Dr. Kazım Bilgeçli
Feed Additives and Animal Health Product Group Manager
Trouw Nutrition Türkiye

The transition period in dairy cows is a critical phase characterized by increased production of reactive oxygen species (ROS) due to elevated metabolic demands and negative energy balance. When antioxidant defense systems fail to counterbalance this increase, oxidative stress develops, predisposing animals to metabolic and infectious diseases and adversely affecting health and productivity. This review evaluates the mechanisms underlying oxidative stress during the transition period and the potential effects of antioxidant support strategies.

INTRODUCTION

The transition period is the metabolic adaptation phase in dairy cows, beginning three weeks before calving and continuing until three weeks after. In the postpartum period, dairy cows may experience not only metabolic diseases such as milk fever, abomasal displacement, fatty liver and ketosis, but also infectious conditions including mastitis, laminitis and reproductive disorders (Arslan, 2010; Wankhade et al., 2017). Stress frequently encountered during this period is directly associated with animal welfare, nutritional status, and metabolic and infectious diseases, and constitutes an important factor in assessing the overall condition of animals. These factors can be evaluated under two broad categories: physiological stress, which arises from crowding, housing conditions and abrupt environmental changes; and other stressors such as hunger, injury and disease (Arslan, 2008). Following calving, the increased energy demand of lactation raises oxygen requirements and consequently elevates the production of reactive oxygen species (ROS). Furthermore, the severity of the Negative Energy Balance (NEB) that dairy cows enter due to the sudden and dramatic rise in milk production at calving is more pronounced in cows with a high body condition score (BCS); as fat mobilization occurs more rapidly in these animals, the metabolic burden on the liver increases and ROS production reaches higher levels.

This review examines how the increased metabolic load during the transition period amplifies free radical (ROS) production, the implications of this for oxidative stress mechanisms, and the resulting effects on the health and performance of dairy cows, while discussing appropriate antioxidant strategies applicable during this process.

FREE RADICALS

Free radicals are defined as molecules or molecular fragments containing one or more unpaired electrons in their outer orbitals (Pala et al., 2008; Özcan et al., 2015). Among all molecules, lipids are the most susceptible to free radical damage. The double bonds of polyunsaturated fatty acids present in cell membranes can react with free radicals and undergo peroxidation. Lipid peroxidation affects membrane permeability, leading to intracellular accumulation of Ca²⁺, which ultimately results in cell swelling and cell death. The presence of stress leads to elevated levels of malondialdehyde (MDA), a product of lipid peroxidation; accordingly, increased MDA levels serve as an indicator of lipid peroxidation and, by extension, of oxidative stress (Simsek, 1999). The susceptibility of proteins to free radical damage depends on their amino acid composition. Proteins containing amino acids with unsaturated bonds or sulfur groups, such as tryptophan, tyrosine, phenylalanine, histidine, methionine and cysteine, are particularly vulnerable to free radical attack (Sezer and Keskin, 2014). At physiological pH and temperature, the auto-oxidation of monosaccharides such as glucose yields H₂O₂, peroxides and oxoaldehydes.

The first line of defense against free radicals in the organism is provided by the enzyme superoxide dismutase (SOD). SOD is an endogenously produced enzyme essential to every cell in the organism; it protects against the harmful effects of oxidants by inhibiting peroxynitrite formation and converting the superoxide radical, which causes cellular damage, into the less harmful hydrogen peroxide and molecular oxygen (Halliwell and Gutteridge, 1999). GSH-Px and CAT are recognized as the primary antioxidant enzymes in the protective mechanism against lipid peroxidation (Kehrer, 1993). GSH-Px mediates the reduction of hydrogen peroxide and lipid hydroperoxides (Kehrer, 1993; Halliwell and Gutteridge, 1999). CAT is an iron-containing enzyme found in all organs, particularly in the liver and erythrocytes; it works in concert with GSH-Px to break down hydrogen peroxide, generated via SOD activity, into oxygen and water (Gutteridge, 1995; Halliwell and Gutteridge, 1999).

Glutathione plays important roles in amino acid transport, DNA synthesis and protein synthesis within cells (Bucak et al., 2010). It is a low molecular weight tripeptide synthesized directly from the amino acids cysteine, glutamic acid and glycine, and is considered the most important soluble antioxidant in the body (Kohen and Nyska, 2002; Valko et al., 2007). Glutathione participates in cell metabolism and is an essential compound for maintaining cellular integrity (Kehrer, 1993). Acting as a non-enzymatic antioxidant, it reduces the toxicity of free radicals through direct reaction. Beyond direct free radical scavenging, glutathione also acts enzymatically in conjunction with GSH-Px and serves as a cofactor for numerous protective enzymes.

HOW OXIDATIVE STRESS DEVELOPS AND HOW IT AFFECTS DAIRY COWS

Oxidative stress is a process arising from environmental or metabolic activities that disrupts the balance between pro-oxidants and antioxidants in the animal body, leading to cellular damage; its effects may vary depending on the capacity of the antioxidant defense system (Valko et al., 2007). Oxidative stress in tissues or cells results from a shift in the equilibrium between free radical production and endogenous antioxidant defense in favor of free radicals. The primary source of free radicals is molecular oxygen. Oxidative stress causes lipid peroxidation, protein nitration, DNA damage and apoptosis (programmed cell death) (Estevez, 2015).

Reactive oxygen species (ROS) and reactive nitrogen species (RNS) are signaling molecules that serve to maintain homeostasis at physiological levels. When produced in excess, they give rise to oxidative stress. ROS are neutralized intracellularly by antioxidant enzymes: SOD (superoxide dismutase), CAT (catalase) and GPx (glutathione peroxidase) (Kurutaş, 2016). RNS are produced in the intestinal mucosa as a byproduct of nitric oxide synthesis; in excess, they damage the mucosa and represent an important class of free radicals that impair nutrient utilization. In addition to environmental factors, mitochondrial metabolic activity, aging and stress conditions all increase free radical production, thereby affecting animal health and productivity (Yavaş et al., 2020).

It has been established that antioxidant deficiency slows uterine contractions and prevents semen from reaching the oviduct. A close relationship has been identified between oxidative stress and mastitis; depending on the severity of inflammation, reductions in milk yield and undesirable changes in milk composition may occur (Jozwik et al., 2004).

Oxidative stress leads to oxidative damage to cell membranes and other cellular components, resulting in cell necrosis and death and, consequently, tissue damage and chronic disease. Given its critical role in the pathogenesis of many diseases, oxidative stress amplifies the severity of illness (Sezer et al., 2014; Tabakoğlu et al., 2013). Dairy cows undergo significant metabolic and physiological adaptations during the transition from pregnancy to lactation. The increased oxygen demand associated with elevated metabolic needs results in greater ROS production. Oxidative stress is an important underlying factor in the immune and inflammatory dysregulation that heightens susceptibility to disease in dairy cows, particularly during the transition period (Sordillo et al., 2009; Sordillo et al., 2013; Abd Ellah, 2016).

In the mitochondria, ROS production may increase due to two related mechanisms: the slowing of electron flow in the mitochondrial electron transport chain by free fatty acids (FFA) and coenzyme A derivatives, and the generation of ROS during beta-oxidation of fatty acids for energy. Both effects are significantly amplified in transition cows owing to the increased utilization of FFAs associated with lipid mobilization, and are more pronounced in cows with a high body condition score compared to those with a low body condition score. ROS and antioxidant (vitamins A and E) concentrations in blood, and lipoperoxides along with vitamins A and E in milk, have been measured in transition dairy cows (Rizzo et al., 2013). The study concluded that supplementation with vitamins A and E during NEB would contribute to the endogenous antioxidant defense of all transition cows. Avcı and Kızıl (2012) demonstrated that oxidative stress in transition cows is associated with an increase in erythrocyte MDA levels and a concurrent decrease in plasma antioxidants (CAT, GSH-Px, vitamin E and vitamin C).

FEED ADDITIVES WITH ANTIOXIDANT PROPERTIES

The defense systems in the body that act to prevent damage caused by ROS are collectively referred to as “antioxidant defense systems.” Antioxidants inhibit lipid peroxidation either by interrupting the peroxidation chain reaction or by scavenging ROS. By reducing the impact of oxidative damage on DNA and limiting abnormal increases in cell division, antioxidants also exert a protective effect against cancer (Sezer and Keskin, 2014).

Antioxidants encounter oxidant substances at low concentrations and delay or inhibit the oxidation of the target molecule (Gutteridge, 1995). Antioxidant enzymes contribute to damage prevention by reducing free radicals (Kızıl et al., 2011), either by capturing them, converting them into less reactive molecules, or by binding free radicals and interrupting or repairing the reaction chain (Traber and Packer, 1995).

Riboflavin indirectly mediates numerous oxidation and reduction reactions involved in energy production in dairy cows Riboflavin has been shown to reduce oxidative stress, which exerts a negative effect on the immune system and reproduction and is elevated during the transition period. Additionally, riboflavin plays an important role in the activation of immune cells, enabling dairy cows to combat infections more effectively.

Magnesium: In transition dairy cows, NEFA metabolites reaching the liver as a result of negative energy balance (NEB) and excessive lipolysis tend to undergo peroxisomal rather than mitochondrial oxidation, thereby generating oxidative stress. Beyond its well-established effects on dietary anion-cation balance during the transition period, the enzymatic support that magnesium provides to antioxidant systems warrants equal consideration.

Manganese: Together with copper and zinc, manganese is incorporated into the structure of superoxide dismutase (SOD), where it plays a role in the antioxidant mechanism responsible for converting mitochondrial superoxides to hydrogen peroxide.

Vitamins facilitate the regeneration of important antioxidants such as vitamins E and C, restoring them to their active forms (Valko et al., 2007). Vitamin E and GSH-Px exert complementary effects against free radicals, with α-tocopherol exhibiting the greatest antioxidant activity (Vinson et al., 1994). It has been concluded that supplementation with minerals containing selenium, copper, zinc and manganese during this period will help reduce oxidative stress (Weiss, 2006). Adequate intake of antioxidant vitamins, including vitamin E, vitamin C and carotenoids, as well as essential trace minerals, is fundamental to this effect (Duthie et al., 1989). These vitamins work synergistically to neutralize harmful reactive oxygen species that contribute to disease and cellular damage. Vitamin E (tocopherols) is among the principal fat-soluble antioxidants, present in all cell membranes, where it protects polyunsaturated fatty acids from oxidation (Diplock, 1998). High-dose dietary supplementation with vitamin E has been reported to confer significant protection against oxidative stress (Reaven et al., 1993).

Phenolic compounds, found particularly in plant-based foods, are also recognized as important antioxidants by virtue of their properties as reducing agents, hydrogen donors, singlet oxygen scavengers and metal chelators (Rice-Evans et al., 1995). Minerals such as selenium, copper, manganese and zinc are likewise essential for the structural integrity and catalytic activity of protective enzymes (Diplock, 1998).

Vitamin E (α-tocopherol) is an effective lipid-soluble antioxidant that functions as a “chain-breaker” during lipid peroxidation in cell membranes and various lipid particles, including low-density lipoprotein (LDL) (Niki, 2014). Vitamin C is a water-soluble free radical scavenger that also regenerates vitamin E in cell membranes in conjunction with compounds capable of donating GSH or reducing equivalents. In terminating the lipid peroxidation chain reaction, vitamin C donates an electron to the lipid radical and is thereby converted to an ascorbate radical (C.Oh et al., 2010; Niki, 1991).

CONCLUSION

The transition period is a critical phase in dairy cows during which metabolic load increases, negative energy balance develops, and reactive oxygen species (ROS) production rises accordingly. When increased ROS production cannot be adequately offset by antioxidant defense systems, oxidative stress ensues, raising the risk of diseases such as mastitis, ketosis, fatty liver, laminitis and reproductive disorders. At the cellular level, oxidative stress adversely affects tissue function through lipid peroxidation, protein damage and DNA damage.

Strengthening antioxidant defenses during the transition period is therefore of critical importance, both for the prevention of metabolic diseases and for sustaining milk yield and reproductive performance. Ensuring adequate levels of antioxidant plant extracts, vitamin E, vitamin C, selenium, and highly bioavailable minerals such as copper, zinc and manganese — together with appropriate regulation of dietary energy balance — are recommended as effective strategies for mitigating the adverse effects of oxidative stress. It is concluded that future studies should comparatively evaluate the effects of different antioxidant combinations on clinical outcomes in transition cows.