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, Reference Shahidi and Zhong2005). 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., Reference Castillo, Pereira, Abuelo and Hernández2013). 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, Reference Lindqvist2012).

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., Reference Topliss, Clark, Ernst, Hufford, Johnston, Rimoldi and Weimann2002). 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, Reference Shahidi2000). 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, Reference Weiser and Vecchi1982). This could be explained by the better bioavailability of RRR-α-tocopherol compared with all-rac-α-tocopherol (Traber et al., Reference Traber, Elsner and Brigelius-Flohé1998). The eight stereoisomers could also vary in their biopotencies with the RRR form being the most active (Weiser and Vecchi, Reference Weiser and Vecchi1982). The bioavailability of these stereoisomers has been further demonstrated to vary in fluids and tissues of different animal species (Jensen and Lauridsen, Reference Jensen and Lauridsen2003). In addition, there are indications that vitamin E is more effective in vivo while vitamin C acts effectively postmortem (Morrissey et al., Reference Morrissey, Sheehy, Galvin, Kerry and Buckley1998; Bou et al., Reference Bou, Guardiola, Grau, Grimpa, Manich, Barroeta and Codony2001).

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., Reference Augustyniak, Bartosz, Cipak, Duburs, Horáková, Luczaj, Majekova, Odysseos, Rackova, Skrzydlewska, Stefek, Strosova, Tirzitis, Venskutonis, Viskupicova, Vraka and Zarkovic2010). 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., Reference Lee, Khor, Shu, Su, Fuentes and Kong2013). 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., Reference Bohn, McDougall, Alegría, Alminger, Arrigoni, Aura, Brito, Cilla, El, Karakaya, Martínez-Cuesta and Santos2015). 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., Reference Augustyniak, Bartosz, Cipak, Duburs, Horáková, Luczaj, Majekova, Odysseos, Rackova, Skrzydlewska, Stefek, Strosova, Tirzitis, Venskutonis, Viskupicova, Vraka and Zarkovic2010). 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., Reference Manach, Scalbert, Morand, Rémésy and Jiménez2004).

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, Reference Shahidi and Zhong2005). 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., Reference Crane, Griffin and Messent2000).

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., Reference Augustyniak, Bartosz, Cipak, Duburs, Horáková, Luczaj, Majekova, Odysseos, Rackova, Skrzydlewska, Stefek, Strosova, Tirzitis, Venskutonis, Viskupicova, Vraka and Zarkovic2010; Błaszczyk et al., Reference Błaszczyk, Augustyniak and Skolimowski2013). 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., Reference Lundebye, Hove, Måge, Bohne and Hamre2010). 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., Reference Bohne, Lundebye and Hamre2008). In contrast, the amount of EQ in foods of animal origin was less than the EU maximum residue level (Aoki et al., Reference Aoki, Kotani, Miyazawa, Uchida, Igarashi, Hirayama, Hakamata and Kusu2010). 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., Reference Błaszczyk, Augustyniak and Skolimowski2013). 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.

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, Reference Fellenberg and Speisky2006; Lykkesfeldt and Svendsen, Reference Lykkesfeldt and Svendsen2007; Surai, Reference Surai2007). 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., Reference Kalam, Singh, Mani, Patel, Khan and Pandey2012). 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., Reference Morrissey, Sheehy, Galvin, Kerry and Buckley1998) result in the onset of lipid deterioration in muscle tissues and consequent oxidative rancidity in meat products (Iglesias et al., Reference Iglesias, Pazos, Andersen, Skibsted and Medina2008).

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, Reference Fellenberg and Speisky2006). 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, Reference Halliwell and Gutteridge1999). 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., Reference Kalam, Singh, Mani, Patel, Khan and Pandey2012).

Table 1 Antioxidants in the biological system of animals (adapted from Weiss, Reference Weiss2010)

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., Reference Yeum, Russell, Krinsky and Aldini2004). 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, Reference Buettner1993). Similarly, Iglesias et al. (Reference Iglesias, Pazos, Andersen, Skibsted and Medina2008) 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, Reference Lykkesfeldt and Svendsen2007), ruminants (Miller et al., 1993Reference Miller, Brzezinska-Slebodzinska and Madsena; Hansen, Reference Hansen2010; Celi, Reference Celi and Chauhan2011) and poultry (Surai, Reference Surai2002; Fellenberg and Speisky, Reference Fellenberg and Speisky2006; Surai, Reference Surai2007).

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., Reference Salami, Majoka, Saha, Garber and Gabarrou2015). 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, Reference Fellenberg and Speisky2006; Salami et al., Reference Salami, Majoka, Saha, Garber and Gabarrou2015). 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, Reference Waldroup2001)

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., Reference Maini, Rastogi, Korde, Madan and Shukla2007). 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. (Reference Taulescu, Mihaiu, Bele, Matea, Dan, Mihaiu and Lapusan2011) 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, Reference Yardibi and Turkay2008; Jena et al., Reference Jena, Panda, Patra, Mishra, Behura and Panigrahi2013). 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., Reference Vossen, Ntawubizi, Raes, Smet, Huyghebaert, Arnouts and De Smet2011). 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., Reference Tavarez, Boler, Bess, Zhao, Yan, Dilger, Mckeith and Killefer2010).

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., Reference Brenes, Viveros, Chamorro and Arija2015). 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., Reference Juin, Decousser and Chicoteau2007). Brenes et al. (Reference Brenes, Viveros, Goni, Centeno, Sáyago-Ayerdy, Arija and Saura-Calixto2008) 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., Reference Loetscher, Kreuzer, Albiker, Stephan and Messikommer2014). The negative feeding value of grape by-products has also been demonstrated. Supplementation of high concentrations of grape seed extract (Chamorro et al., Reference Chauhan, Celi, Leury, Clarke and Dunshea2013) and GP (Goni et al., Reference Goni, Brenes, Centeno, Viveros, Saura-Calixto, Rebolé, Arija and Estevez2007) 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. (Reference Zhu, Zhao, Chen and Xu2012) 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. (Reference Deng, Xu, Yu, He, Zhang, Ding and Chen2010). In addition, there was an increase in serum and liver α-tocopherol levels in pigs supplemented with vitamin E (Ching et al., Reference Ching, Mahan, Wiseman and Fastinger2002). 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. (Reference Fernández-Dueñas, Mariscal, Ramírez and Cuarón2008) 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, Reference Fernández-Dueñas2009).

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., Reference Sharma, Singh, Singh, Pandey and Verma2011). The period is characterized by high metabolic demand and physiological adjustments to the onset of lactation. Abuelo et al. (Reference Abuelo, Hernández, Benedito and Castillo2014) 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., Reference Brzezinska-Slebodzinska, Miller, Quigley, Moore and Madsen1994). 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., Reference Brzezinska-Slebodzinska, Miller, Quigley, Moore and Madsen1994; Bouwstra et al., Reference Bouwstra, Goselink, Dobbelaar, Nielen, Newbold and Van Werven2008 and Reference Bouwstra, Nielen and Van Werven2009). 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., Reference Zeiler, Sauter-Louis, Ruddat, Martin, Mansfeld, Knubben-Schweizer and Zerbe2010). 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., Reference Zeiler, Sauter-Louis, Ruddat, Martin, Mansfeld, Knubben-Schweizer and Zerbe2010).

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., Reference Mudron, Herzog, Höltershinken and Rehage2007). Qu et al. (Reference Qu, Lytle, Traber and Bobe2013) 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 (Reference Chamorro, Viveros, Centeno, Romero, Arija and Brenes2004) 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., Reference Calamari, Petrera, Abeni and Bertin2011). In addition, dietary supplementation of vitamin E improved the semen quality of Aohan fine-wool sheep (Yue et al., Reference Yue, Yan, Luo, Xu and Jin2010) while contrasting failure of fertility improvement was observed when vitamin E and selenium were administered to lactating cows by injection (Paula-Lopes et al., Reference Paula-Lopes, Al-Katanani, Majewski, McDowell and Hansen2003). This difference in observation may be partly attributed to the route of administration, which was suggested by Hansen (Reference Hansen2010) 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, Reference Weiss2010). 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, Reference Weiss2010)

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., Reference Chawla and Kaur2014; Alhidary et al., Reference Alhidary, Shini, Al Jassim, Abudabos and Gaughan2015). 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., Reference Rossi, Compiani, Baldi, Bernardi, Muraro, Marden and Dell’Orto2015).

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., Reference Lettat, Nozière, Silberberg, Morgavi, Berger and Martin2012). 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., Reference Benavides, Martínez-Valladares, Tejido, Giráldez, Bodas, Prieto, Pérez and Andrés2013). Moreover, dietary supplementation of either vitamin E or carsonic acids in concentrate diet corrected the metabolic acidosis in fattening lambs (Morán et al., Reference Morán, Giráldez, Bodas, Benavides, Prieto and Andrés2013).

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, Reference Valenzuela and Nieto1996), especially those high in PUFA such as meats (Morrissey et al., Reference Morrissey, Brandon, Buckley, Sheehy and Frigg1997). Consumption of lipid oxidation products that result from oxidative rancidity of animal products can be very toxic to human health (Esterbauer, Reference Esterbauer1993). 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, Reference Gray and Pearson1987) 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., Reference Liu, Lanari and Schaefer1995).

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., Reference Liu, Lanari and Schaefer1995; Wood and Enser, Reference Wood and Enser1997). 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., Reference Marcinčák, Popelka, Bystrický, Hussein and Hudecová2005). Similarly, dietary α-tocopherol supplementation reduced the TBARS content, lipid oxidation, rancid odor and flavor in raw, cooked and stored meat (Maraschiello et al., Reference Maraschiello, Sarraga and Garcia Regueiro1999; Grau et al., Reference Grau, Guardiola, Grimpa, Barroeta and Codony2001; Ruiz et al., Reference Ruiz, Guerrero, Arnau, Guardia and Esteve-Garcia2001; Tavarez et al., Reference Tavarez, Boler, Bess, Zhao, Yan, Dilger, Mckeith and Killefer2010; Taulescu et al., Reference Taulescu, Mihaiu, Bele, Matea, Dan, Mihaiu and Lapusan2011). Taulescu et al. (Reference Taulescu, Mihaiu, Bele, Matea, Dan, Mihaiu and Lapusan2011) 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. (Reference Mercier, Gatellier, Vincent and Renerre2000) 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, Reference Koreleski and Świątkiewicz2007). 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, Reference Fernández-Dueñas2009). However, Haak et al. (Reference Haak, Raes, Van Dyck and De Smet2008) 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. (Reference Realini, Duckett, Brito, Dalla Rizza and De Mattos2004) 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. (Reference Liu, Lanari and Schaefer1995) 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., Reference Rossi, Compiani, Baldi, Bernardi, Muraro, Marden and Dell’Orto2015).

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, Reference Baldi and Pinotti2008). 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., Reference Castillo, Pereira, Abuelo and Hernández2013). 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 (Reference Politis2012) 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., Reference Baldi, Savoini, Pinotti, Monfardini, Cheli and Orto2000; Politis et al., Reference Politis, Bizelis, Tsiaras and Baldi2004; Machado et al., Reference Machado, Bicalho, Pereira, Caixeta, Knauer, Oikonomou, Gilbert and Bicalho2013). However, antioxidant effect to reduce the SSC in milk has been inconsistent across studies (Sivertsen et al., Reference Sivertsen, Overnes, Osteras, Nymoen and Lunder2005; Waller et al., Reference Waller, Sandgren, Emanuelson and Jensen2007). Nonetheless, the meta-analysis by Zeiler et al. (Reference Zeiler, Sauter-Louis, Ruddat, Martin, Mansfeld, Knubben-Schweizer and Zerbe2010) 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., Reference Politis, Bizelis, Tsiaras and Baldi2004).

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., Reference Gabryszuk, Strzałkowska and Krzyżewski2007). 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., Reference Warly, Nurdin and Rusmana2011). In contrast, there was no effect of vitamin E supplementation on milk composition of lactose, fat and protein (Baldi et al., Reference Baldi, Savoini, Pinotti, Monfardini, Cheli and Orto2000; Politis et al., Reference Politis, Bizelis, Tsiaras and Baldi2004).

Egg

Food enriched in n-3 fatty acids have been widely acknowledged for their health, growth and development benefits in humans (Simopoulos, Reference Simopoulos1991). 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., Reference Meluzzi, Sirri, Manfreda, Tallarico and Franchini2000). However, the role of n-3 fatty acids was positively correlated to lipid peroxidation of tissues (Husvéth et al., Reference Husvéth, Manilla, Gaal, Vajdovich, Balogh, Wagner, Loth and Németh2000), which implies that animal products obtained from such feeding strategies will be more susceptible to rancid spoilage. Meluzzi et al. (Reference Meluzzi, Sirri, Manfreda, Tallarico and Franchini2000) 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., Reference Galobart, Barroeta, Baucells, Codony and Ternes2001). 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., Reference Pappas, Acamovic, Sparks, Surai and McDevitt2005 and Reference Pappas, Acamovic, Sparks, Surai and McDevitt2006).

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).

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., Reference Sadique, Al-Rqobah, Bughaith and El-Gindy1989). 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. (Reference Miller, Rice-Evans, Davies, Gopinathan and Milner1993b) can be used, as well as the ferric reducing ability of plasma (FRAP) value measurement methods described by Benzie and Strain (Reference Benzie and Strain1996). 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 (Reference Aebi1984), GSH-Px activities by the method of Beutler (Reference Beutler1975) and SOD activities by the method of Arthur and Boyne (Reference Arthur and Boyne1985). 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, Reference Palmieri and Sblendorio2007).

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 (Reference Catignani1986). Selenium concentrations in blood and tissues can be measured using the fluorometric method by Rodriguez et al. (Reference Rodriguez, Sanz and Romero1994). The serum uric acid can, however, be determined using a chromogenic system described by Fossati et al. (Reference Fossati, Principe and Berti1980).

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 (Reference Guntherberg and Rost1966), and Griffith (Reference Griffiths1980) 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., Reference Botsoglou, Fletouris, Papageorgiou, Vassilopoulus, Mantis and Trakatellis1994; Jo and Ahn, Reference Jo and Ahn1998; Young et al., Reference Young, Stagsted, Jensen, Karlsson and Henckel2003). Tissue MDA level and plasma MDA based on coupling with TBARS can be assayed according to the methods of Ohkawa et al. (Reference Ohkawa, Ohishi and Yagi1979) and Yagi (Reference Yagi1984), 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., Reference Han, Loukianoff and McLaughlin2000). 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, Reference Roberts and Morrow1994).

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, Reference Griffith2000; Han et al., Reference Han, Loukianoff and McLaughlin2000). 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., Reference Balasubramanian, Du and Zigler1990; Stadtman and Oliver, Reference Stadtman and Oliver1991; Stadtman and Berlett, Reference Stadtman and Berlett1997).

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, Reference Wang and Edens1994; Polla, Reference Polla1998; Goldbaum and Richter-Landsberg, Reference Goldbaum and Richter-Landsberg2001). 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 (Reference Goldbaum and Richter-Landsberg2001). 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. (Reference Yoshiki, Huan, Lida, Kawane, Okubo, Ishizawa and Kawabata1998) 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., Reference Li, Stansbury, Zhu and Trush1999; Barbacanne et al., Reference Barbacanne, Souchard, Darblade, Ilious, Nepveu, Pipy and Arnal2000). The exclusive advantage of this method is its high specificity to interact with the superoxide anion (Allen, Reference Allen1986).

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., Reference Del Rio, Serafini and Pellegrini2002; Collins, Reference Collins2005; Palmieri and Sblendorio, Reference Palmieri and Sblendorio2007). 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., Reference Del Rio, Serafini and Pellegrini2002). 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.