Redox balance

When oxidant activity exceeds the neutralizing capacity of antioxidants it can result in oxidative stress which in ruminants has been associated with several diseases, including conditions that are relevant for animal production and welfare (Celi, Reference Celi, Mandelker and Vajdovich2011). An imbalance between pro-oxidants and antioxidants lead to oxidative stress, therefore the maintenance of redox balance is crucial for optimal ruminant health (Chauhan et al., Reference Chauhan, Celi, Leury, Clarke and Dunshea2014). The main pro-oxidants (reactive oxygen species, reactive nitrogen species) and antioxidants (endogenous and dietary) involved in the maintenance of redox balance in ruminants have been reviewed extensively (Miller et al., Reference Miller, Brzezinska-Slebodzinska and Madsen1993; Lykkesfeldt and Svendsen, Reference Lykkesfeldt and Svendsen2007; Celi, Reference Celi, Mandelker and Vajdovich2011; Chauhan et al., Reference Chauhan, Celi, Leury, Clarke and Dunshea2014; Sordillo and Mavangira, Reference Sordillo and Mavangira2014).

Redox balance plays a key role in the modulation of metabolism, immunity, reproduction, health and welfare. In dairy cattle, redox balance can be disrupted at any time, however, the time around calving (transition period) and the first few weeks of life (neonatal period) are characterized by a higher risk of oxidative stress and incidence of diseases (Ranade et al., Reference Ranade, Talukder, Muscatello and Celi2014; Abuelo et al., Reference Abuelo, Hernández, Benedito and Castillo2019). During these periods the requirement for nutrients and micronutrients, including antioxidants, is considerably increased (Pedernera et al., Reference Pedernera, Celi, García, Salvin, Barchia and Fulkerson2010), therefore, supplementing diets with micronutrients and antioxidants can prove to be useful in maintaining redox balance (Abuelo et al., Reference Abuelo, Hernández, Benedito and Castillo2015; Surai et al., Reference Surai, Kochish, Fisinin and Juniper2019).

A relationship between dairy calf growth rates and antioxidant status has been reported (McGrath, Reference McGrath2016) and it has been proposed that dietary antioxidants can maintain redox balance in young ruminants, reducing the risk of disease and mortality while maintaining productive performance. In light of the observed accelerated growth of dairy calves and its association with increased lifetime productivity of dairy cows (Soberon and Van Amburgh, Reference Soberon and Van Amburgh2013), the use of dietary antioxidants seems to be a valid practical option to increase lifetime performances of dairy cattle (McGrath, Reference McGrath2016).

Redox balance is also implicated in the modulation of reproductive physiology in light of its involvement in the regulation of events such as oocyte maturation, steroidogenesis, regulation of follicular fluid environment, folliculogenesis, corpus luteal function, luteolysis and embryonic mortality (Talukder et al., Reference Talukder, Kerrisk, Gabai and Celi2017). The fact that antioxidant supplementation can improve reproductive outcomes in dairy cattle does not come as a surprise. While it is clear that maintaining redox balance in dairy cattle is crucial to maximize fertility (Talukder et al., Reference Talukder, Kerrisk, Gabai and Celi2017) and health (Abuelo et al., Reference Abuelo, Hernández, Benedito and Castillo2019), a better understanding of the factors involved in the control of redox balance will allow the verification of their success and effectiveness (Chauhan et al., Reference Chauhan, Liu, Leury, Cottrell, Celi and Dunshea2016). More importantly, as antioxidants are routinely supplemented in dairy diets (Lean et al., Reference Lean, VanSaun and DeGaris2013), the question is how much of which formulation(s) of antioxidants should be used to maintain redox balance. There is practical evidence in the literature suggesting that the supplementation of ruminant diets with supra-nutritional levels of antioxidants such as vitamin E and selenium can not only sustain redox balance but also maintain crucial physiological functions like thermoregulation, feed intake, respiratory physiology, rectal temperature, acid-base balance, inflammation and gastrointestinal functionality (Chauhan et al., Reference Chauhan, Celi, Leury, Clarke and Dunshea2014; Celi and Gabai, Reference Celi and Gabai2015).

Skeletal health and redox balance

Skeletal health is another of the many physiological function regulated by redox balance as suggested by epidemiological evidence linking dietary antioxidant intake and bone health (Rao and Rao, Reference Rao, Rao and Flores2013). Redox balance seems to play a role in bone remodeling (Domazetovic et al., Reference Domazetovic, Marcucci, Iantomasi, Brandi and Vincenzini2017). A disruption in redox balance results in decreased bone formation as the result of reduced differentiation and survival of osteoblasts, and activation of osteoclasts resulting in bone reabsorption (Rao and Rao, Reference Rao, Rao and Flores2013). Moreover, antioxidant deficiency has been associated with reduction in bone formation ultimately resulting in osteoporosis (Domazetovic et al., Reference Domazetovic, Marcucci, Iantomasi, Brandi and Vincenzini2017). Antioxidant enzymes such as glutathione peroxidase and catalase are considered markers of antioxidants defence mechanisms against bone resorption and osteoporosis (Rao and Rao, Reference Rao, Rao and Flores2013). Recently, many dietary antioxidants have been reported to restore redox balance and skeletal health (Rao and Rao, Reference Rao, Rao and Flores2013). For example, antioxidants such as vitamin C contribute to the maintenance of skeletal health by suppressing osteoclast activity and promoting osteoblast differentiation. Although vitamin C can be synthesized in tissues by ruminants, it is important to emphasize that vitamin C is an important cofactor for collagen formation and synthesis of hydroxyproline and hydroxylysine, indicating that vitamin C interacts with other nutritional factors such as vitamin E, vitamin D and calcium in the control of skeletal health (Sahni et al., Reference Sahni, Mangano, McLean, Hannan and Kiel2015). Finally, dietary carotenoids have also been reported to play an important role for improving bone health (Sahni et al., Reference Sahni, Mangano, McLean, Hannan and Kiel2015).

Extraskeletal effects of vitamin D

The mechanisms of disrupted skeletal health in dairy cows impairing their ability to maintain effective bone and mineral homeostasis have been recently reviewed (McGrath et al., Reference McGrath, Savage and Godwin2015). The role of vitamin D and its metabolites in maintaining skeletal health and mineral homeostasis are very well established. However, the actions of vitamin D are not confined to the skeleton as vitamin D receptors (VDR) and vitamin D hydroxylases CYP24A1 and CYP27B1 are nearly ubiquitous, advocating for a variety of extraskeletal actions of the vitamin D endocrine system (Bikle, Reference Bikle2016; Bouillon et al., Reference Bouillon, Marcocci, Carmeliet, Bikle, White, Dawson-Hughes, Lips, Munns, Lazaretti-Castro, Giustina and Bilezikian2019). The potential for the bone to now be considered an endocrine organ firmly puts skeletal health of the modern day dairy cow at the forefront of science (McNeill and Anderson, Reference McNeill and Anderson2012; Lean et al., Reference Lean, DeGaris, Celi, McNeill, Rodney and Fraser2014).

In both swine and poultry, the use of vitamin D₃ and 25-OH-D₃ (25-hydroxycholecalciferol) has been shown to improve bone strength, calcification, immunity and muscle content, often resulting in less morbidity and mortality as well as greater productivity (Chou et al., Reference Chou, Chung and Yu2009; Sugiyama et al., Reference Sugiyama, Kusuhara, Chung, Yonekura, Azem and Hayakawa2013). The next frontier of bone health research in dairy cows should be the characterization of the extraskeletal effects, such as the effects on metabolism, reproduction, muscle biology, immunity, and gastrointestinal functionality.

There is substantial evidence from published and new data that supports the hypothesis that in dairy cattle the skeleton has a pivotal role during the homeorhetic adaptation to lactation and that this relationship may be influenced by nutrition. Indeed, recent studies have demonstrated a link between 25-OH-D₃ and energy metabolism in dairy cows (Lean et al., Reference Lean, DeGaris, Celi, McNeill, Rodney and Fraser2014; Rodney et al., Reference Rodney, Martinez, Celi, Block, Thomson, Wijffels, Fraser, Santos and Lean2018). In dairy cows, a dramatic increase in energy and nutrient requirements can be observed during the transition period, exposing them to negative energy and nutrient balance. The metabolic adaptations to negative energy balance require interactions of metabolic fuels and its failure may occur in various tissues resulting in several metabolic diseases (Drackley, Reference Drackley1999). Despite the transition period being the most studied period of the productive life of a dairy cow, the long-term physiological implications on responses like mammary gland function and fertility require a thorough assessment. Essential physiological function such as calcium homeostasis, lipid metabolism, insulin secretion, tissue sensitivity to insulin and the integration of these processes with other functions like redox balance are not fully elucidated yet. It has been observed that nutritional interventions administered before calving can increase milk yield, improve fertility and reduce the risk of metabolic diseases (Lean et al., Reference Lean, DeGaris, Celi, McNeill, Rodney and Fraser2014). It is also worth noting that correlations between energy and bone metabolism have been observed in dairy cows providing evidence to support a homeorhetic role for calcium metabolism in dairy cattle (Lean et al., Reference Lean, DeGaris, Celi, McNeill, Rodney and Fraser2014; Martinez et al., Reference Martinez, Rodney, Block, Hernandez, Nelson, Lean and Santos2018b; Rodney et al., Reference Rodney, Celi, McGrath, Golder, Anderson, McNeill, Fraser and Lean2019). However, it is worth mentioning that vitamin D supplementation around the time of calving has not always succeeded in preventing hypocalcemia (Weiss et al., Reference Weiss, Azem, Steinberg and Reinhardt2015). Considering the complex mechanisms involved in the control of calcium homeostasis in dairy cattle (DeGaris and Lean, Reference DeGaris and Lean2008), further studies are required to evaluate the effectiveness of vitamin D supplementation to reduce hypocalcemia during early lactation. Although it has been reported that vitamin D has a high margin of safety when used at recommended levels (Celi et al., Reference Celi, Williams, Engstrom, McGrath and La Marta2018), excessive intakes of vitamin D can result in intoxication in ruminants and future studies need to evaluate the safety of vitamin D supplementation for long periods of time.

Considering that VDRs are expressed in numerous tissues of the reproductive tract (Dokoh et al., Reference Dokoh, Donaldson, Marion, Pike and Haussler1983; Stumpf et al., Reference Stumpf, Sar and O'Brien1987), reproductive physiology should also be considered in regard to skeletal health. It has been reported that vitamin D has a positive effect on reproductive physiology in cattle (Ward et al., Reference Ward, Marion, Campbell and Dunham1971; Panda et al., Reference Panda, Miao, Tremblay, Sirois, Farookhi, Hendy and Goltzman2001; Kemmis et al., Reference Kemmis, Salvador, Smith and Welsh2006). Indeed, it has been observed that dairy cattle reproductive performance improved after vitamin D supplementation (Ward et al., Reference Ward, Marion, Campbell and Dunham1971), and that calcitriol blood concentration is elevated during pregnancy (O'Brien et al., Reference O'Brien, Li, Cao, Kent, Young, Queenan, Pressman and Cooper2014). A recent study in transition dairy cows has reported that dietary 25-OH-D₃ tended to improve pregnancy rate and reduced the days to pregnancy during the 305-d lactation (Martinez et al., Reference Martinez, Rodney, Block, Hernandez, Nelson, Lean and Santos2018a). The authors argue that metabolites of vitamin D such as 25-OH-D₃ might have directly stimulated the VDRs in reproductive tissues (Lou et al., Reference Lou, Molnár, Peräkylä, Qiao, Kalueff, St-Arnaud, Carlberg and Tuohimaa2010), which might have resulted in positive effects on fertility. Another possibility is that the positive effect on reproductive function might have been indirect as cows fed 25-OH-D₃ presented improved neutrophil function and reduced incidence of inflammatory diseases such as metritis, which are known to decrease fertility (Ribeiro et al., Reference Ribeiro, Gomes, Greco, Cerri, Vieira-Neto, Monteiro, Lima, Bisinotto, Thatcher and Santos2016). Therefore, the observed improved pregnancy rate might have been the consequence of the better health status induced by dietary 25-OH-D₃ (Martinez et al., Reference Martinez, Rodney, Block, Hernandez, Nelson, Lean and Santos2018a).

Considering that VDR and 1-α-hydroxylase are expressed in muscle fibers and myoblasts, it is not surprising that vitamin D plays a direct regulatory role in muscle physiology. Indeed, vitamin D is involved in the regulation of myogenesis, cell proliferation, differentiation, regulation of protein synthesis and mitochondrial metabolism (Montenegro et al., Reference Montenegro, Cruzat, Carlessi and Newsholme2019). Lack of VDR disrupts muscle growth and development, as observed in VDR knockout mice which have smaller muscle fibers and aberrant myogenic regulatory factor expression (Dzik and Kaczor, Reference Dzik and Kaczor2019). Moreover, as the inflammatory processes reduce muscle protein accretion, decreasing the impact of the inflammatory response is a key strategy to sustain production performances. Considering that dairy beef cross cattle production is becoming a financially attractive opportunity for the dairy industry as it allows producers to produce calves that will yield better carcasses than purebred dairy breeds and thus attract higher prices, dietary supplementation of dairy cattle's diet with vitamin D, in light of its the well-known anti-inflammatory effects, represents a great opportunity for the dairy industry to increase profitability and sustainability. Indeed, dairy beef production generates around 33% of the greenhouse gases equivalents per unit weight of meat compared to traditional beef cattle production and thus 25-OH-D₃ supplementation can increase meat yield and lower the carbon footprint of the dairy industry at the same time (Britt et al., Reference Britt, Cushman, Dechow, Dobson, Humblot, Hutjens, Jones, Ruegg, Sheldon and Stevenson2018).

It is well known that both the innate and acquired immune responses are modulated by ligand dependent VDR functions. Indeed, VDR and vitamin D metabolic enzymes can be found in all cells of the innate and adaptive arms of the immune system (Bouillon et al., Reference Bouillon, Marcocci, Carmeliet, Bikle, White, Dawson-Hughes, Lips, Munns, Lazaretti-Castro, Giustina and Bilezikian2019). In cattle, calcitriol augments the production of nitric oxide and β-defensin antimicrobial peptides (Merriman et al., Reference Merriman, Kweh, Powell, Lippolis and Nelson2015), molecules that are toxic to bacteria, indicating a potential for targeted enhancement of defence against bacterial infections via the vitamin D pathway (Nelson et al., Reference Nelson, Reinhardt, Lippolis, Sacco and Nonnecke2012). Macrophages are the main sources of the calcitriol that controls vitamin D-mediated immune responses. In bovine macrophages CYP27B1 is stimulated via toll-like receptor recognition of pathogen associated molecular patterns such as lipopolysaccharide, peptidoglycan, and mycobacterial lipopeptides. In macrophages, CYP27B1 facilitates the conversion of calcidiol to calcitriol, activating vitamin D-mediated immune responses (Nelson et al., Reference Nelson, Reinhardt, Thacker, Beitz and Lippolis2010). Interestingly, CYP27B1 is expressed in the udder during mastitis in dairy cattle (Nelson et al., Reference Nelson, Reinhardt, Thacker, Beitz and Lippolis2010). Although vitamin D treatments does not seem to prevent or cure mastitis, it reduced its negative impact on the cow (Lippolis et al., Reference Lippolis, Reinhardt, Sacco, Nonnecke and Nelson2011). It has been reported that dietary 25-OH-D₃ decreased the incidence of retained placenta and metritis and the percentage of cows with multiple diseases during the first 30 d in milk, which are likely related to the improved measures of immune function evaluated in neutrophils (Martinez et al., Reference Martinez, Rodney, Block, Hernandez, Nelson, Lean and Santos2018a). While it is clear that vitamin D can modulate the immune system in dairy cattle, we still need clarity of understanding of the immunomodulatory effects of dietary 25-OH-D₃ as this will allow the development of nutritional strategies to increase the resilience of dairy cattle to bacterial infection. These strategies have the potential to reduce the use of antibiotics in the dairy industry and thus might contribute to the decrease in antimicrobial resistance.

VDR and vitamin D metabolic enzymes have been localized in virtually all cells of both the innate and adaptive immune system. Moreover, there is now consensus that cells of the immune system produce 1,25(OH)2D₃ locally and that expression of CYP27B1 in the immune system is regulated independently of that in the endocrine system that controls calcium homeostasis (Nelson et al., Reference Nelson, Reinhardt, Lippolis, Sacco and Nonnecke2012). For example, there is evidence that in activated immune cells VDR controls several immune responses (Hewison, Reference Hewison and Gerald2011). It has been observed that in cattle, 1,25(OH)2D₃ increases the production of antimicrobial peptides such as nitric oxide and β-defensin, (Nelson et al., Reference Nelson, Reinhardt, Lippolis, Sacco and Nonnecke2012). Considering that vitamin D metabolism might be quite different in each system (skeletal, immune, digestive, etc.), it is reasonable to argue that the vitamin D requirements for each of these systems may also differ (Nelson et al., Reference Nelson, Lippolis, Reinhardt, Sacco, Powell, Drewnoski, O'Neil, Beitz and Weiss2016). Nevertheless, the optimal 25(OH)D₃ concentration for the dairy cattle immune system has not been established thus far. In vitro studies investigating the effect of different 25(OH)D₃ concentrations on macrophage host defence responses suggest that 100 ng/ml should be an effective level (Nelson et al., Reference Nelson, Reinhardt, Thacker, Beitz and Lippolis2010). However, despite the association between vitamin D deficiency and risk of respiratory infections, calves with a serum 25(OH)D₃ concentration of about 175 ng/ml and experimentally challenged with respiratory syncytial virus (RSV), did not perform better than calves with serum 25(OH)D₃ concentration of about 30 ng/ml in regard to severity the infection (Sacco et al., Reference Sacco, Nonnecke, Palmer, Waters, Lippolis and Reinhardt2012). As reported above, however, there is evidence for dietary 25(OH)D₃ to reduce the incidence of some peripartum diseases in dairy cattle (Martinez et al., Reference Martinez, Rodney, Block, Hernandez, Nelson, Lean and Santos2018a); this apparent discrepancy needs to be addressed with further work investigating the relationship between serum 25(OH)D₃ and infectious disease outcome in dairy cattle.

Vitamin D deficiency has been linked to disrupted gastrointestinal functionality (Christakos, Reference Christakos2012; De Santis et al., Reference De Santis, Cavalcanti, Mastronardi, Jirillo and Chieppa2015), and although vitamin deficiencies do not seem to occur in modern dairy production systems, it should be noted that vitamin requirements of the modern dairy cow may be higher than those currently recommended by the NRC. The current understanding of gastrointestinal functionality encompasses not only the gastro-intestinal (GI) microbiota including pathogens causing diseases, mortality and morbidity in dairy cattle, but also other key components such as diet, effective structure of the gastrointestinal barrier, host interaction with the GI microbiota, effective digestion and absorption of feed and effective maturation and development of innate and acquired immune functions (Celi et al., Reference Celi, Cowieson, Fru-Nji, Steinert, Kluenter and Verlhac2017). This novel definition of gut health focuses on the functionality of the whole GI system and on the complex interactions between all its components mentioned above. With this definition in mind, it is not surprising that skeletal health is also influenced by the GI microbiota and vice versa. Indeed, it has been reported that vitamin D influences the composition of the microbiota (Sun, Reference Sun2018; Waterhouse et al., Reference Waterhouse, Hope, Krause, Morrison, Protani, Zakrzewski and Neale2019), and that the gut microbiota regulates endocrine vitamin D metabolism (Bora et al., Reference Bora, Kennett, Smith, Patterson and Cantorna2018). Vitamin D regulates the expression of tight junction proteins in the intestinal epithelial cells, thereby maintaining optimal intestinal barrier function (De Santis et al., Reference De Santis, Cavalcanti, Mastronardi, Jirillo and Chieppa2015). In the intestinal mucosa, vitamin D, together with vitamin A, supports innate lymphoid cells that produce IL-22, suppressed IFN-γ and IL-17 by T cells, and induces regulatory T cells (Cantorna et al., Reference Cantorna, Snyder and Arora2019). Vitamin D seems to shape the gut microbiota by modulating the intestinal epithelium and mucosal immune system and thus maintaining intestinal homeostasis (Cantorna et al., Reference Cantorna, Snyder and Arora2019). On the other end, the intestinal microbiota may regulate bone metabolism by influencing the relative activities of osteoclasts and osteoblasts through effects on the immune system and host metabolic pathways, as well as through the production of metabolites (Charles et al., Reference Charles, Ermann and Aliprantis2015). It has been proposed that during eubiosis (balance between the gut microbiome and their host), there is a balance between the anti-osteoclastogenic and pro-osteoclastogenic pathways, however, during dysbiosis (disturbance in the balance between the gut microbiome and the host), the gut microbiome may promote osteoclast-mediated bone loss as a result of the increase in inflammatory cytokines which recruit more osteoclast precursors, promote osteoclast activity and reduce anti-osteoclast T cells (Charles et al., Reference Charles, Ermann and Aliprantis2015). Several cytokines are involved in skeletal health and proinflammatory cytokines are believed to have osteoclastogenic effects (Jonsson et al., Reference Jonsson, Fortes, Piper, Vankan, de Cisneros and Wittek2013). For example, tumor necrosis factor α (TNF-α) and IL-1 have been shown to modulate the expression of receptor activator of nuclear factor κ-B (RANK), its ligand (RANKL), and osteoprotegerin (Jonsson et al., Reference Jonsson, Fortes, Piper, Vankan, de Cisneros and Wittek2013). Osteoclastogenesis is the differentiation of osteoclast into multinucleated cells which leads to bone degradation and calcium mobilization. Osteoclastogenesis is initiated by the binding of RANKL to its receptor on osteoclasts. Osteoprotegerin on the other hand, inhibits osteoclastogenesis by acting as a decoy receptor for RANKL (Hatate et al., Reference Hatate, Kawashima, Kayano, Hanada and Yamagishi2020). Changes in cytokine production in tissues and blood seems also to modulate calcium homeostasis during the parturient period, with potential implications for the incidence of peripartum hypocalcemia (Gray et al., Reference Gray, St George and Jonsson2007). While specific studies in this area are lacking in dairy cattle, the literature clearly highlights the biological links between redox balance, skeletal health and gastrointestinal functionality.

In conclusion, future dairy production and its sustainability depends on the development a larger understanding and practical application of concepts related to gastrointestinal functionality that imply complete holistic management of the production system. Nutritional strategies can reduce the incidence of health and welfare issues, but they need to be strategically integrated with management practices and breeding programs. The success of these strategies, however, is often hindered by the complexity of the interactions between cows and their environment. In dairy cattle, effective gastrointestinal functionality is vital in determining health, welfare and productive performance. Optimization of gastrointestinal functionality is crucial to increase nutrient digestibility and thus maximizing value from feed, to sustain host physiological functions such as innate and adaptive immunity and thus increasing resilience to environmental challenges, and finally, to maintain eubiotic conditions. The characterization of the interactions between the antioxidant system, vitamin D metabolism and the rumen microbiome will allow the development of strategic nutritional interventions aimed at improving skeletal health, redox balance and gastrointestinal functionality that would enable achieving optimal lifetime performance.