by the OSI analysis. The measuring instrument, the Rancimat.

Analytical testing considerations in rendering operations

It is common to perform regular analytical testing in a rendering operation as a part of quality control and quality assurance program. There are several methods for testing rancidity in rendering operations. It is important to choose the appropriate method based on the type of product and the desired level of accuracy.

The results of rancidity testing are used to monitor and control the rendering process to prevent or minimize rancidity. This may involve adjusting processing conditions, using antioxidants, or implementing other measures to reduce oxidation.

Table. 1: Analytical testing considerations for rendering

Conclusion

Rancidity is a common problem in rendered animal products. It can have detrimental effects on both the quality and safety of the product. It is caused by the oxidation of fats and oils, leading to the formation of harmful compounds such as free radicals and hydroperoxides. The best way to prevent rancidity is through proper storage, packaging, and handling techniques, as well as the use of antioxidants to slow down the oxidation process. It is important for manufacturers and consumers to be aware of the potential for rancidity in rendered animal products and take the necessary precautions to ensure the safety and quality of the product. 

By Kouji Umeda, Production Director, EW Nutrition Japan

Calves are susceptible to infection by pathogens due to their immature congenital immunity. Bovine rotavirus and bovine coronavirus, pathogenic E. coli, Clostridium, Cryptosporidium, and Eimeria spp are the major pathogens of infectious diarrhea in calves less than one month of age. Bovine rotavirus, the most frequently detected in dairy and beef cattle, is responsible for approximately 40% of diarrhea cases. In addition, 60-70% of cases of diarrhea involving bovine rotavirus occur within the first two weeks of life. Symptoms include fever, anorexia, loss of energy, and acute yellow-white watery diarrhea after 12 to 36 hours post infection, which leads to dehydration and metabolic acidosis. In more severe cases, the disease can lead to death and is considered one of the most severe diarrhea-causing pathogens in newborn calves worldwide.

Rotavirus A is a major causative pathogen of diarrhea in calf

Rotaviruses belong to the family of Reoviridae and are classified into species A to J. The rotaviruses in bovines mainly belong to species A, B, and C, which are the leading infectious agents in cattle. Calf diarrhea is primarily caused by rotavirus A (RVA). This virus is transmitted orally through feces, bedding, utensils, or people contaminated with feces. Significant diarrhea caused by the virus is attributed to

• malabsorption due to the destruction of small intestinal epithelial cells and
• inhibition of water reabsorption by enterotoxin (NSP4) produced by rotaviruses.

Adult cattle and other host animals have an immune system that protects them from infection and the development of various pathogens. As RVA exists in different genotypes, the antibodies must be specifically against this genotype; otherwise, the virus-neutralizing activity, as well as protection against infection and pathogenesis, is significantly reduced.

The classic method to prevent RVA infection

Besides adequate sanitation in the production facilities, farmers try to “improve” the composition of the maternal colostrum by vaccinating the cow. For this purpose, the cows are inoculated with inactivated, previously isolated bovine RVA. However, the immunization of calves through colostrum may not be effective enough. It also may be difficult to prevent the spread of bovine RVA by barn hygiene alone due to the recent increase in the number of cattle being raised and moved from one farm to another.

Calf diarrhea feces contain G and P genotypes of bovine RVA

In general, the three most common G genotypes of bovine RVA detected in calf diarrhea are G6, G8, and G10, and the three most common P genotypes are P[1], P[5], and P[11]. Based on the results of the genotyping survey in Japan from 1987 to 2000 (Fig. 1) and the one from 2017 to 2020 (figure 2) (Animal Health Research Division of the National Institute of Agrobiological Sciences (NIAH) together with IRIG), the bovine RVA genotypes identified as prevalent and endemic in Japan in recent years were G6P[5], G6P[11], and G10P[11]. However, the percentage of genotypes detected differed among cattle breeds (Fig. 3A, Fig. 3B, Fig. 3C).

Fig.1: Genotyping results from 1987-2000

Fig.2: Genotyping results from 2017-2020

Fig. 3A:Percentage of detection in Holstein

Fig. 3B: Detection rate in crossbreeds

Fig. 3C: Detection rate in beef cattle (Wagyu)

Cow colostrum protects the calf, egg yolk the chick AND the calf

A cow provides the calf with colostrum to ensure immunoglobulin delivery (passive immunity). In poultry, hens transfer immunoglobulins to the egg yolks and pass immunoglobulins to their chicks in this way. This biological mechanism of “immune transfer to the egg yolk” in birds can be used to arbitrarily produce yolk immunoglobulin (IgY) against pathogens of enteric infections in livestock (Ikemori et al., 1992; Ikemori et al., 1997; Yokoyama et al., 1998).

For this purpose, hens must get in contact with the respective pathogens. They produce antibodies against these pathogens – which also works with non-poultry-relevant pathogens such as bovine RVA – and transfer them to the egg (⇒IgY). The eggs with accumulated high levels of useful IgY can be collected almost daily. The immunoglobulins can be fed to livestock animals such as calves to protect them in critical times.

Continuous feeding of milk formulas containing IgY allows the IgY to remain in the intestinal lumen for a long time (Nozaki et al., 2019). There, they bind to the target pathogens and prevent infection by inhibiting their attachment to and cell invasion into intestinal epithelial cells.

IgY and genotype of the virus must match

A study verified that anti-bovine RVA IgY consisting of anti-G6P[1], anti-G6P[5], and anti-G10P[11] shows broad-spectrum virus-neutralizing activity against recent field isolates. Separate trials (see table 1) demonstrated that anti-G6 genotype IgY acted best against the RVA genotypes G6P[1] and G6P[5] and showed less activity against the G10 genotype. Anti-G10P[11] IgY worked optimally against the P[11] genotypes. The trials confirmed that either the G or the P genotype must match to achieve a sufficient virus-neutralizing activity. The IgY mixture is not helpful against bovine RVA strains that match neither the G nor the P genotypes (Odagiri et., 2020).

As the genotyping survey of 2017-2020 showed mainly G6 and G10 genotypes, a mixture of anti- bovine RVA G6P[1] IgY, G6P[5], and G10P[11] has strong virus neutralizing activity against bovine RVA that is currently prevalent and spreading in production sites.

Table 1: Virus-neutralizing activity of field-isolated bovine RVA against various genotypic strains

+++：Strong virus neutralizing activity; ++：Moderate virus neutralizing activity; +：Weak virus neutralizing activity; －：No virus neutralizing activity

Anti-bovine RVA IgY supports calves against rotavirus infection

To verify the protective effect of oral passive immunization with anti-bovine RVA IgY against bovine RVA infection, a trial with newborn calves was conducted.

Trial design: Eight calves were separated from their mothers immediately after birth without feeding colostrum and moved to a house with infected animals. From the first day, the calves were fed artificial milk supplemented with anti-bovine RVA IgY (n=4) or non-immune IgY (Control IgY; n=4) three times a day.

The parameters observed were fecal score, bovine RVA excretion, and weight gain; data were collected daily. The fecal score was calculated as the cumulative fecal score during the study period: 0 for normal stools, 1 for soft to muddy stools, and 2 for watery stools. Bovine RVA was isolated from daily fecal samples and evaluated by the total number of days of bovine RVA excretion.

Results: The anti-bovine RVA IgY group was found to be effective in reducing the incidence of diarrhea and shortening the duration of virus excretion in the infection test with the bovine RVA G6 genotype strain and the bovine RVA G10 genotype strain (tables 2 and 3).

Table 2: Efficacy of anti-bovine RVA IgY feeding in bovine RVA G6 genotype strain infection

**： P＜0.01; *： P＜0.05

Table 3: Efficacy of anti-bovine RVA IgY feeding in bovine RVA G10 genotype strain infection

**： P＜0.001

IgY is a valuable tool in rotavirus control

Newborn calves, susceptible to severe diarrhea caused by bovine RVA infection, require passive immunization with antibodies transferred from the colostrum of the mother cow. However, sometimes, calves don’t get enough antibodies which can be the case if

• the calf does not receive enough colostrum or receives it too late
• the cow still has not the farm-specific antibodies because of a too short time of being on the farm

To compensate for this lack of immunity, calves have been fed milk formulas containing anti-bovine RVA IgY for some time. Continuous feeding of anti-bovine RVA IgY, which shows strong virus neutralizing activity against each genotype of bovine RVA isolated from recent cases of calf diarrhea, is expected to provide sufficient immunity and be an effective means of bovine RVA control.

In the case of disease outbreaks, it makes sense to utilize IgY with appropriate mechanisms of action in addition to improving the level of quarantine measures, including hygiene control and vaccination.

References:

Ikemori, Yutaka, Masahiko Kuroki, Robert C. Peralta, Hideaki Yokoyama, and Yoshikatsu Kodama. “Protection of Neonatal Calves against Fatal Enteric Colibacillosis by Administration of Egg Yolk Powder from Hens Immunized with K99-Piliated Enterotoxigenic Escherichia Coli.” Amer. J. Vet. Res. 53, no. 11 (1992): 2005–8. PMID: 1466492.

Ikemori, Yutaka, Masashi Ohta, Kouji Umeda, Faustino C. Icatlo, Masahiko Kuroki, Hideaki Yokoyama, and Yoshikatsu Kodama. “Passive Protection of Neonatal Calves against Bovine Coronavirus-Induced Diarrhea by Administration of Egg Yolk or Colostrum Antibody Powder.” Veterinary Microbiology 58, no. 2-4 (1997): 105–11. https://doi.org/10.1016/s0378-1135(97)00144-2.

Nozaki, I., M. Itoh, F. Murakoshi, T. Aoki, K. Shibano, and K. Yamada. “Effect of an Egg Yolk Immunoglobulin（Igy）Product on Oocyst Shedding and Blood and Fecal Igy Concentrations in Cryptosporidium-Infected Calves.” Japanese Journal of Large Animal Clinics 10, no. 2 (2019): 68–72. https://doi.org/10.4190/jjlac.10.68.

Odagiri, Koki, Nobuki Yoshizawa, Hisae Sakihara, Koji Umeda, Shofiqur Rahman, Sa Van Nguyen, and Tohru Suzuki. “Development of Genotype-Specific Anti-Bovine Rotavirus a Immunoglobulin Yolk Based on a Current Molecular Epidemiological Analysis of Bovine Rotaviruses a Collected in Japan during 2017–2020.” Viruses 12, no. 12 (2020): 1386. https://doi.org/10.3390/v12121386.

Yokoyama, Hideaki, Robert C. Peralta, Kouji Umeda, Tomomi Hashi, Faustino C. Icatlo, Masahiko Kuroki, Yutaka Ikemori, and Yoshikatsu Kodama. “Prevention of Fatal Salmonelosis in Neonatal Calves, Using Orally Administered Chicken Egg Yolk Salmonella-Specific Antibodies.” Amer. J. Vet. Res. 59, no. 4 (1998): 416–20. PMID: 9563623.

By Dr. Inge Heinzl, Editor, and Dr. Ruturaj Patil, Global Product Manager – Phytogenics, EW Nutrition 

For millennia, plants have been used for medicinal purposes in human and veterinary medicine and as spices in the kitchen. Since the ban of antibiotic growth promoters in 2006 by the European Union, they also came into focus in animal nutrition. Due to their digestive, antimicrobial, and gut health-promoting characteristics, they seemed an ideal alternative to compensate for the reduced use of antibiotics in critical periods such as brooding, feed change or gut-related stress.

To optimize the benefits of phytomolecules, it is crucial that

• the phytomolecules levels are standardized for consistent results and synergy
• they show the highest stability during stringent feed processing; being often highly volatile substances, they should not get lost at high temperatures and pressure
• the phytomolecules are preferably completely released and available in the animal to achieve the best effectiveness.

First step: Standardized phytomolecules

Essential oils and other phytogenics are sourced from plants. The composition of the plants substantially depends on genetic dissimilarity within accessions, plant origin, the site conditions, such as weather, soil, community, and harvest time, but also sample drying, storage, and extraction processes (Sadeh et al., 2019; Yang et al., 2018; Ehrlinger, 2007). For example, the oil extracted from thyme can contain between 22 and 71 % of the relevant phenol thymol (Soković et al., 2009; Shabnum and Wagay, 2011; Kowalczyk et al., 2020).

Modern technology enables the production of standardized phytomolecules with the highest degree of purity and lowest possible batch-to-batch variation for high-quality products. It also offers increased environmental and economic sustainability due to reliable and cost-effective sourcing technology.

Using such highly standardized phytomolecules enables the production of phytogenic-based feed supplements of consistently high quality.

Second step: Selection of the most suitable phytomolecules

Phytomolecules have different primary characteristics. Some support digestion (Cho et al., 2006, Oetting, 2006; Hernandez, 2004); others act against pathogens (Sienkiewitz et al., 2013; Smith-Palmer et al., 1998; Özer et al., 2007) or are antioxidants (Wei and Shibamoto, 2007; Cuppett and Hall, 1998). To optimize gut health in animal production, one of the main promising mechanisms is reducing pathogens while promoting beneficial microbes. The decrease of pathogens in the gut not only decreases the risk of enteritis incidence but also eliminates the inconvenient competitors for feed.

In order to find out the best combination serving the intended purpose, a high number of different phytomolecules need to be evaluated concerning their structure, chemical properties, and biological activities first. Availability and costs of the substances are further factors to consider. With the selection of the most suitable phytomolecules, different mixtures are produced and tested for their effectiveness. Here, it is essential to concern synergistic or antagonistic effects.

For an effective and efficient blend of phytomolecules, many steps of selection and tests are necessary – and as a result, possibly only a few mixtures can meet the requirements.

Third step: Protecting the ingredients

Many phytomolecules are inherently highly volatile. So, only having a standardized content of phytogenics in the product can not ensure the full availability of phytomolecules when used through animal feed. Some parts of the ingredients might already get lost in the feed mill due to the stringent feed hygienization process followed by feed millers to reduce pathogenic load. The heating is a significant challenge for the highly-volatile components in a phytomolecule-based product. So, protecting these phytomolecules becomes imperative to guarantee that the phytomolecules put into the feed will reach the animal.

A delicate balancing act is required to ensure the availability and activity of phytomolecules at the right site in the gut. The phytomolecules must not get lost during feed processing but must also be released in the intestine. A carrier with capillary binding of the phytomolecules together with a protective coating can be one of the available effective solutions. It protects the ingredients during feed processing, and ensures the release in the animal.

Study shows excellent stability of Ventar D under challenging conditions

Ventar D is a latest generation phytomolecule-based solution for gut health optimization introduced by ​EW Nutrition, GmbH. A scientific study was conducted to compare the stability of Ventar D, in the pelleting process, with two leading phytogenics competitor feed supplements.

For this trial, feed with the different added phytogenic feed supplements had to undergo a conditioning and pelletization process. The active ingredients were analyzed before and after the pelletization process. All phytogenic feed supplements under testing were added to standard broiler feed at the producer’s recommended inclusion rate. The tests took place under conditioning times of 45, 90, and 180 seconds and pelleting temperatures of 70, 80, and 90°C (158, 176, and 194°F). After cooling, triplicate samples were collected and analyzed. The respective marker substance was analyzed through gas chromatography/mass spectrometry (GC/MS) analysis to measure the recovery rate in the finished feed. The phytomolecule content of the mash feed (before pelletization) found by the laboratory was used as a baseline and set to 100% recovery. The recovery rates of the pelleted feed were evaluated relative to this baseline.

The results are presented in figure 1. Ventar D showed the highest stability of active ingredients with recovery rates of 90% at 70°C/45 sec. or 80°C/90 sec and 84% at 90°C/180 sec. The modern production technology used for Ventar D ensures that the active ingredients are well protected throughout the pelletization process.

Figure 1: Phytomolecule stability under processing conditions, relative to mash baseline (100%)

Another trial was conducted in a feed mill in the US. For this trial, ten samples were collected from different batches of mash feed where Ventar D was added at 110g/t. Conditioning of the mash feed was at 87.8°C (190°F) for 6 minutes and 45 seconds. After the pelleting process, ten samples from the pelleted feed were collected from the continuous flow with a 5 min gap between the samplings to determine Ventar D’s recovery.

The average recovery achieved for Ventar D was 92%.

Trials show improved growth performance

Initial trials showed Ventar D’s complete release in digestion models. To examine the benefit in in-vivo conditions, Ventar D was tested in broilers at an inclusion rate of 100 g/MT.

Several in vitro studies proved the antimicrobial activity of Ventar D. One test also confirms that Ventar D could exhibit differential antimicrobial activity by having stronger activity against common enteropathogenic bacteria while sparing the beneficial ones (Heinzl, 2022). Moreover, Ventar D’s antioxidant and anti-inflammatory activity support better gut barrier functioning. Better gut health leads to higher growth performance and improved feed conversion, which could be demonstrated in several trials with broilers (figures 2 and 3). In the tests, a group fed Ventar D was compared to either a control group with no such feed supplement or groups supplied with competitor products at the recommended inclusion rates.

Compared to a negative control group, the Ventar D group consistently showed a higher average daily gain of 0.3-4.1 g (0.5-8.5 %)  and a 3-4 points better feed conversion. Compared to competitor products, Ventar D provided 1-1.7 g (2-3 %) higher average daily gain and a 3 points better /1 point higher FCR than competitors 2 and 1.

Figure 2: Average daily gain (g) – results of several trials conducted with broilers

Figure 3: FCR – results of several trials conducted with broilers

Standardization and new technologies for higher profitability

Several in vitro and in vivo studies proved that Ventar D takes “phytomolecules’ power” to the next level: Combining standardized phytomolecules and optimal active ingredient protection leads to superior product stability during feed processing. The higher amount of active ingredients arriving in the gut improves gut health and increases the production performance of the animals. Ventar D shows how we can use phytomolecules more effectively and benefit from higher farm profitability.

References:

Cho, J. H., Y. J. Chen, B. J. Min, H. J. Kim, O. S. Kwon, K. S. Shon, I. H. Kim, S. J. Kim, and A. Asamer. “Effects of Essential Oils Supplementation on Growth Performance, IGG Concentration and Fecal Noxious Gas Concentration of Weaned Pigs”. Asian-Australasian Journal of Animal Sciences 19, no. 1 (2005): 80–85. https://doi.org/10.5713/ajas.2006.80.

Cuppett, Susan L., and Clifford A. Hall. “Antioxidant Activity of the Labiatae”. Advances in Food and Nutrition Research 42 (1998): 245–71. https://doi.org/10.1016/s1043-4526(08)60097-2.

Ehrlinger, M. “Phytogenic Additives in Animal Nutrition.” Dissertation, Veterinary Faculty of the Ludwig Maximilians University, 2007.

Heinzl, I. “Efficient Microbiome Modulation with Phytomolecules”. EW Nutrition, August 30, 2022. https://ew-nutrition.com/pushing-microbiome-in-right-direction-phytomolecules/.

Hernández, F., J. Madrid, V. García, J. Orengo, and M.D. Megías. “Influence of Two Plant Extracts on Broilers Performance, Digestibility, and Digestive Organ Size.” Poultry Science 83, no. 2 (2004): 169–74. https://doi.org/10.1093/ps/83.2.169.

Kowalczyk, Adam, Martyna Przychodna, Sylwia Sopata, Agnieszka Bodalska, and Izabela Fecka. “Thymol and Thyme Essential Oil—New Insights into Selected Therapeutic Applications.” Molecules 25, no. 18 (2020): 4125. https://doi.org/10.3390/molecules25184125.

Lindner, , U. “Aromatic Plants – Cultivation and Use.” Düsseldorf: Teaching and Research Institute for Horticulture Auweiler-Friesdorf, 1987.

Oetting, Liliana Lotufo, Carlos Eduardo Utiyama, Pedro Agostinho Giani, Urbano dos Ruiz, and Valdomiro Shigueru Miyada. “Efeitos De Extratos Vegetais e Antimicrobianos Sobre a Digestibilidade Aparente, O Desempenho, a Morfometria Dos Órgãos e a Histologia Intestinal De Leitões Recém-Desmamados.” Revista Brasileira de Zootecnia 35, no. 4 (2006): 1389–97. https://doi.org/10.1590/s1516-35982006000500019.

Sadeh, Dganit, Nadav Nitzan, David Chaimovitsh, Alona Shachter, Murad Ghanim, and Nativ Dudai. “Interactive Effects of Genotype, Seasonality and Extraction Method on Chemical Compositions and Yield of Essential Oil from Rosemary (Rosmarinus Officinalis L”.).” Industrial Crops and Products 138 (2019): 111419. https://doi.org/10.1016/j.indcrop.2019.05.068.

Shabnum, Shazia, and Muzafar G. Wagay. “Essential Oil Composition of Thymus Vulgaris L. and Their Uses”. Journal of Research & Development 11 (2011): 83–94.

Sienkiewicz, Monika, Monika Łysakowska, Marta Pastuszka, Wojciech Bienias, and Edward Kowalczyk. “The Potential of Use Basil and Rosemary Essential Oils as Effective Antibacterial Agents.” Molecules 18, no. 8 (2013): 9334–51. https://doi.org/10.3390/molecules18089334.

Smith-Palmer, A., J. Stewart, and L. Fyfe. “Antimicrobial Properties of Plant Essential Oils and Essences against Five Important Food-Borne Pathogens”. Letters in Applied Microbiology 26, no. 2 (1998): 118–22. https://doi.org/10.1046/j.1472-765x.1998.00303.x.

Soković, Marina, Jelena Vukojević, Petar Marin, Dejan Brkić, Vlatka Vajs, and Leo Van Griensven. “Chemical Composition of Essential Oils of Thymus and Mentha Species and Their Antifungal Activities”. Molecules 14, no. 1 (2009): 238–49. https://doi.org/10.3390/molecules14010238.

Wei, Alfreda, and Takayuki Shibamoto. “Antioxidant Activities and Volatile Constituents of Various Essential Oils.” Journal of Agricultural and Food Chemistry 55, no. 5 (2007): 1737–42. https://doi.org/10.1021/jf062959x.

Yang, Li, Kui-Shan Wen, Xiao Ruan, Ying-Xian Zhao, Feng Wei, and Qiang Wang. “Response of Plant Secondary Metabolites to Environmental Factors”. Molecules 23, no. 4 (2018): 762. https://doi.org/10.3390/molecules23040762.

Özer, Hakan, Münevver Sökmen, Medine Güllüce, Ahmet Adigüzel, Fikrettin Şahin, Atalay Sökmen, Hamdullah Kiliç, and Özlem Bariş. “Chemical Composition and Antimicrobial and Antioxidant Activities of the Essential Oil and Methanol Extract of Hippomarathrum Microcarpum (Bieb.) from Turkey”. Journal of Agricultural and Food Chemistry 55, no. 3 (2007): 937–42. https://doi.org/10.1021/jf0624244.

By Predrag Persak, Regional Technical Manager Europe, EW Nutrition

Imagine you’re at a pub quiz dedicated to feed production, and this question pops up: name a process that returns up to 25 times what was invested in it. Do you know the answer? I’m pretty sure you are probably using it every day: pelleting. For every unit of used energy, pelleting generates up to 25 times more in terms of the nutritional value for animals (mostly metabolizable energy).

The math is simple: while we gain 200 kcal/kg by pelleting broiler mash feed, only 10 Kilowatts are used to produce one ton of broiler feed. This is just one example of how sustainability is at the core of feed production – and has always been, long before it became a buzzword. So, to all those who operate feed mills, who take care of sourcing and quality, and to those behind numbers that represent nutritional values: You are pioneers of sustainability and should be proud of that.

How feed processing can drive sustainability efforts

Besides being proud, we must also be very responsible. Every nutritionist should focus on

1. how processing of feed materials and feed influences the release of nutrients, nutrient density, and exclusion of antinutrients, and
2. how processing can improve these dimensions, making feed more sustainable.

Do we take processing sufficiently into consideration? Do we create formulations in a dynamic or more static way? Not least in an era of precision feeding, the shift from static to dynamic is inevitable.

This is even clearer when we consider how processing can influence digestion, absorption, and the performance of animals. How so? Feed processing makes previously unusable materials suitable for nutrition or improves already usable materials. So, the feed processing itself is a key to sustainability.

Through processing, we alter the physical, chemical, and edible properties of used feed materials, making them usable for animals. Through proper processing, we improve the digestibility of feed materials by up to 20%, enabling a more effective – and thus more sustainable – use of feed resources. In practice, there is room for improvement to make feed processing even more of a sustainability champion.

Moisture optimization is key to energy-efficient pelleting

Let’s take a closer look at pelleting since it requires the most energy within feed processing. How much energy is used? This depends on many factors and can range from 5 KW/h up to 25. Pelleting is mostly used in broiler diets to reduce nutrient segregation and feed sorting and, by extension, feed wastage. Pelleting has also been found to increase the weight gain of individual birds and flock uniformity, and overall feed efficiency is higher.

Pelleting involves the agglomeration of mixed feed into whole pellets through a mechanical process using heat, moisture, and pressure (Falk, 1985). Heat (energy that is transported through steam) has the largest impact on pelleting efficacy. Steam injected during conditioning increases feed moisture and temperature, softens feed particles, extracts natural binders, and reduces friction which leads to greater production rates and pellet quality (Skoch et al., 1981).

The key to an efficient pelleting process is to set the parameters at the levels that will enable proper energy transfer from steam to feed particles. Besides steam quality, the moisture of the feed is a critical factor for efficient energy transfer. Generally, the thermal conductivity of the most used feed materials increases with increasing moisture. A level of 17% moisture in the conditioner is needed for efficient energy transfer. Below 17%, we need more steam (more energy) or more time (more capacity) to achieve the same result. That is why proper moisture optimization is needed to use the energy transferred through steam in the most efficient way.

Reduce shrinkage, improve sustainability

What about shrinkage? Shrinkage is not just a cost factor but a sustainability issue. We must not lose scarce and valuable materials and nutrients. Overall shrinkage tends to be around 1%. For global feed production as a whole, 1% annual shrinkage is equivalent to 15 years of Croatian compound feed production!

We help our industry to keep up sustainability efforts in terms of energy savings and shrinkage reduction by offering SurfAce. It’s a liquid preservative premixture with multiple economic and environmental benefits to the customer. It helps increase pellet output, improves conditioning, enhances the durability of the pelleted feed, reduces the formation of fines, and improves the overall quality of the final feed product. But most importantly, it optimizes feed production costs through energy savings and reduced labor input while also supporting the microbiological quality of the feed.

In the food sector, we have seen vast improvements in non-thermal food processing over the past decade. Examples include ultrasonication, cold plasma technology, supercritical technology, irradiation, pulsed electric field, high hydrostatic pressure, pulsed ultraviolet technology, and ozone treatment. I’m sure some of these technologies will be applied to feed processing one day. Until then, we must keep up our high sustainability standards and make it more efficient by applying all available tools in our feed processing toolbox.

References

Falk, D. “Pelleting Cost Center.” Essay. In Feed Manufacturing Technology III, edited by Robert R. McEllhiney, 167–90. Arlington, VA: American Feed Industry Association, 1981.

Skoch, E.R., K.C. Behnke, C.W. Deyoe, and S.F. Binder. “The Effect of Steam-Conditioning Rate on the Pelleting Process.” Animal Feed Science and Technology 6, no. 1 (1981): 83–90. https://doi.org/10.1016/0377-8401(81)90033-x.

By Dr. Inge Heinzl, Editor, Madalina Diaconu, Produt Manager Pretect D, and Dr. Ajay Awati, Global Category Manager Gut Health & Nutrition, EW Nutrition

Often you have an extensive coccidiosis control program in place. You don’t observe any clinical signs of coccidiosis. However, at the end of the cycle, you record significantly lower body weight and a higher FCR. There is a high probability that your animals have subclinical coccidiosis. This article digs deeper into understanding why birds don’t perform as they should, why subclinical coccidiosis occurs on the farm, and why drug resistance is an important factor.

Subclinical coccidiosis – a silent enemy

Clinical coccidiosis is clearly characterized by severe diarrhea, high mortality rates, reduced feed/water intake, and weight loss. By contrast, subclinical Coccidiosis does not display any visual signs and often remains undetected.

According to De Gussem (2008), the damages caused by subclinical coccidiosis can reach up to 70% of the total cost of coccidiosis control treatments, ranging from US$ 2.3 billion to US$ 13.8 billion/year in 2020 worldwide (De Gussem, 2008; Ferreira da Cunha, 2020; Blake et al., 2020).

Monitoring coccidiosis occurrence on the farm

There are several tools available to evaluate the level of infection. The most common ones are:

Lesion scoring – is used to evaluate the damages caused by coccidiosis in the intestinal tract. Lesion scoring gives insight into the severity of the infection. Furthermore, based on the location of lesions in the GI tract, it is possible to determine the plausible Eimeria spp. responsible for the infection.

OPG (Oocyst per gram) – the number of oocysts per gram of feces indicates the level of shedding of oocysts in the manure, litter, and, eventually, in the farm environment. OPG levels may not give the exact severity of the infection in the bird but certainly provide a clear idea of its likely spread within the flock.

Ways to deal with coccidiosis on the farm

Different tools are widely used to prevent and treat coccidiosis:

Anticoccidials:                  Chemicals, ionophores

Vaccination:                       Natural strains, attenuated strains

Bio-shuttle:                        Vaccine + ionophore

Natural anticoccidials:   Phytomolecules

These coccidiosis control programs are used depending on the farm history and the severity of the infection. Traditionally, treatment was heavily dependent on chemicals and ionophores. However, rampant and unbridled use of ionophores leads to resistance in Eimeria spp. on the farm, the failure of the control program, and significant performance losses, with high mortality due to coccidiosis. Therefore, the tools mentioned above are inserted in rotation or shuttle programs to minimize the generation of resistances. In a rotation program, the anticoccidial changes from flock to flock. In a shuttle program, the anticoccidial changes within one cycle according to the feed (Chapman, 1997).

However, this strategy is often not 100% effective due to a lack of diversity and overuse of certain tools within programs. The rigorous financial optimization of the program leads to the use of cost-effective but marginally effective solutions. These factors over the period weaken the program, which seems to work well but leads to resistance to anticoccidial drugs and sets up subclinical coccidiosis.

Resistances have been reported in the US (Jeffers, 1974, McDougald, 1981), South America (McDougald, 1987; Kawazoe and Di Fabio, 1994), Europe (Peeters et al., 1994; Bedrník et al., 1989; Stephan et al., 1997), Asia (Lan et al., 2017; Arabkhazaeli et al., 2013), and Africa (Ojimelukwe et al., 2018). Chapman and co-workers (1997) even stated that resistances were documented for all anticoccidial drugs employed at this time, and new products have not been approved for decades.

Resistance and subclinical coccidiosis can be approached naturally

When an anticoccidial has lost its effectiveness due to excessive use, some resistant coccidia survive. They can cause a mild course of the disease, subclinical coccidiosis, driving the costs high. Reducing the occurrence of resistance and subclinical coccidiosis can significantly decrease the expenses of coccidiosis control programs and, eventually, the cost of production.

Increasing consumer pressure to reduce the overall usage of drugs in animal production has driven innovation efforts to find natural solutions that can be effectively used within coccidiosis control programs. However, this shift was not easy for the producers. Lack of reliable data, poor understanding of the mode of action, lack of quality optimization, and unsubstantiated claims led to the failure of many earlier-generation natural solutions.

However, the consumer-driven movement to find natural solutions to animal gut health issues has recently led to relentless innovation in this area. Knowledge, research, and technological developments are now ready to offer solutions that can be an effective part of the coccidia control program and open opportunities to make poultry production even more sustainable by reducing drug dependency.

For centuries, phytomolecules have been used for their medicinal properties and effects on the health and well-being of animals and humans. In the case of coccidiosis, tannins and saponins have been proven to support animals in coping with this disease. Tannic acids and tannic acid extracts strengthen the intestinal barrier by reducing oxidative stress and inflammation (Tonda et al., 2018). On the other hand, saponins lessen the shedding of oocysts, improve the lesion score, and, in the case of an acute infection, the occurrence of bloody diarrhea (Youssef et al., 2021).

These natural substances can be integrated into shuttle or rotation programs to reduce the use of anticoccidials and, therefore, minimize resistance development.

Pretect D: Coccidiosis programs can be strengthened naturally!

In an EU field trial conducted with more than 200 000 birds, Pretect D (a natural phytogenic-based product designed to increase the efficacy of coccidiosis control) was used in the shuttle program together with ionophores. The trial provided excellent results on zootechnical performance (figures 1-4).

Figures 1-4: Zootechnical performance of broilers with Pretect D included in the shuttle program

Trials show that Pretect D supports the efficiency of coccidiosis control programs by impairing the Eimeria development cycle when used in combination with vaccines, ionophores, and chemicals as part of the shuttle or rotation program:

• It protects the epithelium from inflammatory and oxidative damage
• It promotes the restoration of the mucosal barrier function

Table 1 exemplifies one way of including a natural solution (Pretect D) in actual coccidiosis control programs.

Table 1: Exemple of including Pretect D into coccidiosis control programs

Natural solutions suit both farmers and consumers

With phytomolecules partly replacing anticoccidials in rotation or shuttle programs, the use of anticoccidials in poultry production can be decreased. On the one hand, this answers consumers’ demand; on the other hand, it leads to a push-back of resistances in the long run. The returning effectiveness of the anticoccidials can reduce subclinical coccidiosis, leading to lower costs spent on this disease and a higher profit for the farmers.

References:

Arabkhazaeli, F., M. Modrisanei, S. Nabian, B. Mansoori, and A. Madani. “Evaluating the Resistance of Eimeria spp. Field Isolates to Anticoccidial Drugs Using Three Different Indices.” Iran J Parasitol. 8, no. 2 (2013): 234–41.

Bedrník, P., P. Jurkovič, J. Kučera, and A. Firmanová. “Cross Resistance to the IONOPHOROUS Polyether Anticoccidial Drugs IN Eimeria Tenella Isolates from Czechoslovakia.” Poultry Science 68, no. 1 (1989): 89–93. https://doi.org/10.3382/ps.0680089

Blake, Damer P., Jolene Knox, Ben Dehaeck, Ben Huntington, Thilak Rathinam, Venu Ravipati, Simeon Ayoade, et al. “Re-Calculating the Cost of Coccidiosis in Chickens.” Veterinary Research 51, no. 1 (2020). https://doi.org/10.1186/s13567-020-00837-2

Chapman, H. D. “Biochemical, Genetic and Applied Aspects of Drug Resistance in Eimeria Parasites of the Fowl.” Avian Pathology 26, no. 2 (1997): 221–44. https://doi.org/10.1080/03079459708419208.

De Gussem, M., and S. Huang. “The Control of Coccidiosis in Poultry.” International Poultry Production 16, no. 5 (2008): 7–9.

Ferreira da Cunha, Anderson, Elizabeth Santin, and Michael Kogut. “Editorial: Poultry Coccidiosis: Strategies to Understand and Control.” Frontiers in Veterinary Science 7 (2020). https://doi.org/10.3389/fvets.2020.599322

Jeffers, T. K. “Eimeria Acervulina and E. Maxima: Incidence and Anticoccidial Drug Resistance of Isolants in Major Broiler-Producing Areas.” Avian Diseases 18, no. 3 (1974): 331. https://doi.org/10.2307/1589101

Kawazoe, Urara, and J. Di Fabio. “Resistance to DICLAZURIL in Field Isolates OfEimeriaspecies Obtained from Commercial BROILER Flocks in Brazil.” Avian Pathology 23, no. 2 (1994): 305–11. https://doi.org/10.1080/03079459408418998

Lan, L.-H., B.-B. Sun, B.-X.-Z. Zuo, X.-Q. Chen, and A.-F. Du. “Prevalence and Drug Resistance of Avian Eimeria Species in Broiler Chicken Farms of Zhejiang PROVINCE, CHINA.” Poultry Science 96, no. 7 (2017): 2104–9. https://doi.org/10.3382/ps/pew499

McDougald, L. R. “Anticoccidial Drug Resistance in the Southeastern United STATES: POLYETHER, IONOPHOROUS Drugs.” Avian Diseases 25, no. 3 (1981): 600. https://doi.org/10.2307/1589990

McDougald, Larry R., Jose Maria Silva, Juan Solis, and Mauricio Braga. “A Survey of Sensitivity to Anticoccidial Drugs in 60 Isolates of Coccidia from Broiler Chickens in Brazil and Argentina.” Avian Diseases 31, no. 2 (1987): 287. https://doi.org/10.2307/1590874

Ojimelukwe, Agatha E., Deborah E. Emedhem, Gabriel O. Agu, Florence O. Nduka, and Austin E. Abah. “Populations of Eimeria Tenella Express Resistance to Commonly Used Anticoccidial Drugs in Southern Nigeria.” International Journal of Veterinary Science and Medicine 6, no. 2 (2018): 192–200. https://doi.org/10.1016/j.ijvsm.2018.06.003

Peeters, Johan E., Jef Derijcke, Mark Verlinden, and Ria Wyffels. “Sensitivity of AVIAN EIMERIA Spp. to Seven Chemical and Five Ionophore Anticoccidials in Five Belgian INTEGRATED Broiler Operations.” Avian Diseases 38, no. 3 (1994): 483. https://doi.org/10.2307/1592069

Stephan, B., M. Rommel, A. Daugschies, and A. Haberkorn. “Studies of Resistance to Anticoccidials IN Eimeria Field Isolates and Pure Eimeria Strains.” Veterinary Parasitology 69, no. 1-2 (1997): 19–29. https://doi.org/10.1016/s0304-4017(96)01096-5

Tonda, RM, J.K. Rubach, B.S. Lumpkins, G.F. Mathis, and M.J. Poss. “Effects of Tannic Acid Extract on Performance and Intestinal Health of Broiler Chickens Following Coccidiosis Vaccination and/or a Mixed-Species Eimeria Challenge.” Poultry Science 97, no. 9 (2018): 3031–42. https://doi.org/10.3382/ps/pey158

Youssef, Ibrahim M., Klaus Männer, and Jürgen Zentek. “Effect of Essential Oils or Saponins Alone or in Combination on Productive Performance, Intestinal Morphology and Digestive Enzymes’ Activity of Broiler Chickens.” Journal of Animal Physiology and Animal Nutrition 105, no. 1 (2020): 99–107. https://doi.org/10.1111/jpn.13431

By Dr. Inge Heinzl, Editor, and Dr. Ajay Bhoyar, Global Technical Manager Poultry, EW Nutrition

Faster growth of breast muscle in broilers may lead to increased incidences of different types of muscle degeneration. Downgrading the affected breast fillets results in high economic losses for the poultry meat industry.

The article discusses the three important myopathies impairing the breast muscles, their impact on the meat industry, influencing factors, and how to cope with these challenges.

Muscle degeneration heaps up with faster broiler growth

According to Sirri and co-workers (2016), breast fillets from broilers with 3.9 kg live weight carry a higher risk for myopathic lesions. Studies in different countries revealed that myopathies in broilers are not neglectable:

Country	Myopathy	Number of breasts examined	Conditions	Occurrence	Reference
Italy	WS	28,000 broilers	commercial	12 %	Petracci et al., 2013
Italy	WS	70 flocks; always 500 of 35,000 breasts randomly examined	commercial	43%, with 6.2% considered severe	Lorenzi et al., 2014
Italy	WS	57 flocks	commercial	70.2 % (medium)-82.5 % (heavy-weight)	Russo et al., 2015
Italy	WS	16,000 samples	commercial	9 % moderate22 % severe	Petracci in Baldi et al., 2020
Brazil	WS	25,520	commercial	10 %	Ferreira et al., 2014
USA	WS	960 (week 6)+ 960 (week 9)	experimental	Score 1: 78.4 % (wk 6) 29.9 % (wk 9) Score 2: 14.0 % (wk 6) 53.9 % (wk 9) Score 3:0 % (wk 6) 15.1 % (wk 9)	Kuttapan et al., 2017
Brazil	WB		commercial	10-20 %	Carvalho, in Petracci et al., 2019
Italy	WB	16,000 samples	commercial	42 % moderate 18 % severe	Petracci, in Baldi et al., 2020
China	WB	1,135 breast fillets	commercial	61.9%	Xing et al., 2020
USA	WB	960 (week 6)+ 960 (week 9)	experimental	Score 1: 32.5 % (wk 6) 33.2 % (wk 9) Score 2: 7.9 % (wk 6) 36 % (wk 9) Score 3: 1.96 % (wk 6) 15.6 % (wk 9)	Kuttapan et al., 2017
Italy	SM	16,000 samples	commercial	4 % moderate 17 % severe	Petracci in Baldi et al., 2020
Brazil	SM	5,580 samples	commercial	10 %	Montagna et al., 2019

 

Figure 1: Different myopathies in broilers (R. Baileys)

As the appearance of products is one of the most important arguments for the purchase decision, these myopathies are serious issues; the downgrading of the breast quality results in a lower reward for the producer. Kuttapan et al. (2016) estimated that 90 % of the broilers are affected by wooden breast and white striping (see below), causing about $200 million to $1 billion of economic losses to the U.S. poultry industry per year.

Wooden Breast (WB), a result of the proliferation of connective tissues

The muscle affected by the wooden breast is bulging and hard, is covered with clear, viscous fluid, and shows petechiae (see figure 2). The myopathy of the pectoralis major is “pale expansive areas of substantial hardness accompanied by white striation” (Kuttapan, 2016; Huang and Ahn, 2018; Sihvo et al., 2013). It is characterized by microscopically visible polyphasic myodegenerations with fibrosis in the chronic phase. At approximately two weeks of age, it appears as a focal lesion but then develops as a widespread fibrotic injury (Papah et al., 2017). WB can be detected by palpating the breast of the live bird.

Figure 2: Comparison of a severe wooden breast (on the left) and a healthy breast fillet (on the right)

Source: Kuttapan et al., 2016

According to Kuttapan et al. (2016), the anomaly is caused by circulatory insufficiency and increased oxidative stress resulting in damage and degeneration. Its occurrence rose with increasing growth and slaughter weights of the birds. Wooden breast is more common in male than female broilers as they show an increased expression of genes related to the proliferation of connective tissues (Baldi et al., 2021).

The hardness of the meat, a 1.2 – 1.3 % higher fat content (Soglia et al., 2016, Tasoniero et al., 2016), and the worse appearance lead to a degradation of the fillet quality (Kuttappan et al., 2012). The reduction in the water holding capacity of muscle results in toughness before and after cooking.

White Striping (WS), a result of fiber degeneration

The characteristics of WS are white striations parallel to the muscle fibers. A microscopic examination of these white stripes reveals an accumulation of lipids and a proliferation of connective tissue occurring in breast fillets and thighs (Kuttappan et al., 2013a; Huang and Ahn, 2018). Kuttapan et al. (2016) adapted a scoring system for the evaluation of the severity of WS, which he had established earlier (Kuttapan et al., 2012)(see picture 1). It was concluded that broilers fed a diet with high energy content led to higher and more efficient growth (improved feed conversion, higher live and fillet weights) but also to a higher percentage of fillets showing a severe degree of white striping.

Figure 3: Different degrees of white striping

• 0 = normal (no distinct white lines)
• 1 = moderate (small white lines, generally < 1 mm thick)
• 2 = severe (large white lines, 1-2 mm thick, very visible on the fillet surface)
• 3 = extreme (thick white bands, > 2 mm thickness, covering almost the entire surface of the fillet
• (scoring and image source: Kuttapan, 2016)

Moreover, the WB and WS can simultaneously occur in the same muscle (Cruz et al., 2016; Kuttappan, Hargis, & Owens, 2016; Livingston, Landon, Barnes, & Brake, 2018).

Spaghetti Meat (SM), a result of decreased collagen linking

The condition of Spaghetti Meat was first mentioned by Bilgili (2015) under “Stringy-spongy”. SM is characterized by an insufficient bonding of the muscles due to an immature intramuscular connective tissue in the pectoralis major. The fiber bundles composing the breast muscle detach, and the muscle gets soft and mushy and resembles spaghetti pasta (Baldi et al., 2021). Probably due to the reduced collagen-linking degree, the texture of SM fillets is smoother after cooking (Baldi et al., 2019). In contrast to wooden breast, SM cannot be noticed in the living animal. Meat severely impacted by SM is downgraded and can only be used in further processed products, whereas slightly affected meat can be sold in fresh retailing (Petracci et al., 2019).

Another possible explanation for this myopathy may be the strong development of the breast muscle. The thickness of its upper section might reduce muscular oxygenation by compressing the pectoral artery (Soglia et al., 2021). The spaghetti structure generally appears mainly in the superficial layer and less in the deep ones.

Oxidative stress – one link in the chain of causes for myopathies

Oxidative stress is a result of impaired blood supply

Oxidative stress is one key factor of myopathies in breast muscle. As the faster growth is connected with an increase in muscle fiber diameter, the higher pressure of the surrounding fascia on the muscle tissue compresses the blood vessels, leading to a decreased blood flow, resulting in insufficient oxygen supply (hypoxia) and limited removal of metabolic by-products (Lilburn et al., 2019) from the muscle tissue. Hypoxia as – well as hyperoxia – plus the deficient removal of metabolic waste, promote the generation of free radicals (Kähler et al., 2016; Strapazzon et al., 2016; Petrazzi et al., 2019). If the endogenous antioxidant system cannot efficiently eliminate these ROS by using endogenous and exogenous antioxidants, the ultimate effect is increased oxidative stress.

Soglia and co-workers (2016) reported higher TBARS (Thiobarbituric acid reactive substances) and protein carbonyl levels, signs of oxidative stress, in severe wooden breast muscle tissue. The oxidative stress hypothesis was also supported by gene transcription analysis conducted by Mutryn et al. (2015) and Zambonelli et al. (2017).

Oxidative stress causes damage

ROS (reactive oxygen species) or free radicals are highly reactive. They can cause damage to the DNA, RNA, proteins, and lipids in the muscle cells (Surai et al., 2015), leading to inflammation and metabolic disturbances, and, in the end, the degeneration of muscle fibers (Kuttapan et al., 2021). If the regenerative capacity of the muscle cells does not countervail against the damages caused by oxidative stress, fibrous tissue and fat accumulate and lead to myopathies such as wooden breast (Petracci et al., 2019)

Oxidative stress can be managed

To support the animals in coping with oxidative stress, combining two approaches, an external and an internal, makes sense. This entails protecting feed at the same time as protecting the animal.

Chemical antioxidants preserve feed quality and prevent oxidation

Chemical antioxidants such as ethoxyquin, BHA, and BHT efficiently prevent feed oxidation. These antioxidants prevent the oxidation of unsaturated fats/oils and maintain their energy value. They are scavengers for free radicals, protect trace minerals like Zn, Cu, Mg, Se, and Vit E from oxidation and spare them to be used in the body for different metabolic processes as well as for the endogenous antioxidant system.

However, keep in mind that chemical antioxidants are strictly regulated, depending on type, concentration, and region. Ethoxyquin has a challenging status in the EU, for instance, due to a ruling that excludes it for the use of long-living or reproductive animals and that sets safety levels at a maximum total concentration of 50 mg ethoxyquin/kg complete feed for all animal species, except dairy ruminants.

Phy­tomolecules act as natural antioxidants and reduce lipid oxidation in breast muscles

Inside the body, phy­tomolecules help to mitigate oxidative stress by the direct scavenging of ROS and the activation of antioxidant enzymes. Phytogenic compounds like Carvacrol and thymol possess phenolic OH-groups that act as hydrogen donors (Yanishlieva et al., 1999). These hydrogens can “neutralize” the peroxy radicals produced during the first step of lipid oxidation and, therefore, retard the hydroxyl peroxide formation. The increase in serum antioxidant enzyme activities and a resulting lower level of malondialdehyde (MDA) can be caused by cinnamaldehyde (Lin et al., 2003). MDA is a highly reactive dialdehyde generated as a metabolite in the degradation process of polyunsaturated fatty acids.

Antioxidant capacity of phytomolecules demonstrated in broilers

A trial with 480 Cobb male chicks (3 treatments, 8 replicates) was conducted at the University of Viçosa (Brazil). The breast muscles of the birds fed a blend of phy­tomolecules showed lower MDA levels and thus reduced lipid oxidation compared to the negative control, but also to the birds fed an antibiotic.

The impact of breast muscle degradation in broilers can be mitigated

The downgrading of broiler meat due to increased incidence of breast muscle myopathies is a common issue, resulting in the significant economic losses to the broiler meat producers. Oxidative stress caused due to due faster growth rate and various other stressors, including the oxidation of feed and feed ingredients, can contribute to increased incidence of woody breast and white striping. Different nutritional and management strategies are employed to reduce WB and WS in broiler production. The inclusion of synthetic antioxidants to control the oxidation in feed as well as phytomolecules to support the endogenous antioxidant system can be a part of promising tools to mitigate the impact of breast myopathies and reduce economic losses in broiler production.

References:

Alnahhas, Nabeel, Cécile Berri, Marie Chabault, Pascal Chartrin, Maryse Boulay, Marie Christine Bourin, and Elisabeth Le Bihan-Duval. “Genetic Parameters of White Striping in Relation to Body Weight, Carcass Composition, and Meat Quality Traits in Two BROILER Lines Divergently Selected for the Ultimate PH of the Pectoralis Major Muscle.” BMC Genetics 17, no. 1 (2016). https://doi.org/10.1186/s12863-016-0369-2.

Baldi, Giulia, Francesca Soglia, and Massimiliano Petracci. “Current Status of Poultry Meat Abnormalities.” Meat and Muscle Biology 4, no. 2 (2020). https://doi.org/10.22175/mmb.9503.

Baldi, Giulia, Francesca Soglia, and Massimiliano Petracci. “Spaghetti Meat Abnormality in Broilers: Current Understanding and Future Research Directions.” Frontiers in Physiology 12 (2021). https://doi.org/10.3389/fphys.2021.684497.

Baldi, Giulia, Francesca Soglia, Luca Laghi, Silvia Tappi, Pietro Rocculi, Siria Tavaniello, Daniela Prioriello, Rossella Mucci, Giuseppe Maiorano, and Massimiliano Petracci. “Comparison of Quality Traits among Breast Meat Affected by Current Muscle Abnormalities.” Food Research International 115 (2019): 369–76. https://doi.org/10.1016/j.foodres.2018.11.020.

Boerboom, Gavin, Theo van Kempen, Alberto Navarro-Villa, and Adriano Pérez-Bonilla. “Unraveling the Cause of White Striping in Broilers Using Metabolomics.” Poultry Science 97, no. 11 (May 28, 2018): 3977–86. https://doi.org/10.3382/ps/pey266.

Carvalho, Leila M, Josué Delgado, Marta S Madruga, and Mario Estévez. “Pinpointing Oxidative Stress behind the White Striping Myopathy: Depletion of Antioxidant Defenses, Accretion of Oxidized Proteins and Impaired Proteostasis.” Journal of the Science of Food and Agriculture 101, no. 4 (2020): 1364–71. https://doi.org/10.1002/jsfa.10747.

Editors, AccessScience. “U.S. Bans Antibiotics Use for Enhancing Growth in Livestock.” Access Science. McGraw-Hill Education, January 1, 1970. https://www.accessscience.com/content/u-s-bans-antibiotics-use-for-enhancing-growth-in-livestock/BR0125171.

Ferreira, T.Z., R.A. Casagrande, S.L. Vieira, D. Driemeier, and L. Kindlein. “An Investigation of a Reported Case of White Striping in Broilers.” Journal of Applied Poultry Research 23, no. 4 (2014): 748–53. https://doi.org/10.3382/japr.2013-00847.

Hashemipour, H., H. Kermanshahi, A. Golian, and T. Veldkamp. “Effect of Thymol and Carvacrol Feed Supplementation on Performance, Antioxidant Enzyme Activities, Fatty Acid Composition, Digestive Enzyme Activities, and Immune Response in Broiler Chickens.” Poultry Science 92, no. 8 (2013): 2059–69. https://doi.org/10.3382/ps.2012-02685.

Huang, Xi, and Dong Uk Ahn. “The Incidence of Muscle Abnormalities in Broiler Breast Meat – a Review.” Korean journal for food science of animal resources 38, no. 5 (October 2018): 835–50. https://doi.org/10.5851/kosfa.2018.e2.

Kolakshyapati, Manisha. “Proteoglycan and Its Possible Role in ” Wooden Breast ” Condition in Broilers.” Nepalese Journal of Agricultural Sciences 13 (September 1, 2015): 253–60.

Kuttappan, V.A., B.M. Hargis, and C.M. Owens. “White Striping and Woody Breast Myopathies in the Modern Poultry Industry: A Review.” Poultry Science 95, no. 11 (2016): 2724–33. https://doi.org/10.3382/ps/pew216.

Kuttappan, V.A., C.M. Owens, C. Coon, B.M. Hargis, and M. Vazquez-Añon. “Incidence of Broiler Breast Myopathies at 2 Different Ages and Its Impact on Selected Raw Meat Quality Parameters.” Poultry Science 96, no. 8 (2017): 3005–9. https://doi.org/10.3382/ps/pex072.

Kuttappan, V.A., H.L. Shivaprasad, D.P. Shaw, B.A. Valentine, B.M. Hargis, F.D. Clark, S.R. McKee, and C.M. Owens. “Pathological Changes Associated with White Striping in Broiler Breast Muscles.” Poultry Science 92, no. 2 (2013): 331–38. https://doi.org/10.3382/ps.2012-02646.

Kuttappan, V.A., Y.S. Lee, G.F. Erf, J.-F.C. Meullenet, S.R. McKee, and C.M. Owens. “Consumer Acceptance of Visual Appearance of Broiler Breast Meat with Varying Degrees of White Striping.” Poultry Science 91, no. 5 (2012): 1240–47. https://doi.org/10.3382/ps.2011-01947.

Kuttappan, Vivek A., Megharaja Manangi, Matthew Bekker, Juxing Chen, and Mercedes Vazquez-Anon. “Nutritional Intervention Strategies Using Dietary Antioxidants and Organic Trace Minerals to Reduce the Incidence of Wooden Breast and Other Carcass Quality Defects in Broiler Birds.” Frontiers in Physiology 12 (2021). https://doi.org/10.3389/fphys.2021.663409.

Kuttappan, Vivek A., Megharaja Manangi, Matthew Bekker, Juxing Chen, and Mercedes Vazquez-Anon. “Nutritional Intervention Strategies Using Dietary Antioxidants and Organic Trace Minerals to Reduce the Incidence of Wooden Breast and Other Carcass Quality Defects in Broiler Birds.” Frontiers in Physiology 12 (2021). https://doi.org/10.3389/fphys.2021.663409.

Kähler, Wataru, Frauke Tillmans, Sebastian Klapa, Inga Koch, Julia Last, and Andreas Koch. “Oxidativer Stress Durch Hyperbare Hyperoxie Und Dessen Wirkung Auf Periphere Mononukleäre Zellen (PBMC) – Eine Übersicht Über Den Aktuellen Forschungsstand Am Schifffahrtmedizinischen Institut Der Marine.” Wehrmedizinische Monatsschrift 60, no. 2 (2016): 50–55.

Lake, Juniper A., and Behnam Abasht. “Glucolipotoxicity: A Proposed Etiology for Wooden Breast and Related Myopathies in Commercial Broiler Chickens.” Frontiers in Physiology 11 (2020). https://doi.org/10.3389/fphys.2020.00169.

Lilburn, M.S., J.R. Griffin, and M Wick. “From Muscle to Food: Oxidative Challenges and Developmental Anomalies in Poultry Breast Muscle.” Poultry Science 98, no. 10 (2019): 4255–60. https://doi.org/10.3382/ps/pey409.

Lin, Chun-Ching, Sue-Jing Wu, Cheng-Hsiung Chang, and Lean-Teik Ng. “Antioxidant Activity of Cinnamomum Cassia.” Phytother. Res. 17 (August 7, 2003): 726–30. https://doi.org/ https://doi.org/10.1002/ptr.1190.

Linden, Jackie. “Deep Pectoral Myopathy (Green MUSCLE Disease) in Broilers.” The Poultry Site, August 9, 2021. https://www.thepoultrysite.com/articles/deep-pectoral-myopathy-green-muscle-disease-in-broilers.

Lorenzi, M., S. Mudalal, C. Cavani, and M. Petracci. “Incidence of White Striping under Commercial Conditions in Medium and Heavy Broiler Chickens in Italy.” Journal of Applied Poultry Research 23, no. 4 (2014): 754–58. https://doi.org/10.3382/japr.2014-00968.

Martindale, L., W.G. Siller, and P.A.L. Wight. “Effects of Subfascial Pressure in Experimental Deep Pectoral Myopathy of the Fowl: An Angiographic Study.” Avian Pathology 8, no. 4 (1979): 425–36. https://doi.org/10.1080/03079457908418369.

Montagna, FS, G Garcia, IA Nääs, NDS Lima, and FR Caldara. “Practical Assessment of Spaghetti Breast in Diverse Genetic Strain Broilers Reared under Different Environments.” Brazilian Journal of Poultry Science 21, no. 2 (2019). https://doi.org/10.1590/1806-9061-2018-0759.

Mutryn, Marie F, Erin M Brannick, Weixuan Fu, William R Lee, and Behnam Abasht. “Characterization of a Novel Chicken Muscle Disorder through DIFFERENTIAL Gene Expression and Pathway Analysis Using Rna-Sequencing.” BMC Genomics 16, no. 1 (2015). https://doi.org/10.1186/s12864-015-1623-0.

National Chicken Council. “Per Capita Consumption of Poultry and LIVESTOCK, 1965 to Forecast 2022, in Pounds.” National Chicken Council, June 28, 2021. https://www.nationalchickencouncil.org/about-the-industry/statistics/per-capita-consumption-of-poultry-and-livestock-1965-to-estimated-2012-in-pounds/.

Papah, Michael B., Erin M. Brannick, Carl J. Schmidt, and Behnam Abasht. “Evidence and Role Of Phlebitis and LIPID Infiltration in the Onset and Pathogenesis of Wooden Breast Disease in MODERN Broiler Chickens.” Avian Pathology 46, no. 6 (2017): 623–43. https://doi.org/10.1080/03079457.2017.1339346.

Petracci, M., F. Soglia, M. Madruga, L. Carvalho, Elza Ida, and M. Estévez. “Wooden-Breast, White Striping, and Spaghetti MEAT: Causes, Consequences and Consumer Perception of Emerging Broiler MEAT ABNORMALITIES.” Comprehensive Reviews in Food Science and Food Safety 18, no. 2 (2019): 565–83. https://doi.org/10.1111/1541-4337.12431.

Petracci, M., S. Mudalal, A. Bonfiglio, and C. Cavani. “Occurrence of White Striping under Commercial Conditions and Its Impact on Breast Meat Quality in Broiler Chickens.” Poultry Science 92, no. 6 (2013): 1670–75. https://doi.org/10.3382/ps.2012-03001.

Ruberto, Giuseppe, and Maria T Baratta. “Antioxidant Activity of SELECTED Essential Oil Components in Two Lipid Model Systems.” Food Chemistry 69, no. 2 (2000): 167–74. https://doi.org/10.1016/s0308-8146(99)00247-2.

Russo, Elisa, Michele Drigo, Corrado Longoni, Raffaele Pezzotti, Paolo Fasoli, and Camilla Recordati. “Evaluation of White Striping Prevalence and Predisposing Factors in Broilers at Slaughter.” Poultry Science 94, no. 8 (2015): 1843–48. https://doi.org/10.3382/ps/pev172.

Schulze, Dieter. “Aktuelles Aus Der Broilermast .” Presentation – 7. Tagung des VET Arbeitskreises Geflügelforschung Tiergesundheit beim Nutzgeflügel, Rust, 2018.

Sihvo, H.-K., K. Immonen, and E. Puolanne. “Myodegeneration with Fibrosis and Regeneration in the Pectoralis Major Muscle of Broilers.” Veterinary Pathology 51, no. 3 (May 26, 2014): 619–23. https://doi.org/10.1177/0300985813497488.

Sirri, F., G. Maiorano, S. Tavaniello, J. Chen, M. Petracci, and A. Meluzzi. “Effect of Different Levels of Dietary Zinc, Manganese, and Copper from Organic or Inorganic Sources on Performance, Bacterial Chondronecrosis, Intramuscular Collagen Characteristics, and Occurrence of Meat Quality Defects of Broiler Chickens.” Poultry Science 95, no. 8 (2016): 1813–24. https://doi.org/10.3382/ps/pew064.

Soglia, Francesca, Luca Laghi, Luca Canonico, Claudio Cavani, and Massimiliano Petracci. “Functional Property Issues in Broiler Breast Meat Related to Emerging Muscle Abnormalities.” Food Research International 89 (2016): 1071–76. https://doi.org/10.1016/j.foodres.2016.04.042.

Strapazzon, Giacomo, Sandro Malacrida, Alessandra Vezzoli, Tomas Dal Cappello, Marika Falla, Piergiorgio Lochner, Sarah Moretti, Emily Procter, Hermann Brugger, and Simona Mrakic-Sposta. “Oxidative Stress Response to Acute Hypobaric Hypoxia and Its Association with Indirect Measurement of Increased Intracranial Pressure: A Field Study.” Scientific Reports 6, no. 1 (2016). https://doi.org/10.1038/srep32426.

Surai F, Peter. “Antioxidant Systems in Poultry Biology: Superoxide Dismutase.” Journal of Animal Research and Nutrition 01, no. 01 (2016). https://doi.org/10.21767/2572-5459.100008.

Tasoniero, G., M. Cullere, M. Cecchinato, E. Puolanne, and A. Dalle Zotte. “Technological Quality, Mineral Profile, and Sensory Attributes of Broiler Chicken Breasts Affected by White Striping and Wooden Breast Myopathies.” Poultry Science 95, no. 11 (2016): 2707–14. https://doi.org/10.3382/ps/pew215.

United Nations. “How Your Company Can Advance Each of THE SDGS: UN Global Compact.” How Your Company Can Advance Each of the SDGs | UN Global Compact. Accessed August 31, 2021. https://www.unglobalcompact.org/sdgs/17-global-goals.

Xing, T., X. Zhao, L. Zhang, J.L. Li, G.H. Zhou, X.L. Xu, and F. Gao. “Characteristics and Incidence of Broiler Chicken Wooden Breast Meat under Commercial Conditions in China.” Poultry Science 99, no. 1 (2020): 620–28. https://doi.org/10.3382/ps/pez560.

Zambonelli, Paolo, Martina Zappaterra, Francesca Soglia, Massimiliano Petracci, Federico Sirri, Claudio Cavani, and Roberta Davoli. “Detection of Differentially Expressed Genes in Broiler Pectoralis Major Muscle Affected by White Striping – Wooden Breast Myopathies.” Poultry Science 95, no. 12 (2016): 2771–85. https://doi.org/10.3382/ps/pew268.

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By Technical Team, EW Nutrition

A storm has been brewing.

Even before the invasion of Ukraine in late February, global growth was expected to trend significantly downward, from 5.5-5.9% in 2021 to 4.1-4.4% in 2022 and 3.2% in 2023. The causes are similar across industries:

• rising inflation around the world
• supply chain issues stretching long into the foreseeable future, including exponentially higher freight costs
• pandemic restrictions and long-lasting effects
• rising raw material prices

In early 2022, this “perfect storm” quickly stifled the moderate optimism of Q4 2021. Of course, the worst was yet to come.

What causes sustained price increases?

With the ongoing crisis in Eastern Europe, economic perspectives are tilting down to a new level of uncertainty. The new variables now thrown into the mix are crude oil and natural gas prices, as well as added concerns over other raw materials coming out of Russia and Ukraine.

Source: tradingeconomics.com, March 2022

Russia accounts for 25% of the global natural gas market and 11% of the crude oil market. It is also the largest wheat exporter (China and India are still the largest producers, but Russia exports appreciably more). Together with Ukraine, also a powerhouse of agricultural exports, the two now enemies account for 29% of international annual wheat sales.

Source: ING, March 2022

Wheat prices were already nearly double the five-year average shortly before the invasion; after February 24, they rose by another 30%. Today we are at a staggering 53% increase in wheat prices in just the last few months. We are at a 14-year peak. And the countries that import the most from Russia and Ukraine (such as Egypt or Indonesia) will bear the brunt of this crisis.

Together, Russia and Ukraine’s exports account for 12% of the world’s traded calories. The two countries account for almost 30 percent of global wheat exports, almost 20 percent of corn exports, and more than 80 percent of the world supply of sunflower oil. However, the compounded effect of embargo and devastation in the two countries will surely exert tremendous influence on the global economic outlook for years to come.

What are the perspectives?

Agriculture was already hurting before February 24^th. Poor harvests caused by extreme weather conditions, continued losses along the production chain, supply chain issues, and abnormal pandemic buying patterns combined to sink global wheat stocks one third lower than the five-year average. Reserves, in other words, are low – and will be significantly lower.

We need to be realistic about the coming months and years. Corn (where Ukraine accounts for 13% of global exports) and wheat will be severely hit by the war and its aftermath. This will compound all the pre-existing factors (transportation costs, supply chain slowdown, continuing weather disruptions, energy costs), none of which will trend down. Fertilizer prices have also gone up exponentially, and Russia – the largest exporter – has banned fertilizer exports at the beginning of March. The effects will be ultimately reflected in the cost of raw materials.

Ukraine and Russia have all but banned grains exports – either for security reasons or to protect internal needs. On top of this, the last harvests collected in Ukraine are now sitting in bins where ventilation and temperature controls have been affected by power cuts.

Source: World Bank, March 2022

At the end of February, World Bank data already showed upward movement for nearly all categories; whatever was not trending up at that time is catching up fast. The last time things looked like this, experts warn, was in 2008-2009 – and social unrest followed around the world, to serious global consequences.

However, the perspective is not catastrophic and there is room to conserve profitability. The essential is to intervene with fast, targeted action that favors smart optimization, localization, and long-term planning.

What can feed producers do?

 Most feed producers will be caught in the middle of all rising costs, from raw materials to transport and energy. Where, then, can they look for shelter when the storm hits?

Optimize feed costs without losing performance

One of the first things feed producers will focus on will be cutting down feed costs. At this point, it is essential that this basic optimization does not impact animal health and performance. Here is what should be kept in mind.

Preserve feed material and feed quality

Whatever raw materials you choose to use, minimizing losses and maintaining quality should be the first step. Losses caused by storage are often the easiest to mitigate.

Quick intervention #1: Use mold inhibitors and mitigate the impact of mycotoxins

Compensate for lost nutrients (protein content, digestibility)

Freight costs will continue to cause pressure on transported raw materials, driving producers to local/regional options. When you replace one feed ingredient with a cheaper one, the first effects will be on the active principle and on the digestibility of the feed. Often something you are taking out of the diet cannot be replaced 1:1.

Quick intervention #2: Maximize the use of enzymes to ensure high feed digestibility; for poultry, pigments can replace corn-derived coloration (to control color variability)

Compensate for stress caused by diet changes

Adjusting the feed composition doesn’t only have effects on paper.

Even if you choose the best replacements, adjust the balance, compensate for loss of digestibility and optimize everything in every possible way, one thing remains:

The animal receives a new diet.

New diets are textbook stressors. But sometimes the nutritionist or the producer is so stressed that it is easy to overlook the stress placed inside the animal. Since animal efficiency is key for productivity, it is essential that the effects of diet stress are mitigated for the animal.

Quick intervention #3: Precautionary use of gut-health mitigating additives; also consider palatable feed materials and taste enhancers

Optimize production costs without losing quality

To optimize costs on the production floor, there are three essential areas where feed producers can act:

• Saving on energy costs and reducing the carbon footprint
• Reducing losses on the production floor
• Increasing throughput without increasing manpower

To answer these challenges, there are solutions that can operate individually. More importantly in such times, there are products that can impact all three areas without negatively influencing the quality of output. One such solution, for instance, can decrease energy costs, increase throughput and pellet quality, and reduce fines.

Quick intervention #4: Choose a solution that satisfies 3/3 of your issues

Conclusion

Climate change will continue to wreak havoc on the predictability of harvests. Freight costs are projected to keep rising. And the costs of war and (hopefully) reconstruction will take a toll on the cost of living and cost of doing business around the world, for years to come.

In the storm that has already started, it is unwise to take shelter for a while and hope for good weather soon. Cutting down on ingredients here and additives there won’t keep profitability high in the long run. Feed producers must look at all aspects – from feed storage and composition to process improvement – and consider holistic measures that protect animals and profitability at the same time.

Multiple mycotoxins contaminate animal feed – problems and solutions

Mycotoxins pose an exceptional challenge for feed and animal producers. Generated by common molds, they occur in a great variety and numbers. Difficult to diagnose, mycotoxicosis in farm animals shows in a range of acute and chronic symptoms: decreased performance, feed refusal, poor feed conversion, reduced body weight gain, immune suppression, reproductive disorders, and residues in animal food products.

Regulatory mycotoxin thresholds don’t account for interactions

Regulatory thresholds for permissible mycotoxin levels in feed are derived from toxicological data on the effects of exposure of a certain species, at a certain production stage, to a single mycotoxin. This makes practical sense: while aflatoxins are carcinogens, fumonisins attack the pulmonary system in swine, for example. Mycotoxins also affect poultry in a different way than cattle, and broilers in a different way than breeders or laying hens, to mention more cases.

The problem is that, in reality, individual mycotoxin challenges are the exception. Animal diets are usually contaminated by multiple mycotoxins at the same time (Monbaliu et al., 2010; Pierron et al., 2016). Since 2014, EW Nutrition has conducted more than 50,000 mycotoxin tests on both raw material and finished feeds samples, across the globe. 85% of these samples were contaminated with more than one mycotoxin and one third positive for four or more mycotoxins.

How does contamination with multiple mycotoxins occur in animal feed?

The concurrent appearance of mycotoxins in feed can be explained as follows: each mold species has the capacity to produce several mycotoxins simultaneously. Each species, in turn, may infest several raw materials, leaving behind one or more toxic residue. In the end, a complete diet is made up of various raw materials with individual mycotoxin loads, resulting in a multitude of toxic challenges for the animals.

If animals were exposed to only one mycotoxin at a time, following the regulatory guidelines on maximum challenge levels would usually be enough to keep them safe. However, several studies have shown that the effects of exposure to multiple mycotoxins can differ greatly from the effects observed in animals exposed to a single mycotoxin (Alassane-Kpembi et al., 2015 & 2017). The simultaneous presence of mycotoxins may be more toxic than one would predict based on the known effects of the individual mycotoxins involved. This is because mycotoxins interact with each other. The interactions can be classified into three main different categories: antagonistic, additive, and synergistic  (Grenier and Oswald, 2011).

Types of mycotoxin interactions

• Additivity occurs when the effect of the combination equals the expected sum of the individual effects of the two toxins.
• Synergistic interactions of two mycotoxins lead to a greater effect of the mycotoxin combination than would be expected from the sum of their individual effects. Synergistic actions may occur when the single mycotoxins of a mixture act at different stages of the same mechanism. A special form of synergy, sometimes called potentiation, occurs when one or both of the mycotoxins do not induce significant effects alone but their combination does. Fumonisin alone, for example, requires high levels to exerts effects on broiler performance. When aflatoxin is also in the feed, the effects are higher than those of aflatoxin alone (Miazzo et al., 2005)
• Antagonism can be observed when the effect of the mycotoxin combination is lower than expected from the sum of their individual effects. Antagonism may occur when mycotoxins compete with one another for the same target or receptor site. In an in-vitro study using human colon carcinoma cells (HCT116), Bensassi and collaborators (2014), found that DON and Zearalenone individually caused a marked decrease of cell viability in a dose-dependent manner; when combined, the effect was drastically reduced.

Most of the mycotoxin mixtures lead to additive or synergistic effects. The actual consequences for the animal will depend on its species, age, sex, nutritional status, the dose and duration of exposure as well as environmental factors. What is clear is that mycotoxin interactions pose a significant threat to animal health and critically impede risk assessment.

From awareness to action: risk assessment and toxin binders

Given their complex interactions, the toxicity of combinations of mycotoxins cannot merely be predicted based upon their individual toxicities. Mycotoxin risk assessments have to consider that even low levels of mycotoxin combinations can harm animal productivity, health, and welfare. Feed and animal producers need to be aware of which raw materials are likely to be contaminated with which mycotoxins, be able to accurately link them to the risk they pose for the animal and consequently take actions before the problems appear in the field.

Trials demonstrate effectiveness of toxin mitigation solutions

Toxin binders that are effective against a broad spectrum of mycotoxins significantly reduce the risks of mycotoxin exposure. In vitro trial data shows that EW Nutrition’s cost-effective toxin-mitigating product Solis Max shows a high mitigation capacity, even at low inclusion rates (Figure 1). Importantly, Solis Max helps to reduce various mycotoxins’ negative effects on performance without any negative effects on nutrient absorption.

In another trial with 480 Ross 308 broilers, Solis Max 2.0 has demonstrated its ability to support animals coping with multiple mycotoxin challenges. For broilers challenged with 30 ppb AFB1 and 500 ppb OTA, Solis Max 2.0 supplementation resulted in a significantly 16% higher weight gain, an 18.5% higher final weight, and a 22% better FCR than the challenged group (Figure 2), which means a total recovery of the performance when compared with the non-challenged control.

Liver health also improved: after 42 days of mycotoxin exposure, broilers receiving Solis Max 2.0 showed lower AST (-13%) and ALT (-44%) levels compared to the challenged group.

Proactive management: tackle multiple mycotoxin challenges head on

Mycotoxins interactions are the norm, not the exception. Yet, regulatory standards currently only cover the effects of individual mycotoxins, leaving productions exposed to risks of additive and synergistic mycotoxin interactions animals’ health and performance. Luckily, management options are available: Careful risk evaluation explicitly includes the threat of multiple contaminations. And producers can proactively ensure better health, welfare and productivity of their animals by investing in the right toxin mitigation solution for their business.

References

Alassane-Kpembi, Imourana, Olivier Puel, and Isabelle P. Oswald. “Toxicological Interactions between the Mycotoxins Deoxynivalenol, Nivalenol and Their Acetylated Derivatives in Intestinal Epithelial Cells.” Archives of Toxicology 89, no. 8 (August 2015): 1337–46. https://doi.org/10.1007/s00204-014-1309-4.

Alassane-Kpembi, Imourana, Gerd Schatzmayr, Ionelia Taranu, Daniela Marin, Olivier Puel, and Isabelle Paule Oswald. “Mycotoxins Co-Contamination: Methodological Aspects and Biological Relevance of Combined Toxicity Studies.” Critical Reviews in Food Science and Nutrition 57, no. 16 (November 2017): 3489–3507. https://doi.org/10.1080/10408398.2016.1140632.

Bensassi, Fatma; Gallerne, Cindy; Sharaf el dein, Ossama; Rabeh Hajlaoui, Mohammed; Lemaire, Christophe and Bacha, Hassen. “In vitro investigation of toxicological interactions between the fusariotoxins deoxynivalenol and zearalenone” Toxicon 84 (2014): 1-6. https://doi.org/10.1016/j.toxicon.2014.03.005.

Grenier, B., and I. Oswald. “Mycotoxin Co-Contamination of Food and Feed: Meta-Analysis of Publications Describing Toxicological Interactions.” World Mycotoxin Journal 4, no. 3 (May 5, 2011): 285–313. https://doi.org/10.3920/wmj2011.1281.

Miazzo, R., M.F. Peralta, C. Magnoli, M. Salvano, S. Ferrero, S.M. Chiacchiera, E.C.Q. Carvalho, C.A.R. Rosa, and A. Dalcero. “Efficacy of Sodium Bentonite as a Detoxifier of Broiler Feed Contaminated with Aflatoxin and Fumonisin.” Poultry Science 84, no. 1 (January 2005): 1–8. https://doi.org/10.1093/ps/84.1.1.

Monbaliu, Sofie, Christof Van Poucke, Christ’l Detavernier, Frédéric Dumoulin, Mario Van De Velde, Elke Schoeters, Stefaan Van Dyck, Olga Averkieva, Carlos Van Peteghem, and Sarah De Saeger. “Occurrence of Mycotoxins in Feed as Analyzed by a Multi-Mycotoxin LC-MS/MS Method.” Journal of Agricultural and Food Chemistry 58, no. 1 (2010): 66–71. https://doi.org/10.1021/jf903859z.

Pierron, Alix, Imourana Alassane-Kpembi, and Isabelle P. Oswald. “Impact of Mycotoxin on Immune Response and Consequences for Pig Health.” Animal Nutrition 2, no. 2 (2016): 63–68. https://doi.org/10.1016/j.aninu.2016.03.001.