Monika Leukert
Product Manager
Lallemand Animal Nutriton

Oxidative stress is a significant issue that negatively impacts growth, health, and productivity in animal production. To reduce these effects and improve animal performance, it is essential to deeply understand the causes of the problem and adopt targeted strategies.

INTRODUCTION

Oxidative stress—a term often heard in discussions about health, welfare, and well-being—goes beyond just free radicals and antioxidants. It’s a complex phenomenon that causes chaos in all cells of the body. Production of reactive oxygen species (ROS) is a natural consequence of life. It arises within the cells of animals as a byproduct of metabolic processes, especially in the mitochondria, where energy is produced. Normally, the body has a finely tuned system to maintain a balance between ROS production and the action of antioxidants. Oxidative stress is the result of an imbalance between ROS and the body’s ability to counteract or neutralize their harmful effect with antioxidants. This imbalance can occur due to an overproduction of ROS or a depleted antioxidant system. In this whitepaper, we will investigate the world of oxidative stress, exploring its intricacies and how it influences animal health.

THE DUAL NATURE OF ROS

The cell is exposed to a large variety of ROS from both exogenous and endogenous sources (Figure 1). ROS encompasses a group of molecules like superoxide radicals (O2•-) and hydrogen peroxide (H2O2). ROS, often labeled as “free radicals,” are highly reactive and can cause cellular damage. However, not all ROS are harmful. ROS also act as cellular messengers, involved in various signaling pathways. At moderate levels, ROS play a role in cell growth, immune response, and other essential functions. Excessive ROS can trigger abnormal signaling. The dual nature of ROS makes their regulation vital.

Figure 1- Exogenous and endogenous sources of reactive oxygen species (ROS) (Kohen and Nyska, 2002) & Tab. Overview of ROS and their characteristics (adapted from Domej et al., 2014).

THE MITOCHONDRION: THE ENDOGENOUS PRODUCTION SITE OF ENERGY AND ROS

Every living organism relies on energy production, and a significant part of this process occurs within tiny structures called mitochondria. These cellular powerhouses generate energy in the form of ATP (adenosine triphosphate). In doing so, they also produce ROS as natural byproducts of normal cell activity (Figure 2). During this process, superoxide radicals are produced at several sites in the mitochondria. Under normal physiological conditions, mitochondria are protected from ROS by a defense system including antioxidants such as glutathione peroxidases (GPXs), superoxide dismutases (SODs), catalase (CAT) and others. If there is an overproduction of ROS in the mitochondria, or if antioxidants are depleted, excess free radicals will be released from the mitochondria, initiating damages of different cell compartments including to the mitochondria themselves.

Figure 2 - Link between energy production and ROS production inside of the mitochondrion:
(1) Mitochondria provide cell energy and control programmed cell death (apoptosis).
(2) Electronic losses arise along the respiratory chain giving rise to ROS.
(3) If antioxidant defenses are insufficient, excess ROS initiate the free radical cascade

The number of mitochondria in a cell can vary widely depending on the type of cell and its energy needs. Some cells have a few hundred mitochondria, while others may have thousands. For example:

• Muscle cells: Muscle cells are highly energy demanding, and they can contain thousands of mitochondria. This is because they need a substantial amount of energy to contract and perform their functions.
  
• Liver cells: Liver cells, which perform various metabolic functions, also have a significant number of mitochondria, ranging from several hundred to over a thousand.
• Nerve cells: Nerve cells, or neurons, typically have fewer mitochondria compared to muscle or liver cells because they are more focused on transmitting electrical signals rather than generating energy.
• Red blood cells: Mature red blood cells lack mitochondria entirely, as their main role is to transport oxygen without using it for energy production.
• Oocytes: Mature oocytes can contain around 100,000 mitochondria. No other mammalian cell contains a higher number of mitochondria.

THE ANTIOXIDANT DEFENSE SYSTEM

To counteract the damaging effects of free radicals, cells have a network of antioxidants (Figure 3), both enzymatic (for instance, superoxide dismutase, catalase and glutathione peroxidase) and non-enzymatic (such as vitamins A, C, E and polyphenols). Enzymatic antioxidants (also called primary antioxidants) are endogenously produced and can be found in the cytoplasm but also in the mitochondria. These enzymes directly neutralize ROS by converting them into less harmful substances. For instance, SOD converts superoxide radicals into hydrogen peroxide, which is then detoxified by catalase or glutathione peroxidase into water and oxygen (Figure 4). Direct superoxide radicals converting (quenching) by primary antioxidants prevent or minimize the generation of hydroxyl radicals (OH°) responsible for initiating the free radical cascade and associated oxidative injuries.

Figure 3 - Classification of antioxidants (adapted from Lakovou & Kourti, 2022).

In comparison, non-enzymatic antioxidants (also called secondary antioxidants) can be endogenously produced or come from dietary sources. They indirectly combat oxidative stress by scavenging hydroxyl radicals (OH°), hence terminating the propagation of the free radical cascade. Accordingly, they also play a role in protecting cellular structures like cell membranes and DNA. While primary (enzymatic) antioxidants can metabolize ROS continuously, secondary antioxidants cannot. It is one secondary antioxidant molecule that can neutralize only one free radical molecule.

Together, primary and secondary antioxidants build the antioxidant defense system.

Figure 4 - Pathway of endogenous ROS production and role of primary and secondary antioxidants at the cellular level.

THE CONSEQUENCES OF OXIDATIVE STRESS

A disruption of the delicate balance between ROS and antioxidants sets off a chain of events within the cell, resulting in chaos at the cellular level. Here’s how oxidative stress causes cellular chaos. 

Mitochondria, as energy generators, are particularly vulnerable to oxidative stress. Oxidative damage to mitochondria can lead to energy production dysfunction, which can affect cell health, organ function and animal vitality. Cell membranes, which consist of a continuous double layer of lipid molecules can be destabilized by lipids damage as a result of oxidative stress, affecting cellular integrity and function. This lipid peroxidation is a chain reaction process involving three main stages (Figure. 5).

Figure 5 - Overview lipid peroxidation in three steps (adapted from Mortensen et al., 2023).

Lipid peroxidation typically begins with the initiation stage where free radicals, such as hydroxyl radicals (•OH) attack polyunsaturated fatty acids (PUFAs) within the lipid bilayer of cell membranes. This attack results in the formation of a lipid radical (L•) and a reactive oxygen species. In the propagation stage, the newly formed lipid radical reacts with molecular oxygen (O2 ), producing a lipid peroxyl radical (LOO•). This peroxyl radical is highly reactive and can attack neighboring PUFA, leading to a chain reaction. This process generates more lipid radicals and lipid peroxyl radicals, continuing the oxidation of lipids. The termination stage involves the termination of the chain reaction. This occurs when two lipid radicals combine, forming a non-radical product. Alternatively, antioxidants such as vitamin E, vitamin C, glutathione or antioxidant enzymes can neutralize free radicals, halting the propagation of lipid peroxidation.

Protein oxidation can lead to misfolding and aggregation, impairing the cells function. DNA damage can result in mutations and genomic instability. Excessive oxidative stress can induce controlled and uncontrolled cell death.

Oxidative stress can trigger inflammation, as the body’s immune system responds to cellular damage. While inflammation is a natural defense mechanism, chronic inflammation resulting from persistent oxidative stress can contribute to various diseases and further disrupt cellular function.

In summary, oxidative stress disrupts cellular function, setting off a cascade of events that damages cellular components and impairs cellular functions and integrity. The cumulative effect of these disturbances leads to a state of cellular chaos, ultimately contributing to various diseases and pathological conditions (Figure 6).

Figure 6 - Disease in case of uncontrolled oxidative stress (adapted from Lykkesfeldt & Svendsen, 2007).

STRATEGIES TO MITIGATE THE IMPACT OF OXIDATIVE STRESS IN ANIMAL PRODUCTION AND TO SUPPORT ANTIOXIDANT DEFENSE

Mitigating oxidative stress in animal production is essential for ensuring animals’ health and welfare. Here are some strategies:

• Minimize stress: implement stress-reduction measures, including optimal housing conditions, reduced crowding and appropriate handling.
• Environment management: control environmental factors that contribute to oxidative stress, such as exposure to pollutants or harsh weather conditions (such as high temperatures). 
• Selection for resilience: select animals for breeding based on their genetic resilience to oxidative stress. This can be a long-term strategy to improve animal health.

Supporting antioxidant defense through nutrition:

A. SECONDARY ANTIOXIDANTS

Today, exogenous, secondary antioxidants are added into almost all animal diets. For example, vitamin E, polyphenols and carotenoids are well known for their ability to neutralize free radicals. They scavenge the free radicals by donating an electron and maintaining a chemical balance. However, these dietary agents get saturated easily, as one molecule of secondary antioxidant can neutralize only one free radical molecule. The less free radicals are generated, the less secondary antioxidants have to be used, leading to a transfer of those to critical tissues and to accumulation for nutritional/commercial values (e.g. more vitamin E in eggs or carotenoids in salmon fillet). This can also contribute to better preservation and quality of the final product (less drip loss, cooking loss and/or improved texture) in particular by fighting lipid peroxidation and destabilization of the lipid cell layer and cellular integrity post-mortem.

B. BIOAVAILABLE TRACE MINERALS

Another strategy can be to directly support endogenous antioxidants in the cells. Trace minerals are supplemented as essential important co-factors of the primary antioxidants (SOD needs copper, zinc and manganese; CAT needs iron, and GPx needs selenium). Without those trace minerals, antioxidant enzymes cannot function. It is recommended to supplement trace minerals in the most bioavailable form. In the case of selenium, a source of highly bioavailable organic selenium is selenium-enriched yeast. After absorption, a significant proportion of the cell’s selenium is used to produce GPx, an important antioxidant enzyme.

C. UPREGULATION OF ANTIOXIDANT ENZYME PRODUCTION

Another possibility is to directly up-regulate the antioxidant defense (primary antioxidants). Several authors have demonstrated that supplementing exogenous SOD has the ability to increase the endogenous production of SOD, CAT and GPx. Studies in different animal species have demonstrated this increase of antioxidant enzymes in the cells of different tissues. This mechanism supports the antioxidant defense system directly in the mitochondria (the first line of defense), where secondary antioxidants cannot act. It also brings an extra layer of protection to the vulnerable mitochondria that are important for the energy production in the cells. Supporting the first line of defense in the mitochondria can help to reduce the production and release of the hydroxyl-radical (OH°), consequently lowering the demand for scavenging antioxidants to counteract oxidative damage.

A SOURCE OF SUPEROXIDE DISMUTASE

Melofeed contains dried melon juice produced from a specific melon variety, which is naturally rich in the antioxidant enzyme superoxide dismutase (SOD = min. 2,600,000 IU/kg). The dried melon juice is protected by a specific coating that prevents the digestion of its active compounds along the digestive tract. Figure 7 shows the resistance of the coated dried melon juice along the GIT in comparison to nonprotected dried melon juice (in a mouse model). Without the coating, the SOD in the melon juice is degraded like any other protein and is then eliminated via the bladder after 24 hours. Thanks to the coating that opens in the upper intestine, the SOD remains intact when arriving in the ileum (last part of the small intestine), where specific receptors get stimulated (Figure 7).

Figure 7 - Blue spots are imagery artefacts; the radioactivity is represented in yellow and red. A reagent has been injected to visualize the digestive tract in gray in addition to the skeleton, unlike other organs, which are not visible.

This receptor stimulation in the ileum is crucial to induce an activation cascade in the entire body to produce antioxidant enzymes in the cells (SOD, CAT and GPx). Supplementing MELOFEED has been shown to help increase expression of antioxidant enzymes in different tissues of various animal species (for example, reproductive tract of layers (Figure 8), gill of fish, liver, heart and fat tissue of rodents). This, in turn, was shown to positively modulate markers of immune functions and antioxidant defenses with a positive effect on animals’ resilience against oxidative stress, performance, reproduction and end-products quality (milk, meat and/or eggs).

Figure 8 - In laying hens fed MELOFEED for 6 weeks, the expression of endogenous antioxidant enzymes increased by 25-30% in all tissues of the reproductive tract (superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) compared to a non-supplemented control (Barbé et al., 2015; Carillon et al., 2016).

A PREMIUM SOURCE OF ORGANIC SELENIUM

ALKOSEL is an inactivated yeast rich in organic selenium (Se). Its production process mimics the natural path of organic selenium incorporation into plants. Like plants, the specific yeast strain (Saccharomyces cerevisiae) used to produce ALKOSEL absorbs inorganic selenium and converts it into organic selenium. This makes selenized yeast a natural solution for animals, containing a selenium-rich biomass with around 100 different organic selenium compounds — selenomethionine (SeMet) representing the biggest proportion. The combination of several organic selenium forms in ALKOSEL guarantees optimal Se bioavailability and functionality. Indeed, some forms such as SeMet are initially stored by incorporation into the animal’s structural protein such as muscles where they are then passively and progressively released as proteins and naturally turned over. In comparison, some other Se forms, such as Se-cysteine (SeCys), are directed into the functional Se-pool to act as essential cofactors of various bioactive seleno-enzymes such as the primary antioxidant GPx. Animals fed with selenized yeasts are shown to elevate their antioxidant status during challenging situations, increasing their resilience to stress events.

Glutathione peroxidases (GPx) are a selenium-containing group of antioxidant enzymes. An inappropriate activity of GPxs correlates with the nutritional deficiency in selenium. These antioxidant enzymes are selenoproteins involved in the detoxification of hydrogen peroxide (H2 O2 ) and lipid hydroperoxides (LOOH), among other types of free radicals. In these reactions, glutathione (GSH) is used as a reducing agent (Figure 9), which is restored by the enzyme glutathione reductase.

Figure 9 - Toxification pathway of Hydrogen peroxide and lipid hydroperoxides by GPx (adapted from Ellwanger et al., 2016).

CONCLUSION

Oxidative stress is a complex phenomenon with far-reaching consequences for animal health and welfare. Understanding the roles of primary and secondary antioxidants is essential in defining an optimal strategy to help address this challenge. By implementing management practices that reduce oxidative stress — including minimizing exposure to stressors, adequate nutrition and careful selection of an antioxidant strategy — we can preserve the health, longevity and performance of livestock and companion animals alike.

TIPS: HOW TO MEASURE OXIDATIVE STRESS

No single biomarker will measure oxidative stress, inflammation and antioxidant status.

It is recommended to associate 1 marker of oxidative stress and 1 marker of antioxidant status:

• Sampling overtime and individual measurements
• ROS are nearly never analyzed (very short half-time, instability, etc.)
• To be associated with APP1 and inflammatory markers in case of immune challenge
• To be interpreted in parallel with health criteria
• To be linked with parameters of products quality (meat, milk and eggs)
• To be linked with parameters of organ health and function (e.g. gamete, liver)
• Markers specific to the challenge: KRL for heat stress, GPx activity/haptoglobin/ lipid peroxides for vaccination, etc.

Typical biomarkers of antioxidant status:

• Antioxidant enzyme activity in blood or different organs
• Genomic/proteomic analysis of antioxidant enzymes
• Ratio GSH/GSSG (reduced and oxidized glutathione)
• General biomarkers: total antioxidant status (TAS), KRL, etc.
• Selenium levels in serum

Typical biomarkers of oxidative stress:

• Oxidized proteins (carbonyls)
• Oxidized lipids (MDA, TBARS, lipid peroxides)
• Oxidized DNA (8-oxoG)
• General biomarker: total oxidant status (TOS)
• Haptoglobin

1 APP: Acute phase proteins // KRL: Kit Radicaux Libres // MDA : Malondialdehyd// TBARS : Thiobarbituric acid reactive substances // 8-oxoG: 8-Oxoguanine

ABOUT LALLEMAND ANIMAL NUTRITION

Lallemand Animal Nutrition is a prominent player in the science of fermentation and a primary producer of yeast and bacteria. The company harness microorganisms to optimize animal well-being and performance, forage management, and the animal environment. It remains unwavering in its commitment to helping the industry partners and farmers sustainably feed a growing global population through improved animal performance – and enhancing the wellbeing of livestock and companion animals. The company provides innovative microbial products, services and solutions for customers around the world. It delivers tailor-made services according to specific needs and offer expert technical support to ensure the optimal application and efficacy of its solutions.