Review: In vivo and postmortem effects of feed antioxidants in livestock: a review of the implications on authorization of antioxidant feed additives

Abstract

The pivotal roles of regulatory jurisdictions in the feed additive sector cannot be over-emphasized. In the European Union (EU), antioxidant substances are authorized as feed additives for prolonging the shelf life of feedstuffs based on their effect for preventing lipid peroxidation. However, the efficacy of antioxidants transcends their functional use as technological additives in animal feeds. Promising research results have revealed the in vivo efficacy of dietary antioxidants for combating oxidative stress in production animals. The in vivo effect of antioxidants is significant for enhancing animal health and welfare. Similarly, postmortem effect of dietary antioxidants has been demonstrated to improve the nutritional, organoleptic and shelf-life qualities of animal products. In practice, dietary antioxidants have been traditionally used by farmers for these benefits in livestock production. However, some antioxidants particularly when supplemented in excess could act as prooxidants and exert detrimental effects on animal well-being and product quality. Presently, there is no exclusive legislation in the EU to justify the authorization of antioxidant products for these in vivo and postmortem efficacy claims. To indicate these efficacy claims and appropriate dosage on product labels, it is important to broaden the authorization status of antioxidants through the appraisal of existing EU legislations on feed additives. Such regulatory review will have major impact on the legislative categorization of antioxidants and the efficacy assessment in the technical dossier application. The present review harnesses the scientific investigations of these efficacy claims in production animals and, proposes potential categorization and appraisal of in vivo methodologies for efficacy assessment of antioxidants. This review further elucidates the implication of such regulatory review on the practical application of antioxidants as feed additives in livestock production. Effecting these regulatory changes will stimulate the innovation of more potent antioxidant products and create potential new markets that will have profound economic impacts on the feed additive industry. Based on the in vivo efficacy claims, antioxidants may have to contend with the legislative controversy of either to be considered as veterinary drugs or feed additives. In this scenario, antioxidants are not intended to diagnose or cure diseases as ascribed to veterinary products. This twisted distinction can be logically debated with reference to the stipulated status of feed additives in Commission Regulation (EC) No 1831/2003. Nonetheless, it is imperative for relevant stakeholders in the feed additive industry to lobby for the review of existing EU legislations for authorization of antioxidants for these efficacy claims.

Implications

A multitude of studies have shown that dietary antioxidants can alleviate oxidative stress in livestock and improve the quality of animal products. This review highlights the need for appraisal of feed additive legislations in the European Union to exclusively ascribe these efficacy claims to the authorization of antioxidants. This will give broader and defined marketing perspectives to the uses of antioxidants with consequent impacts on the feed additive industry and market. Inclusion of these claims and required dosages on product labels will better guide feed manufacturers, nutritionists and livestock producers in their purchasing decisions and application of antioxidants.

Introduction

Historically, antioxidants have been added to commercial feeds to prevent lipid peroxidation and oxidative rancidity during production, processing and storage of feeds. More importantly, the current trend of formulating diets with polyunsaturated fatty acids (PUFAs)-rich ingredients has heightened the use of antioxidants in animal feeds. Indeed, PUFA-rich foods and feeds are highly susceptible to lipid peroxidation (Decker et al., 2012). The use of exogenous antioxidants in commercial feeds helps to preserve the sensory qualities of the feed and prevent the destruction of critical nutrients such as pigments and vitamins (Calabotta and Shermer, 1985). Interestingly, the biological activities of antioxidants have been well-established in both humans and animals aside from their activities in foods and feeds (Halliwell and Gutteridge, 1999; Surai, 2007; Kalam et al., 2012). As illustrated in Figure 1, the biological effects of supplemental antioxidants could accumulate into in vivo and postmortem benefits by preventing oxidative stress and oxidative rancidity in production animals and animal food products, respectively (Fellenberg and Speisky, 2006).

Figure 1 Schematic concept of in vivo and postmortem benefits of antioxidants in livestock. PUFA=polyunsaturated fatty acid.

There is an important global focus on the legislative frameworks regulating the authorization of feed additive products due to the overwhelming significance of feed additives in modern livestock production. There are vast disparities in the regulatory status of antioxidants as feed additives when considered across different countries. Commission Regulation (EC) No 1831/2003 (Commission Regulation, 2003) guides the authorization of feed additives in the European Union (EU). Feed additives can be authorized, based on their efficacy claims, into one of the five existing functional groups (i.e. technological, sensory, nutritional, zootechnical, coccidiostats and histomonostats additives). Presently, antioxidant substances are authorized as feed additives for prolonging the shelf life of feedstuffs based on their effect for preventing lipid peroxidation. Antioxidant substances are exclusively categorized under the ‘technological additives’ functional group. Moreover, vitamins and trace minerals are categorized as a functional group in the category of ‘nutritional additives’ even though certain vitamins and trace minerals could exhibit antioxidative activity. It is crystal clear that the present EU regulations for authorization of antioxidants as feed additives lack exclusive consideration for the in vivo and postmortem effects of antioxidants in livestock. Indeed, regulatory recognition of the in vivo and postmortem efficacies of antioxidant products may be a major stumbling block for the global feed additive industry and market.

In recent years, extensive scientific reviews have elucidated the in vivo and/or postmortem benefits of antioxidant nutrition in production animals (Fellenberg and Speisky, 2006; Celi and Chauhan, 2013; Castillo et al., 2013; Salami et al., 2015). This review is exclusively aimed at describing the roles of antioxidants as feed additives with respect to animal well-being and product quality. This review further exploits how these efficacy claims could stimulate the appraisal of legislations guiding the authorization of antioxidants as feed additives in the EU.

Feed antioxidants

Halliwell and Gutteridge (1999) defined antioxidants as substances that when present in feeds and foods at a concentration lower than that of an oxidizable substrate will significantly interrupt or avert the oxidation of the substrate. Hilton (1989) and Decker (1998) noted that substances that could exhibit antioxidative capacity include deactivators of peroxides and other reactive oxygen species; quenchers of secondary lipid oxidation products that produce rancid odors; oxygen scavengers; free radical scavengers and metal chelates. Antioxidants used in animal feeds are functionally considered to be similar to those used in food and they can be broadly categorized into natural and synthetic ones. Several authors (Shahidi, 2000; Shahidi and Zhong, 2005; Augustyniak et al., 2010) have provided detailed overview as well as the regulatory status of natural and synthetic antioxidants in foods and feeds; thus, a concise discussion will be provided in this review.

Natural antioxidants

These are antioxidants that exist naturally in plant-based materials and predominantly include vitamins and polyphenolic compounds. Albeit there are quite a number of antioxidants in nature, only few are commercially used as additives for combating feed peroxidation. Vitamin E (tocopherols and structurally related tocotrienols) and vitamin C (ascorbic acid) are the most significant natural antioxidants used in feeds (Shahidi and Zhong, 2005). Others include vitamin A (retinol) and carotenoids. Substantial quantities of these natural antioxidants can be present in feed ingredients including vegetable oil, legumes and cereals. Animals fed forages could also have access to substantial quantities of these vitamins as well as polyphenolic compounds (Castillo et al., 2013). Factors such as the type of forage species, conservation methods and the forage maturity status could, however, affect the concentration of vitamins in forages; thereby demanding for exogenous supply of dietary antioxidants (Lindqvist, 2012).

Commercial forms of vitamins can be produced either by fermentation, chemical synthesis or extraction from natural sources. Synthetic derivatives of vitamins can be largely similar in chemical structures to their corresponding naturally occurring forms in plants (Topliss et al., 2002). The antioxidant stability and activity of tocopherols depend on temperature and vary with their chemical structure. The antioxidant activity of tocopherols in foods decreases in the order of δ->γ->β->α-tocopherol (Shahidi, 2000). However, the biological efficacy of the synthetic derivatives could differ from their natural forms. For instance, the biological activity of the synthetic derivative of vitamin E, all-rac-α-tocopherol (a mixture of eight stereoisomers) is lower than that of its natural form RRR-α-tocopherol in a given ratio of 1 : 1.36 (Weiser and Vecchi, 1982). This could be explained by the better bioavailability of RRR-α-tocopherol compared with all-rac-α-tocopherol (Traber et al., 1998). The eight stereoisomers could also vary in their biopotencies with the RRR form being the most active (Weiser and Vecchi, 1982). The bioavailability of these stereoisomers has been further demonstrated to vary in fluids and tissues of different animal species (Jensen and Lauridsen, 2003). In addition, there are indications that vitamin E is more effective in vivo while vitamin C acts effectively postmortem (Morrissey et al., 1998; Bou et al., 2001).

Furthermore, botanicals rich in polyphenols such as rosemary extract, grape pomace (GP), grape seed extract, green tea and olive oil, have been tested and still undergoing extensive evaluation as natural antioxidants in feeds. The in vitro and in vivo antioxidant protection of phytochemicals could be mediated by direct scavenging of free radicals, which could be partly influenced by their low values of standard one-electron reduction potential (Augustyniak et al., 2010). In addition, it could indirectly involve the complex mechanism of activating the nuclear factor erythroid-2, nuclear-related factor 2 (Nrf2) and Kelch-like ECH associated protein 1 (Keap1) complex. Activation of the Nrf2-Keap1 complex could then induce a cytoprotective mechanism against free radicals (Lee et al., 2013). Several factors could influence the physiological functions of phytochemicals. These include the composition of the raw material, processing methods, location of bioactive compounds within the tissue as well as factors influencing solubilization, micelle formation, transporter for uptake and factors influencing in vivo metabolism (Bohn et al., 2015). The major drawbacks associated with the development of botanicals as natural antioxidants include the assessment of their antioxidant potency as well as the separation of their individual phytochemicals (Augustyniak et al., 2010). Moreover, many of the phytochemicals have low bioavailability and there is extensive need to identify phytochemicals with high bioavailability to enhance their biological activities (Manach et al., 2004).

Indeed, natural antioxidants have green image and are becoming more acceptable to consumers than their synthetic counterparts. Similarly, the safety assessment of natural antioxidants is less-stringent compared with synthetic antioxidants and most of them are enlisted with GRAS (generally recognized as safe) status for authorization.

Synthetic antioxidants

These are chemically synthesized and are required at low concentrations to stabilize oil, fat and lipid-containing feeds. They are mostly phenolic and nitrogen compounds and the phenolic derivatives contain more than one methoxy or hydroxyl groups. The most commonly used synthetic phenolic compounds include butylated hydroxytoluene (BHT), butylated hydroxyanisole (BHA), tert-butylhydroquinone (TBHQ) and propyl gallate (Shahidi and Zhong, 2005). Phenolic compounds execute their antioxidant function by capturing free radicals and halting oxidation chain reaction. Examples of nitrogen compounds include ethoxyquin (EQ), capsaicin and vanillylamide, of which EQ remains the most efficacious. Synthetic antioxidants are generally perceived to be more effective than equal quantities of natural antioxidants and can better resist processing losses (Crane et al., 2000).

The use of EQ and other synthetic antioxidants for in vivo benefits must be extensively tested for the absence of carcinogenicity and other toxic effects in their pure and oxidized forms as well as their reaction products with feed components (Augustyniak et al., 2010; Błaszczyk et al., 2013). In addition, the residues of synthetic antioxidants and their metabolites in animal foods may hamper the use of synthetic antioxidants for postmortem benefits. Consumption of 300 g fillet was shown to contribute significantly to the acceptable daily intake of BHT as well as EQ and its oxidation products, when included in the diets of farmed Atlantic salmon (Lundebye et al., 2010). Moreover, the feed dosage of these synthetic antioxidants and their duration of exposure in animals could be directly related to the amount of their residues in animal foods as shown for EQ in the fillet of Atlantic salmon (Bohne et al., 2008). In contrast, the amount of EQ in foods of animal origin was less than the EU maximum residue level (Aoki et al., 2010). Indeed, there is crucial need for regulatory monitoring of both EQ and its oxidation products as well as that of other synthetic antioxidants in animal foods considering their potential toxicity in humans (Błaszczyk et al., 2013). Nonetheless, the US Food and Drug Administration regulated for maximum inclusion level of 150 ppm for EQ, 200 ppm for both BHT and BHA in animal feeds and similar regulatory levels have been adopted by many other countries including the EU.

Effects of supplemental antioxidants in livestock

Mechanisms of antioxidant protection

The mechanisms of antioxidant protection in the biological system of animals have been extensively reviewed by several authors (Fellenberg and Speisky, 2006; Lykkesfeldt and Svendsen, 2007; Surai, 2007). Thus, a succinct detail will be provided in this review. In reality, the animal is naturally endowed with an overwhelming biological antioxidant system to combat the free radicals that are continuously produced as a result of several metabolic activities in the body. Free radicals include reactive oxygen species and reactive nitrogen species such as superoxide anion, hydroxyl radical and hydrogen peroxide (Kalam et al., 2012). However, there is a certain limit to the protection that could be offered by the endogenous antioxidant barrier. This limit is further compromised by the presence of factors that could trigger excessive production of free radicals and/or weaken the efficiency of the biological antioxidant system, thereby causing oxidative stress. Such factors include: consumption of high-PUFA or rancid diet; intake of mycotoxins, heavy metals, fungicides and pesticides; nutritional deficiency such as selenium; pathogenic infections; stress-related practices such as weaning, vaccination and transportation; exposure to ionizing radiation; animal’s production status such as early lactation; and heat stress. Furthermore, once animals are slaughtered, there is concomitant loss of efficiency in the biological antioxidant system, which together with other post-slaughter conditions (Morrissey et al., 1998) result in the onset of lipid deterioration in muscle tissues and consequent oxidative rancidity in meat products (Iglesias et al., 2008).

Free radicals are unstable and highly reactive chemical species with an unpaired electron which induces them to trap electron from biological macromolecules such as DNA, lipids and proteins, in order to neutralize themselves. The reaction of free radicals with biological molecules results in oxidative damage of such macromolecules and potential cellular damage (Fellenberg and Speisky, 2006). In a counter protective response, antioxidants act by either directly scavenging the free radicals or stabilizing the free radicals by donating the electron needed (Figure 2). As presented in Table 1, the biological antioxidant system consists of both the enzymatic (superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), catalase (CAT), etc.) and non-enzymatic (selenium, vitamins E, C and A, etc.) components. In essence, oxidative stress is the deteriorative condition, which results from the imbalance between the endogenous generation of free radicals and the biological antioxidant defense systems in the body (Halliwell and Gutteridge, 1999). In situations of excess free radical production, there is a keen need for exogenous intake of antioxidants to prevent potential cellular damage.

Figure 2 Mechanism of action of antioxidants (adapted from Kalam et al., 2012).

Table 1 Antioxidants in the biological system of animals (adapted from Weiss, 2010)

Based on the nature of antioxidants, they can be grouped into water-soluble (e.g. ascorbic acid) and lipid-soluble (e.g. vitamin E and carotenoids) antioxidants. The former and the latter are located in the hydrophilic and lipophilic compartments of the cell, respectively (Yeum et al., 2004). There are emerging indications that redox cooperation exist between these two groups of cellular antioxidants, which accumulate to antioxidant synergism. Example of such redox cooperation is the ability of terminal hydrophilic ascorbic acid to repair oxidized tocopheroxyl radical of vitamin E in order to allow vitamin E perform its antioxidant function again (Buettner, 1993). Similarly, Iglesias et al. (2008) demonstrated that exogenous phenolic compound, grape procyanidins, had the ability to repair oxidized α-tocopherol and delay the depletion of ascorbic acid in the muscle tissues of fish. Thus, this highlights the importance of supplementing livestock with both groups of antioxidants to enhance duality of action which has proven to have synergistic effects.

In vivo efficacy in livestock

A multitude of published studies have highlighted the benefits of antioxidants on the health and production of livestock. Thus, the information provided in this review should be considered as an overview. Several comprehensive review papers have elucidated the deteriorative role of oxidative stress and the in vivo benefits of antioxidant nutrition in farm animals (Lykkesfeldt and Svendsen, 2007), ruminants (Miller et al., 1993a; Hansen, 2010; Celi, 2011) and poultry (Surai, 2002; Fellenberg and Speisky, 2006; Surai, 2007).

Poultry

Several factors which could be of nutritional, pathological, physiological or environmental origins could induce oxidative stress and impair the performance of chickens (Salami et al., 2015). Consequently, dietary antioxidants have been shown to counteract the negative effects of oxidative stress in poultry and extensive reviews have been provided for broiler chickens (Fellenberg and Speisky, 2006; Salami et al., 2015). Table 2 depicts the recommended dietary antioxidant nutrients in poultry.

Table 2 Recommended dietary antioxidant nutrients for various classes of poultry per ton of complete feed (adapted from Waldroup, 2001)

MIU=million international unit; TIU=thousand international unit; mg= milligram.

Mint leaves, amla electrolyte and vitamin E supplemented as dietary antioxidants, enhanced the antioxidant status of broilers reared under heat stress (Maini et al., 2007). Glutathione (GSH) concentration was higher with mint leaves and electrolyte supplements while SOD activity was found highest in brain, liver and heart with amla electrolyte and vitamin E. Taulescu et al. (2011) reported that supplementation of vitamin E and selenium (Se) positively influenced the BW gain in broilers fed oxidized lipids but no effect was observed on the carcass characteristics of the birds. Increased SOD and CAT activity, higher ferric reducing antioxidant power (FRAP) and significantly lower malondialdehydes (MDA) were reported in heat stress layer and breeder hens supplemented with vitamins E and C (Yardibi and Turkay, 2008; Jena et al., 2013). Conversely, feeding singly or a combination of natural antioxidant supplements of grape seed extracts, tomato extracts, rosemary extracts, green tea extracts and natural tocopherols did not affect the oxidative status and lipid oxidation of plasma in broilers (Vossen et al., 2011). Though low inclusion dose was suggested as a possible cause for the observed tenuous antioxidant effect. In a study evaluating synthetic antioxidants, EQ and propyl gallate decreased liver thiobarbituric acid reactive substances (TBARS) levels when broilers were fed diet containing oxidized oil (Tavarez et al., 2010).

There may be potential to complement the antioxidant activity of dietary vitamin E with that of phenolic extracts even though there are limited data in this regard. It was recently highlighted that polyphenol-rich grape by-products could partially replace costly vitamin E in monogastric diets by finding the right proportion to combine them (Brenes et al., 2015). There was significant positive effect on the growth performance of broilers when grape extracts were supplemented in combination with 100 ppm of vitamin E (Juin et al., 2007). Brenes et al. (2008) concluded that the antioxidant potency of GP was as effective as that of vitamin E and supplementing up to 60 g/kg dosage of GP did not impair broiler performance. Moreover, dietary replacement of vitamin E by sage leaves did not affect the performance and meat yield of spent hens (Loetscher et al., 2014). The negative feeding value of grape by-products has also been demonstrated. Supplementation of high concentrations of grape seed extract (Chamorro et al., 2013) and GP (Goni et al., 2007) impaired the growth rate and/or protein and amino acid digestibility of broilers. Current levels of dietary vitamin E, particularly in pig and poultry production, is a safety margin to ensure the protection of animals in oxidative stress condition. It is imperative to acknowledge that the partial replacement of dietary vitamin E level with phenolic extracts require further research to avoid compromising the protective margin of the feed. This is emphatically important given that phenolic extracts exhibits their antioxidant activity via different mechanism compared with vitamin E as highlighted in previous sections in this review.

Swine

Zhu et al. (2012) indicated that antioxidant blend of vitamins C and E, tea polyphenols, lipoic acid and microbial antioxidants has the potential to prevent free radical-induced damage in pigs and suppress oxidative stress by modulating the expression of tumor protein 53 and PGC-1α genes post-weaning. The immuno-modulatory potential of tea polyphenol in piglets subjected to oxidative stress was supported by Deng et al. (2010). In addition, there was an increase in serum and liver α-tocopherol levels in pigs supplemented with vitamin E (Ching et al., 2002). However, as the dietary levels of vitamins A and E increased, there was contrasting interaction that resulted in the decline of the α-tocopherol levels in the tissues. Fernández-Dueñas et al. (2008) observed no effect on the antioxidant status measured in terms of TBARS concentration and GSH-Px activity in weaned pigs supplemented with vitamin C and β-carotene. Furthermore, TBHQ and EQ improved performance, decreased lipid oxidation and boosted the biological antioxidant system such as GSH-Px, SOD and CAT activities when pigs were fed oxidized corn oil (Fernández-Dueñas, 2009).

Ruminants

The transition or periparturient period is generally considered as a crucial time during which dairy cows are highly susceptible to oxidative stress (Sharma et al., 2011). The period is characterized by high metabolic demand and physiological adjustments to the onset of lactation. Abuelo et al. (2014) recently provided a review of the in vivo benefits of dietary antioxidants on udder health, uterine health and reproductive performance, and incidence of production diseases of periparturient cows. Insufficient dietary antioxidants during this period were suggested to possibly increase oxidative stress and occurrence of retained placenta in dairy cows (Brzezinska-Slebodzinska et al., 1994). However, supplementing transition cows and periparturient heifers with vitamin E resulted in improved signs of oxidative status with regards to higher serum α-tocopherol level, decreased lipid peroxidation and reduced oxidative damage in liver (Brzezinska-Slebodzinska et al., 1994; Bouwstra et al., 2008 and 2009). A meta-analysis of 19 experiments suggested that dietary addition of vitamin E and Se could decrease the average relative risk of mastitis by 34% (Zeiler et al., 2010). However, individual supplementation of Se was more potent in reducing the risk of mastitis compared with the individual supplementation of vitamin E (40% v. 30%). Moreover, dietary addition of vitamin E and Se apparently increased milk yield with mean of 1 kg milk/animal per day and this effect was greater for vitamin E than Se (Zeiler et al., 2010).

Furthermore, left displaced abomasum (LDA) is a significant health problem in dairy herd, especially during early lactation. Veterinary surgery aimed at repositioning the abomasum usually results in stress reaction that was found to be positively correlated to high level of plasma TBARS (Mudron et al., 2007). Qu et al. (2013) further found that the depletion of serum vitamin E preceded the occurrence of LDA and persisted after LDA correction. These observations provided better insights to the role of oxidative stress in LDA cows both before and after surgical correction. However, Chawla and Kaur (2004) observed that plasma α-tocopherol, retinol and β-carotene levels at parturition can be increased by supplementing cows with these vitamins during dry period in order to augment their immunity status. Supplementing heat-stressed lactating dairy cows with dietary selenium boosted the preventive antioxidant system of the animals (Calamari et al., 2011). In addition, dietary supplementation of vitamin E improved the semen quality of Aohan fine-wool sheep (Yue et al., 2010) while contrasting failure of fertility improvement was observed when vitamin E and selenium were administered to lactating cows by injection (Paula-Lopes et al., 2003). This difference in observation may be partly attributed to the route of administration, which was suggested by Hansen (2010) as a factor that contributes to the effective concentration of antioxidant in tissues. It has been suggested that the present NRC requirement of dairy cows for antioxidant mineral nutrients should be considered as the minimum dietary requirement (Weiss, 2010). Therefore, necessary adjustments should be made when formulating diets to allow for expected differences due to intake, environment and feed composition (Table 3).

Table 3 Suggested dietary concentration (dry matter basis) of antioxidant mineral nutrients¹ (adapted from Weiss, 2010)

¹ Values are for a Holstein cow with an average BW for various stages of lactation and gestation. Pre-fresh is for cows in the last 2 weeks of gestation. Fresh is for cows in the first 3 weeks of lactation.

Environmental challenges such as heat stress, especially during summer period, could constitute serious welfare problems, which could impair the performance and health of livestock. Induction of oxidative stress has been suggested as major mechanism through which heat stress exert its negative effect on animals. There are indications that supplementation of vitamin E and selenium could attenuate some of the negative effects mediated by heat stress by improving the antioxidant status of sheep (Chauhan et al., 2014; Alhidary et al., 2015). However, the source of selenium (organic or inorganic) did not influence the performance of non-stressed beef heifers during the fattening phase (Rossi et al., 2015).

Ruminal acidosis is a metabolic disorder associated with feeding high quantity of readily fermentable carbohydrates to ruminants and it could impair animal productivity and cause detrimental health problems such as laminitis (Lettat et al., 2012). There are existing data that suggest the potential of dietary antioxidants to enhance rumen protection. Dietary inclusion of quercetin (a flavonoid with antioxidant capacity) in lamb diet decreased the level of parakeratosis, which is an indicator of subacute ruminal acidosis (Benavides et al., 2013). Moreover, dietary supplementation of either vitamin E or carsonic acids in concentrate diet corrected the metabolic acidosis in fattening lambs (Morán et al., 2013).

In general, despite the intriguing in vivo benefits of antioxidant supplementations in livestock species, there are some inconsistent results particularly with the supplementation of botanicals. The inconsistent effect of polyphenols may be related to the fact that in vitro studies were largely used to demonstrate their antioxidant properties. Most of these data may not be relevant to in vivo situation because of the uncertainty that phenolic compounds would be delivered to target tissues in concentrations that could elicit direct free radical scavenging effects. Moreover, the inconsistent effect of dietary antioxidants may be partly attributed to insufficient methodological approaches used for measuring biomarkers of oxidative stress in animals. Such discrepancies could be further attributed to experimental factors such as the loss of dietary antioxidants during feed processing and storage, dosage of antioxidants, antioxidant status of the animals before the experiment, age of animals, route of antioxidant administration, production traits, level of stress challenges and the level of natural antioxidants already present in the diets before supplementation. There is a need to seek adequate and standardized analytical methodologies to accurately measure biomarkers of oxidative stress in animals and subsequent effect of antioxidant supplementation. In addition, different elements of the experimental conditions particularly the animal factors should be clearly and extensively stated when reporting research results relating to antioxidant supplementations.

Postmortem efficacy in livestock

Oxidative rancidity imparts negatively on the sensory, nutritional and shelf-life qualities of food products (Valenzuela and Nieto, 1996), especially those high in PUFA such as meats (Morrissey et al., 1997). Consumption of lipid oxidation products that result from oxidative rancidity of animal products can be very toxic to human health (Esterbauer, 1993). Though, the addition of antioxidants in food processing has an age-long history; targeting antioxidant supplementation in animal diets may be a more potent strategy for enhancing the oxidative stability, sensory qualities and nutritional antioxidant content of animal products. In support of this assertion, lipid oxidation process was thought to be initiated at the subcellular membrane level of muscle foods (Gray and Pearson, 1987) and dietary antioxidants can better incorporate into tissue membranes due to inherent in vivo metabolism rather than the superficial contact made by the postmortem addition of exogenous antioxidants (Liu et al., 1995).

Meat

Improvement in meat color and lipid oxidative stability as well as drip loss prevention by dietary antioxidant supplementations were previously reviewed (Liu et al., 1995; Wood and Enser, 1997). Supplementation of vitamin E and rosemary powder for broiler chickens increased the oxidative stability of fats in meat measured in terms of the MDA content (Marcinčák et al., 2005). Similarly, dietary α-tocopherol supplementation reduced the TBARS content, lipid oxidation, rancid odor and flavor in raw, cooked and stored meat (Maraschiello et al., 1999; Grau et al., 2001; Ruiz et al., 2001; Tavarez et al., 2010; Taulescu et al., 2011). Taulescu et al. (2011) further observed an increased concentration of α-tocopherol in meat due to supplementation with vitamin E or vitamin E with Se, which implies an enriched antioxidant status of the meat for benefits in human nutrition. Mercier et al. (2000) suggested that dietary α-tocopherol exhibits its antioxidant mechanism via increase in the free sulfhydryl present in muscle cell which then helps to trap free radicals from the cells, thereby protecting the cell membrane from oxidative degeneration during storage. On the contrary, dietary addition of marigold xanthophylls reduced the oxidative stability of meat as indicated by the increased TBARS content (Koreleski and Świątkiewicz, 2007). This suggests that there should be careful consideration for the use of new antioxidant substances, particularly plant extracts, with respect to their effect on meat quality.

Furthermore, the shelf life of pork from pigs fed oxidized corn oil supplemented with TBHQ and EQ was positively impacted by decreased discoloration and lipid oxidation (TBARS) after 0, 7, 14 and 21 days in retail display (Fernández-Dueñas, 2009). However, Haak et al. (2008) observed no effect on color and protein oxidation of pork when α-tocopherol and rosemary were supplemented in the diets of pigs. In contrast to rosemary supplementation that further depicts lack of effect on lipid oxidation in both raw and cooked pork, there was decrease in lipid oxidation in α-tocopherol supplemented raw pork with exception to the cooked one.

Realini et al. (2004) observed that vitamin E supplementation of concentrate-fed steers increased lipid stability of ground beef and steaks but not color stability. On the other hand, vitamin C addition to ground beef improved color stability without altering lipid oxidation. Liu et al. (1995) reviewed that dietary vitamin E supplementation could have greater positive impacts in preventing discoloration as well as lipid oxidation in ground and frozen beef than cooked beef. However, the authors further noted that the adoption of 500 IU/steer per day of vitamin E based on results from accumulated studies, may pose a threat to the cost effectiveness of the American beef industry unless adequate quantitative strategy can be developed for detecting at slaughter, beef fed such amount of vitamin E. Moreover, changing beef heifers from inorganic to organic selenium during the last 2 months of fattening could be an effective way to enhance the qualities of meat from beef heifers (Rossi et al., 2015).

Milk

Dietary supplementation of antioxidants in dairy cows could be an effective way of fortifying milk and dairy products with antioxidant nutrients such as vitamins and minerals, while also promoting animal health (Baldi and Pinotti, 2008). The quality of milk can be compromised due to high somatic cell counts (SSC) resulting from incidence of mammary gland infection predominantly mastitis (Castillo et al., 2013). More importantly, SSC is one of the significant factors that determine milk price as it is considered as a gauge for the hygienic quality of milk. A review by Politis (2012) suggested that dietary vitamin E can improve milk quality either by directly enhancing the oxidative stability of milk or by indirectly reducing the level of SSC and plasmin activity in milk. Dietary antioxidants including vitamin E, Se and other trace minerals could reduce the occurrence of intramammary infection and thus decrease the SSC in milk (Baldi et al., 2000; Politis et al., 2004; Machado et al., 2013). However, antioxidant effect to reduce the SSC in milk has been inconsistent across studies (Sivertsen et al., 2005; Waller et al., 2007). Nonetheless, the meta-analysis by Zeiler et al. (2010) showed that supplementation of vitamin E and Se could decrease SSC by 24 000 cells/ml milk. Vitamin E supplementation has also been shown to decrease plasmin by 30%, a proteolytic enzyme that could compromise the processing quality of milk (Politis et al., 2004).

Antioxidant supplementations could also have positive influence on milk composition. Pre- and postpartum intramuscular injections of Se, Zn and vitamin E improved the lipid profile of ovine milk (Gabryszuk et al., 2007). Similarly, antioxidant blend of pineapple rind, Zn and Cu had positive effect on blood and milk cholesterol and lactose content of goat milk (Warly et al., 2011). In contrast, there was no effect of vitamin E supplementation on milk composition of lactose, fat and protein (Baldi et al., 2000; Politis et al., 2004).

Egg

Food enriched in n-3 fatty acids have been widely acknowledged for their health, growth and development benefits in humans (Simopoulos, 1991). It is clearly evident that increasing the content of these fatty acids in animal diets will simultaneously increase their availability in animal products including eggs (Meluzzi et al., 2000). However, the role of n-3 fatty acids was positively correlated to lipid peroxidation of tissues (Husvéth et al., 2000), which implies that animal products obtained from such feeding strategies will be more susceptible to rancid spoilage. Meluzzi et al. (2000) observed that dietary vitamin E prevents alteration in the fatty acid profile of the yolk after 28 days of storage while also enriching the table eggs with α-tocopherol as the level of dietary vitamin E increases. In a comparative study involving supplementation of vitamin E and rosemary extract, similar positive antioxidant effect of vitamin E was found but rosemary extract had no effect on peroxidation of fresh eggs and when subjected to iron-induced lipid oxidation (Galobart et al., 2001). Though the tenuous effect observed for rosemary extract may be attributed to the low dietary dosage used in the study. In addition, dietary organic selenium was suggested to improve egg fertility and hatching quality of stored eggs obtained from broiler breeders fed PUFA-rich diets (Pappas et al., 2005 and 2006).

Regulatory review for authorization of antioxidants as feed additives

In most countries, a company intending to place a feed additive product on the market is legally required to obtain prior authorization via a process termed marketing authorization. To accomplish the authorization process for a product, a technical dossier application must be submitted to the relevant regulatory authorities according to the required standards and regulations. Figure 3 depicts the authorization process for feed additives in the EU. The regulatory jurisdictions for the technical dossier application of feed additives differs across countries. Commission Regulation (EC) No 1831/2003 authorized antioxidant substances as feed additives mainly for their efficacy in preventing oxidative damage and preserving the quality of feedstuffs and feed materials (Commission Regulation, 2003). Commission Regulations No 1831/2003 and 429/2008 (Commission Regulation, 2003 and 2008). detailed the established procedures for submitting the dossier application for feed additives in the EU. To consider the in vivo and postmortem effects of antioxidants, the review of existing EU legislations will have significant implication on the legislative classification of antioxidants and the efficacy assessment in the dossier application.

Figure 3 Authorization scheme for feed additives in the European Union (adapted from Jans D., personal communication).

Implication on the categorization of antioxidants

An unequivocal feature of the regulatory jurisdictions in the EU, Canada, Brazil, China, Japan, South Africa and United States is that the claim or function of an ingredient or additive can change the regulatory category of such ingredient (Smedley, 2013). Hence, regulatory review for consideration of in vivo and postmortem efficacy claims of antioxidants may require the establishment of new functional category in the EU legislation for feed additives.

Potential category based on in vivo efficacy claims

Increasing number of efficacy claims has been attributed to feed additives such as enzymes, probiotics, prebiotics and phytogenics. Consequently, different proposals have been presented by the feed additive industry to urge for the amendment of the existing EU legislations for feed additives. It is very obvious that increasing consumers’ interests in issues like animal welfare and environmental sustainability are rapidly shaping the livestock sector in the EU. As such, the dynamics of this tremendous influence should encompass the entire livestock chain including the feed additive sector. Thus, there is need to propose a new category in the regulatory framework to explicitly accommodate feed additives that could ‘favorably affect the welfare of animals’. In this potential category, there are two functional groups under which antioxidants can be proposed: (1) antioxidants as substances that will positively influence the immune function of animals (i.e. immuno-modulators), (2) antioxidants as substances that will act within the animal to correct undesired consequences of nutritional origin (i.e. metabolic regulators).

Potential category based on postmortem efficacy claims

The quality of animal products encompasses the nutritional, sensory, safety and shelf-life values of the product. The existing category for ‘sensory additives’ can be reviewed to generally include feed additives used to ‘improve animal product qualities’. Thus, ‘sensory additives’ should be stipulated as a functional group under a potential category for feed additives used to ‘improve animal product qualities’. Three potential functional groups can be proposed for antioxidants in this new category: (1) antioxidants as substances intended to improve the sensory characteristics and product acceptance of animal products (i.e. sensory additives), (2) antioxidants as substances intended to improve the nutritional characteristics of animal products (i.e. nutrition enhancers), (3) antioxidants as substances used for prolonging the shelf life of animal products (i.e. shelf-life extenders).

Implication on efficacy assessment for in vivo effects

The demonstration of product efficacy is often considered as one of the most demanding and expensive requirements in the dossier application for authorization of feed additives. Apparently, failure to adequately demonstrate the efficacy of a product in the dossier application by any applicant (i.e. feed additive manufacturer) will hamper the success of the application and result into denial of market approval for such product. As opposed to the routine in vitro trials that are currently used for demonstrating the efficacy claims of antioxidants in dossier applications, in vivo and postmortem efficacy of antioxidants will keenly require documentation of in vivo trials. The extrapolation of in vivo effects of dietary antioxidants from in vitro trials could have some limitations due to lack of consideration for antioxidant uptake from the gastrointestinal tract and subsequent metabolism (Collins, 2005; Papić and Poljšak, 2012). However, measurements of oxidative stress and in vivo efficacy of antioxidants is presently one of the greatest challenges confronting oxidation research. Palmieri and Sblendorio (2007) extensively reviewed the assays involved in the measurements of oxidative stress and antioxidant intervention in biological organisms. As such, this review will only provide an overview of the commonly used methodologies.

Indirect measurements of in vivo efficacy

These consist of methods that measure the effect of free radicals on a biological system including direct damage to cell membranes.

Membrane stability assay

The correlation between antioxidant status of the animal and the stability of its cells’ membranes is evident and this has led to the development of an analytical approach aimed at measuring the ability of the cell membrane to resist hemolysis (Sadique et al., 1989). This method provides simple and rapid measurements of oxidative stress and the in vivo efficacy of dietary antioxidant supplementation in animals.

Measurement of total antioxidant activity

Measuring the overall antioxidant status of the biological system may be more important than the measurement of any single antioxidant. Methods described by Miller et al. (1993b) can be used, as well as the ferric reducing ability of plasma (FRAP) value measurement methods described by Benzie and Strain (1996). The FRAP method is principally based on the reduction of the ferric-tripyridyltriazine (Fe³⁺-TPTZ) complex to the ferrous (Fe²⁺) form at low pH.

Measurement of antioxidant enzymes

Changes in antioxidant enzyme activity in erythrocytes have also been used to measure oxidative stress. The enzymes commonly measured include SOD, CAT and GSH-Px. CAT activities in the erythrocyte and tissue can be measured according to the method of Aebi (1984), GSH-Px activities by the method of Beutler (1975) and SOD activities by the method of Arthur and Boyne (1985). It is crucial to understand that the fate of free radicals on antioxidant enzymes could confound the interpretation of results. Free radicals could either depress the concentration/activity of these antioxidant enzymes by activating their damage or increase their concentration by stimulating their induction via endogenous protective mechanism (Palmieri and Sblendorio, 2007).

Measurement of non-enzymatic antioxidants

Vitamins A, C and E, selenium, uric acid and to a lesser extent β-carotene, are principal non-enzymatic components of the biological antioxidant system. Vitamins A and E in serum and liver can be analyzed by HPLC following the procedures described by Catignani (1986). Selenium concentrations in blood and tissues can be measured using the fluorometric method by Rodriguez et al. (1994). The serum uric acid can, however, be determined using a chromogenic system described by Fossati et al. (1980).

Measurement of GSH levels

GSH level in the biological system of an animal is an important biomarker for oxidative stress and likewise for indicating the antioxidant status of an animal. Specifically, reduced GSH to oxidized GSH ratio decreases under oxidative conditions. Quite a number of assays including that of Guntherberg and Rost (1966), and Griffith (1980) are utilized to measure the GSH levels in biological tissues.

Measurement of products of lipid peroxidation

Biomarkers of lipid peroxidation in biological systems include MDA, lipid hydroperoxides (LOOH), isoprostanes and conjugated dienes. MDA and TBARS are probably the most commonly applied test system for lipid peroxidation in livestock (Botsoglou et al., 1994; Jo and Ahn, 1998; Young et al., 2003). Tissue MDA level and plasma MDA based on coupling with TBARS can be assayed according to the methods of Ohkawa et al. (1979) and Yagi (1984), respectively. TBARS is a well-recognized and established method for quantifying lipid peroxides. However, it has been largely criticized for its reactivity towards compounds other than MDA which consequently contribute to the unreliability of the results obtained from these assays. LOOH are formed earlier in the pathway leading to MDA. HPLC (chemiluminescence) and enzymatic methods can be utilized to detect LOOH in blood and tissues (Han et al., 2000). MDA determination could therefore be coupled with the HPLC method to improve the selectivity of the MDA assays. In addition, F2-isoprostanes and prostaglandin-like compounds in biological tissues can be measured as biomarkers of oxidative stress using gas chromatography-mass spectroscopy (Roberts and Morrow, 1994).

Measurement of protein and DNA oxidation

Changes in conjugated protein carbonyls can also serve as useful biomarkers for oxidative stress. When reactive oxygen species attack amino acids, carbonyl groups are produced and variety of assays has been developed to measure protein oxidation. These include HPLC and ELISA procedures (Griffiths, 2000; Han et al., 2000). However, many of these methods have been criticized for being unreliable and non-specific, coupled with the controversial debate of whether or not carbonyls represent good markers of protein oxidation in vivo (Balasubramanian et al., 1990; Stadtman and Oliver, 1991; Stadtman and Berlett, 1997).

Measurement of stress or heat shock proteins (HSP)

Expressions of HSP are regarded as manifestation of the endogenous protective mechanism against free radical damage. Stress protein synthesis has been explored as biomarker to assess the effect of oxidative stress on cellular defense system, and the counter-effect of antioxidant interventions (Wang and Edens, 1994; Polla, 1998; Goldbaum and Richter-Landsberg, 2001). Immunoblot technique can be used for analyzing stress proteins such as HSP70, HSP60, HSP32, HSP25 and αB-crystallin at cytological level as described by Goldbaum and Richter-Landsberg (2001). However, the interpretation of results can be very complicated as the expression of HSP could be induced by several stressor stimuli including hyperoxia, heat shock and oxidative stress.

Direct measurements of in vivo efficacy

Few techniques have also been developed to directly measure the level of specific reactive oxygen species present in a biological system. While these assays are robust enough to provide more precise quantification of the antioxidant status of a biological organism, their technical complexity and exorbitant analytical costs have undermined their frequency of use in oxidation research.

Electron spin resonance spectroscopy method

Collaborative utilization of this method with the spin trapping techniques can be used to directly measure free radical species in the blood and tissue samples. It is currently considered as the most sensitive direct measure of free radicals. Yoshiki et al. (1998) provided a description of how electron spin resonance could be used to measure superoxide free radicals.

Lucigenin-derived chemiluminescence (LDCL) method

This method is considered as one of the most sensitive techniques for superoxide free radical anion ( $${\rm O}_{{{\rm 2} ^{\cdot {{\rm {\minus}}}} }} $$ ) detection. The LDCL has been used to reliably measure the $${\rm O}_{{{\rm 2} ^{\cdot {{\rm {\minus}}}} }} $$ produced in isolated mitochondria and cultured endothelial cells (Li et al., 1999; Barbacanne et al., 2000). The exclusive advantage of this method is its high specificity to interact with the superoxide anion (Allen, 1986).

Indeed, several biochemical reactions are involved in the prooxidant-antioxidant balance of the biological organism. Critical evaluation of the above methodologies has suggested that many of the different assays and biomarker measurements should be applied simultaneously and the related information should be combined in order to obtain more precise results (Del Rio et al., 2002; Collins, 2005; Palmieri and Sblendorio, 2007). For accurate interpretation of the results, the relevance of the biomarkers needs to be understood in relation to their pathological and physiological significance (Del Rio et al., 2002). To reduce the cost of trials required for the efficacy assessment in the dossier application, in vivo results obtained from what are defined as major species can be extrapolated to other physiologically similar species. For instance, results obtained in laying hens may be extrapolated to other poultry species. Thus, it would be worthwhile to focus on research efforts aimed at developing accurate extrapolation models from in vivo studies between physiologically similar species.

Implication on efficacy assessment for postmortem effects

Postmortem effects of antioxidants can be measured using routine methods currently utilized for testing product quality efficacy of feed additives. Oxidative stability of animal products measured in terms of TBARS, water binding capacity of meat, product color measurement and antioxidant nutrient content in meat, egg and milk, are all important parameters to be measured to demonstrate the postmortem efficacy of antioxidant.

Major challenge: antioxidants as feed additives or veterinary drugs?

The in vivo efficacy claims may entangle antioxidants in the legislative controversy of either to be considered as veterinary drugs or feed additives. If antioxidants are to be considered as veterinary drugs, they will be subjected to stricter authorization procedures relevant to veterinary products and not those for feed additives. This may require extensive resources to document the safety and efficacy assessment for dossier application. Subsequently, this may significantly increase the duration and cost of securing authorization for such antioxidant products. However, there seems to be a twisted distinction between the description of feed additives and those of veterinary products in Commission Regulation (EC) 1831/2003. This distinction is essentially based on the requirement for zootechnical additives to ‘affect favorably the performance in good health’ contrary to veterinary products used to treat specific disorders (Smedley, 2013). Although this distinction is not as fundamental as it may first appear, such intrinsic opportunities can be debated since antioxidants are not intended to diagnose or cure diseases as ascribed to veterinary products.

Implication on application of antioxidant feed additives

Though antioxidants are currently not market-authorized for their in vivo and postmortem efficacy claims, they have been traditionally used in livestock practices to reduce stress conditions in animals and for improving animal product quality. Most of these antioxidant products are currently marketed and used under the labels of ‘vitamin and mineral additives’ as well as ‘zootechnical additives’ in the case of plant extracts and herbs. However, some dietary antioxidants could act as prooxidants particularly when supplemented in excess dosage. Prooxidants can induce oxidative stress in biologic system by increasing the production of reactive free radicals or depleting the antioxidant defense system to cause cellular damage (Palozza, 1998). Thus, prooxidants could exert detrimental effects on animal health and product quality in contrast to antioxidants. There are available data that showed the potential prooxidant effect of vitamin E (Pearson et al., 2006; Ouchi et al., 2009), vitamin C (Podmore et al., 1998), carotenoids (Palozza, 1998), synthetic antioxidants (Kahl et al., 1990) and phenolic compounds (Fukumoto and Mazza, 2000; Simić et al., 2007). It is noteworthy that the in vivo prooxidant effect of carotenoids and vitamins C and E has triggered much criticisms (Bland, 1998; Young and Lowe, 2001; Hathcock et al., 2005) and their prooxidant activity is yet to be convincingly proven due to sparse and conflicting data. Moreover, antioxidant substances such as vitamins are currently market-authorized without maximum inclusion limit in the feed. Nonetheless, there is need for in-depth research to ascertain the in vivo relevance of high dosage of antioxidant substances with regard to their potential to exhibit prooxidant effect.

Future use of antioxidants in livestock production will be driven tremendously by the increasing trend for intensive livestock production which simultaneously will elevate the exposure of animals to oxidative stress conditions. Similarly, increasing consumers’ demand for high quality and functional animal foods would continue to escalate the demand for antioxidants in animal nutrition. The exclusive authorization of antioxidants based on their in vivo and postmortem benefits in livestock will stimulate the innovation of more potent antioxidant products and help to avoid indiscriminate use of antioxidants due to specification of claims and dosage on the product labels. In addition, it will better guide animal nutritionists in making dietary recommendations and enable livestock farmers to make better purchasing decisions that will improve their profitability. Appraisal of antioxidant registration system may also open potential new markets for feed antioxidants. However, legislative questions on antioxidant dosage and dose restriction need to be answered and may have to be demonstrated in the safety assessment of the dossier application.

Conclusion

With respect to the opinions presented in this review paper, it is anticipated that future feed additive legislations in the EU and possibly including other countries, would recognize the in vivo and postmortem efficacy of antioxidants. Appraisal of EU legislations on antioxidant registration require adequate data to address many of the knowledge gaps identified in this review. Thus, there is esteem need for additional investment in antioxidant-related research in production animals. Moreover, extensive future research should aim at developing more potent dietary antioxidant products as well as feeding strategies for effective delivery of antioxidant solutions to livestock. For these optimisms to be achieved, relevant stakeholders in the feed additive industry should dedicate concerted efforts to antioxidant research and lobby for the review of existing EU legislations guiding the authorization of antioxidants. Organizations such as the EU Feed Additives and Premixtures Association and the European Manufacturers of Feed Minerals Association (EMFEMA) could play major roles in this regard.