1. Introduction
Lignans are polyphenolic, phytoestrogenic compounds known to display a wide range of biological functions, including weak estrogenic and cardioprotective activities, as well as antiestrogenic, antioxidant, anti-inflammatory, and anticarcinogenic properties [
1,
2,
3]. The weak and antiestrogenic effects of lignans are caused by distinct transactivation activities of estrogen receptors between the enterolignans enterodiol (ED) and enterolactone (EL) [
4]. There is a growing interest in promoting the consumption of lignan-rich foods because of the potential benefits to human health. The outer fibrous-containing layers of flaxseed (
Linum usitatissimum L.) is the richest source of the lignan secoisolariciresinol diglucoside (SDG) [
5], which accounts for over 95% of the total lignans found in flax [
6]. In ruminants, the rumen appears to be the main site for conversion of SDG into the mammalian lignans ED and EL [
7,
8,
9,
10]. However, only EL was detected in milk of dairy cows fed flaxseed meal (FM) [
11] possibly because of ruminal dehydrogenation reactions converting ED to EL like those occurring in humans [
12]. This suggests that EL-enriched milk can be used as a source of lignans for humans due to the following reasons: (1) milk is consumed by a large part of the world population despite regional differences in per capita consumption [
13], (2) global consumption of milk is projected to increase by 60% between 2005/2007 and 2050, particularly in regions where the population traditionally consumes less milk such as East and North Africa, sub-Saharan Africa, and South and East Asia [
14], and (3) a poor and variable consumption of plant lignans worldwide [
15].
Hulls, meal, and whole seeds are flaxseed products that have been used as sources of the lignan SDG to improve the concentration of EL in milk of dairy cows [
11,
16,
17,
18,
19]. It is important to note that other ingredients (e.g., forages, cereal grains, protein supplements) used in diets of dairy cows also provide lignans. Therefore, comparison of milk EL concentrations across experiments should consider the contribution of lignans from non-flaxseed feedstuffs. Diets containing sources of nonstructural carbohydrates (NSC) with different ruminal degradability (e.g., ground corn vs. liquid molasses) also have been shown to affect the EL concentration in milk of dairy cows fed FM [
18]. Despite the growing knowledge regarding the impact of flaxseed supplementation on milk EL concentration in the last 10 years, little is known about how dietary manipulation affects the ruminal microbiome and EL production in dairy cows. Research in this area is needed to unravel dietary strategies suitable to modulate the concentration of EL in dairy cows’ milk.
In addition to human health benefits, flaxseed lignans can be also used as natural antioxidants to improve animal health via upregulation of antioxidant enzymes. Newborn dairy calves and periparturient dairy cows are prone to oxidative stress and immune depression [
20,
21]. Previous research revealed that the antioxidant activity of plant enterolignans is stronger than that of vitamin E [
22]. Furthermore, weanling albino rats receiving 10% flaxseed (1.5 g/kg of body weight) during 14 d followed by a challenge with a toxin (i.e., carbon tetrachloride) known to downregulate the hepatic expression of antioxidant enzymes were able to restore the activities of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) by 95, 182, and 136%, respectively, compared with the control treatment [
23]. Altogether, these results are encouraging and open new opportunities to explore the use of flaxseed products or flaxseed-derived lignans as bioactive sources to mitigate oxidative stress in newborn, growing, and adult dairy cattle.
The primary objective of this review is to present an in-depth summary and evaluation of research that have investigated the impacts of flaxseed hulls (FH), FM, and whole seeds flaxseed (WF) on milk EL concentration and animal health. We also covered the metabolism of lignans in the gastrointestinal tract of humans and animals and the pharmacokinetics of milk EL consumed by newborn dairy calves, which may have implications to ruminants and humans’ health.
2. Metabolism of Lignans in the Gastrointestinal Tract
The flaxseed lignans SDG, secoisolariciresinol (SECO), pinoresinol, lariciresinol, and matairesinol are converted by the gut microbiota of humans [
6,
24] and ruminants [
7,
8,
9,
10] to the enterolignans ED and EL. In contrast, the lignan isolariciresinol, also derived from flaxseed, is not converted to ED and EL [
25]. Enterodiol and EL are named mammalian lignans or enterolignans because they are produced in the gut of humans and other mammals and not found in plant tissues [
26]. A simplified pathway highlighting the conversion of plant lignans to enterolignans in humans is presented in
Figure 1. Consortia of gut microorganisms appear to be involved in the sequential catalytic reactions reported in
Figure 1, including 28 bacterial species belonging to 12 different genera such as
Bacteroides,
Clostridium,
Bifidobacterium, and
Ruminococcus among others according to previous research [
12,
27,
28,
29,
30,
31,
32,
33]. After conversion of lignans into ED and EL, these enterolignans are absorbed in the large intestine followed by conjugation as glucuronides and sulfates based on in vitro work using human colon epithelial cells [
34]. Conjugated EL and ED undergo extensive first-pass metabolism and enterohepatic recirculation [
34,
35], as well as deconjugation by colonic bacterial β-glucuronidases and sulfatases followed by reabsorption [
36]. It has also been shown that conjugation of EL takes place not only in the colon, but also in the small intestine and liver microsomes of humans and rats according to in vitro enzymatic kinetic analysis of EL glucuronidation [
35].
An investigation of the relationship among the gut microbial community, urinary EL excretion, and diet from a 3-d food record of 115 premenopausal American women (40–45 years old) revealed a significant positive association between EL excretion and either the gut microbial community or its diversity [
37]. They also demonstrated that the gut microbial community associated with high EL production was distinct and enriched in
Moryella,
Acetanaerobacterium,
Fastidiosipila spp., and
Streptobacillus spp. [
37]. Interestingly, these 4 bacterial genera were not part of those typically related to the sequential pathway of lignans catabolism [
12,
27,
28,
29,
30,
31,
32,
33]. However, despite these genera not being previously linked to EL production, they are closely related to those involved in the metabolism of lignans [
37]. Recently, the complete metabolic pathway of pinoresinol and lariciresinol was unraveled using comparative genomics and transcriptional profiling (RNAseq) prepared from stool samples, thus indicating that the conversion of dietary lignans to bioactive enterolignans is a common route adopted by the gut microbiota of humans [
38]. These results are an important step for advancing the molecular genetic understanding of the gut bioactivation of lignans and other plant secondary compounds to downstream metabolites relevant to humans’ health [
38].
In ruminants, it is conceivable that deglycosylation, demethylation, dehydroxylation, and dehydrogenation reactions like those reported in humans (
Figure 1) are also involved in the metabolism of lignans, but little is known about which ruminal bacteria species or consortia participate in these reactions. Lignans present in FH and WF were both converted to mammalian lignans by ruminal and fecal microbiota of dairy cows during in vitro incubations [
7]. While EL was the major enterolignan produced by the ruminal microbiota, the fecal counterpart yielded primarily ED [
7]. In a study conducted using ruminally-cannulated goats, the concentrations of SDG, ED, and EL increased significantly in both rumen and serum following ruminal infusion of SDG (1 mg/kg of body weight) [
9]. These authors also observed that the ruminal and serum concentrations of EL were approximately 2-fold greater than those of ED [
9], indicating that EL is the predominant enterolignan in the rumen, which agree with results from another study [
7]. The role of the ruminal microbiota and the effects of flaxseed oil (FO) in the metabolism of flaxseed-lignans and concentrations of EL in biological fluids have been also investigated [
8]. Flaxseed oil is a rich source of polyunsaturated fatty acids (PUFA) [
39], which are known to be toxic for certain species of ruminal microorganisms [
40,
41]. Therefore, feeding sources rich in PUFA may interfere with the ruminal metabolism of flaxseed-lignans and ultimately affect the concentrations of EL in biological fluids. The concentrations of EL increased by an average of 1,755% in urine, 238% in plasma, and 925% in milk of cows administered with FH in the rumen compared with FO and FH infused in the abomasum [
8]. However, no significant differences in the concentrations of EL in urine, plasma, and milk were observed when FO was administered in the rumen and FH infused in the abomasum [
8], which confirm that rumen is the major site for conversion of SDG to EL. In addition, the ruminal concentration of EL increased linearly and a strong correlation (r = 0.76) between EL concentrations in ruminal fluid and milk was observed in dairy cows fed incremental amounts of FM [0, 5, 10, and 15% of the diet dry matter (DM)] [
10,
42], further reinforcing the key role of the ruminal microbiota in the metabolism of flaxseed-SDG.
It appears that in ruminants, ED and EL are absorbed in the rumen and intestines [
10,
43,
44], possibly as conjugated forms like other phytoestrogens including formononetin, daidzein, and equol [
43]. Interestingly, sheep had a greater conjugative activity than cattle in most parts of the gastrointestinal tract evaluated (i.e., rumen, reticulum, omasum) except in the small intestine [
43]. In humans, deconjugation performed by gut microbial β-glucuronidases and sulfatases is known to enhance the reabsorption of ED and EL [
36,
45,
46]. Studies conducted with lactating dairy cows showed no relationship between flaxseed supplementation (FH or FM) and activity of microbial β-glucuronidase in the rumen [
8,
10,
47], thus suggesting that this enzyme has little or no involvement in the ruminal absorption of EL, possibly because conjugation occurs during or after cell uptake of enterolignans [
43]. In fact, when the ruminal activity of microbial β-glucuronidase decreased in dairy cows fed FH [
48], the concentrations of EL in rumen, plasma, urine, and milk increased compared with the control diet. However, additional research is needed to elucidate the actual mechanisms involved in the absorption of enterolignans in ruminant animals. Likewise, research investigating the potential effects of intestinal β-glucuronidases on deconjugation of enterolignans before reabsorption in the large intestine of ruminants is warranted.
Studies in which oil (FO or sunflower) was administered in the rumen or infused in the abomasum also helped to shed light on the gastrointestinal tract metabolism of lignans in dairy cows. Oil sources rich in n-3 PUFA such as FO are known to inhibit the growth of ruminal microorganisms involved in fiber degradation (e.g.,
Butyrivibrio,
Ruminococcus) and methanogenesis (e.g.,
Methanobrevibacter) [
40,
41]. β-glucuronidase activity in humans has been attributed to colonic bacteria belonging to the genera
Ruminococcus,
Bacteroides,
Bifidobacterium, and
Eubacterium [
49], which are also found in the rumen [
50,
51]. Thus, it is conceivable that FO may inhibit ruminal bacteria with β-glucuronidase activity. In fact, FO reduced microbial β-glucuronidase activity when it was administered in the rumen, but not during abomasal infusion in lactating dairy cows [
8]. These results [
8] imply that ruminal bacteria with predominant β-glucuronidase activity may be more susceptible to the toxic effects of FO than those primarily involved in the conversion of SDG to EL as the concentration of EL in the rumen was not affected by the site of FO supplementation (rumen or abomasum). Compared with the control treatment, fecal β-glucuronidase activity tended to increase in dairy cows fed FH and no change was detected with abomasal infusion of FO in another experiment [
48]. In contrast, it was found that feeding FM and infusing sunflower oil (n-6 PUFA source) in the abomasum of lactating dairy cows decreased fecal β-glucuronidase activity relative to the control treatment [
47]. It has been shown that the ruminal microbiota can be modulated by modifying the dietary PUFA profile and similar processes may also take place in the large intestine of ruminants, which may explain to a certain extent these inconsistent results in fecal β-glucuronidase activity [
8,
47,
48]. Changes (increase or decrease) in 16S rRNA copy numbers of ruminal microorganisms such as
Butyrivibrio, ciliate protozoa, methanogens,
Selenomonas ruminantium, and
Streptococcus bovis were detected during an in vitro rumen simulation technique study in which fermenters were dosed with diets rich in n-6 PUFA (i.e., sunflower oil) or a n-6/n-3 PUFA mix (i.e., sunflower oil plus fish and algae oil) [
52]. Overall, ruminal or fecal microbiota β-glucuronidase activity appears to have limited biological importance for the absorption of EL in lactating dairy cows fed different flaxseed products or abomasally-infused with n-3 or n-6 PUFA oil sources.
As mentioned earlier, there is scarce information about the role of ruminal microbiota species in the metabolism of plant-derived lignans. Ruminal supplementation of SDG stimulated the growth of the bacterium
Ruminococcus gnavus, which is likely involved with glucuronidase activity in the rumen [
9]. In fact,
R. gnavus E1, an anaerobic bacterium belonging to the dominant human gut microbiota, expresses the gene
gnus known to encode for the β-glucuronidase enzyme [
49]. In a more recent study, the concentration of total ruminal bacteria 16S rRNA obtained using qPCR did not differ in cows fed incremental amounts of FM [
42]. However, additional PCR-DGGE and DNA extraction analyses using bands from cows fed 15% FM showed that several genera contributed to the metabolism of lignans, particularly
Prevotella spp. [
42]. Moreover, a follow-up in vitro pure culture assay revealed that 11 ruminal bacteria species were able to metabolize SDG to SECO, with bacteria from the genus
Prevotella being the most efficient followed by
Butyrivibrio fibrisolvens and
Peptostreptococcus anaerobius, whereas
Ruminococcus albus,
Eubacterium ruminantium,
Butyrivibrio proteoclasticus, and
Ruminococcus flavefaciens showed the least conversion efficiency [
42]. Their data also suggested that intermediate compounds between the SDG to EL pathway were formed during in vitro pure culture incubations due to the presence of unidentified peaks in the chromatograms [
42]. Overall, the genus
Prevotella appears to be the most relevant in the metabolism of plant lignans to enterolignans in ruminants. However, the current knowledge regarding ruminal microbiota diversity and function in young and adult ruminants fed different sources of flaxseed is limited and warrants further research.
4. Pharmacokinetics of Milk EL and Potential Implications on Animal and Human Health
Elevated blood concentrations of ED and EL have been associated with reduced risk of coronary diseases and colorectal adenoma in humans [
82,
83,
84]. A dose-response relationship between flaxseed intake and serum concentrations of ED or EL was observed in a study conducted with healthy young women [
85]. Moreover, a 5-fold increase in the urinary excretion of EL was found in rats fed pure EL compared with those fed plant lignans [
86]. These authors [
86] hypothesized that EL may be passively absorbed along the intestinal tract, while plant lignans must be first converted to EL by colonic microorganisms followed by absorption in a limited segment of the gut. A large interindividual variation in the blood concentration of enterolignans has been observed in humans, thus revealing differences in the capacity of the colonic microbiota in converting plant lignans to ED and EL [
46,
85,
87]. Therefore, EL-enriched milk has potential to be used as an enterolignan source for improving human health, particularly because EL appears to be more bioavailable than plant lignans [
86]. Periparturient dairy cows, as well as newborn and nursing dairy calves could also benefit from the antioxidant properties of EL due to their susceptibility to oxidative stress and depressed immune system [
20,
21]. However, there is limited information regarding the pharmacokinetics of EL derived from milk and we are not aware of any published research that have instigated the effects on EL-enriched milk on human or animal health.
Recently, we investigated the pharmacokinetics of EL in newborn dairy calves fed milk replacer or EL-enriched milk [
58]. In newborn calves, suckling stimulates the reflex closure of the esophageal groove so that ingested milk or milk replacer bypass the reticulo-rumen down to the abomasum [
50]. Thus, calves may be used as a translational model to make inferences about the pharmacokinetics of EL in simple-stomach mammals including humans. We hypothesized that the area under the curve and plasma concentration of EL would be greater in Holstein calves fed a single bolus of EL-enriched milk versus milk replacer [
58]. The EL-enriched milk was collected from a Jersey cow fed 15% FM. On d 5 of life, 20 calves (10 males and 10 females) were administered 2 L of milk replacer (low-EL treatment: 123 n
M of EL) or 2 L of EL-enriched milk (high-EL treatment: 481 n
M of EL) during the morning feeding. The area under the curve for the plasma concentration of EL, which was determined using the trapezoidal rule between 0 and 12 h after treatment administration was greater in high- (26 n
M × h) than low-EL calves (4.30 n
M × h). Similarly, the maximum concentration of EL in plasma was greater in high- (5.06 n
M) versus low-EL calves (1.95 n
M). Furthermore, the time after treatment administration to reach maximum plasma concentration of EL was faster in the high- (4.31 h) compared with the low-EL (4.44 h) treatment. Our results showed that newborn calves were able to absorb EL, suggesting that EL-enriched milk can potentially be used as a natural source of antioxidants to pre-weaned ruminants. We also calculated the apparent efficiency of EL absorption between 0 and 12 h after the oral administration of treatments; calves fed EL-enriched milk tended to have lower apparent efficiency of EL absorption than those fed milk replacer (1.31 vs. 1.80%, respectively). In a study in which 12 healthy volunteers (6 men and 6 women) ingested a single dose of purified SDG (1.31 µmol/kg of body weight), ED and EL reached their maximum plasma concentrations at 14.8 and 19.7 h after intake of SDG, respectively [
87]. In addition, the area under the curve of EL (mean = 1762 n
M × h) increased by 2-fold compared with that of ED (mean = 966 n
M × h), indicating a greater systemic exposure to EL than ED [
87]. Although our study shed some light in the metabolism of milk EL in vivo [
58], future research using humans or animal models that better represent the anatomy and physiology of humans’ gastrointestinal tract is warranted to provide further insights about the pharmacokinetics of EL consumed through milk.
An association between serum EL concentration ≥ 10 n
M and decreased mortality risk (i.e., all-causes and breast cancer-specific) after breast cancer surgery has been reported in women [
88]. Milk concentration of EL averaged 395 n
M in two studies in which Jersey cows received 15–16% FM [
18,
19]. Thus, 1 daily serving (250 mL) of EL-enriched milk with a concentration of 395 n
M of EL would result in 1.3 n
M of EL in plasma assuming an apparent efficiency of absorption of 1.31% based on our previous work [
58]. These results imply that EL-enriched milk needs to be consumed in combination with other lignan-rich foods to reach EL concentration in blood that has been linked to decreased mortality and positive health outcomes in humans [
88]. However, our inferences should be interpreted cautiously because calves were fed milk as the sole dietary source [
58], which may have increased digesta passage rate ultimately limiting the intestinal absorption of EL.
5. Antioxidant Activity of Flaxseed Products and Dairy Cow Health
Periparturient dairy cows mobilize triacylglycerols from the adipose tissue to support elevated energy demand during early lactation [
59,
89]. As lactation advances, dairy cows also experience extensive metabolic adaptations for maintenance and high milk production [
90]. This increased metabolic activity requires more oxygen consumption, which stimulates production of reactive oxygen species (ROS) [
91]. When ROS generation exceeds the endogenous antioxidant defense capacity, animals are susceptible to oxidative damage to DNA, lipids, protein, and other cellular components [
92]. Oxidative stress may also impair the immune system of dairy cows [
91,
93] so that they are likely more vulnerable to a variety of metabolic disorders, including udder edema, milk fever, retained placenta, mastitis, and reproductive issues [
90,
91]. It has been shown that newborn calves had greater blood concentration of free radicals than pregnant cows, suggesting that they undergo a more severe oxidative stress [
20]. Therefore, mitigation of oxidative stress has great potential to improve dairy cattle health and profitability of dairy enterprises. In recent years, several studies were conducted to investigate the effects of flaxseed products on the activity of antioxidant enzymes in plasma and erythrocytes, and their gene expression in mammary and hepatic tissues and results are discussed below.
Superoxide dismutase, CAT, and GP
X are antioxidant enzymes commonly involved in combating free radicals in animals’ blood and tissues. Superoxide dismutase catalyzes the reaction of highly reactive superoxides to form less reactive peroxides [
94]. Peroxides can then be converted to water and oxygen under the catalyzation of CAT [
95]. Glutathione peroxidase is an enzyme that facilitates reduction reactions of hydroperoxides such as organic hydroperoxides and peroxides [
94]. According to previous work [
96], CAT mainly works against free radicals when animals experience severe oxidative stress, whereas GP
X protects those with less oxidative stress pressure.
The activity of antioxidant enzymes in lactating dairy cows fed different flaxseed products are summarized in
Table 3. Overall, the activities of SOD, CAT, and GPx in plasma, erythrocytes, and mammary and hepatic tissues were not affected by supplementation of FH, FM, WF, and whole linola (see
Table 3). Linola is a cultivar of flaxseed containing approximately 70% linoleic acid [
97]. A potential explanation for the inability of flaxseed products to modify the activity of antioxidant enzymes in most studies listed in
Table 3 may be due to the use of mid-lactation dairy cows experiencing low oxidative stress. Contrarily, a study [
98] reported that inclusion of 12.4% FM lowered plasma CAT activity and tended to elevate that of erythrocytes. Likewise, a tendency for increased activity of SOD in mammary tissues was observed with feeding 9.88% FH [
56]. It is also important to note that significant treatment by sampling time interactions were found for plasma CAT and GPx activity with FM supplementation [
99]. Plasma CAT and GPx activity responded quadratically and cubically to increasing amounts of FM (0, 5, 10, 15%) when blood samples were collected before feeding, but no treatments effect was observed with sampling 3 h post-feeding [
99]. These interactions were probably caused by a longer-lasting supply of antioxidants from the diet with the greatest intake of SDG (i.e., 15% FM) compared with the lower levels [
99].
The effect of flaxseed products on mRNA abundance of antioxidant enzymes genes in the mammary gland of lactating dairy cows are summarized in
Table 4. Feeding 9.88% FH [
56] and incremental amounts of FM (0, 5, 10, and 15%) [
99] increased mRNA abundance of CAT gene, whereas no changes were observed with inclusion of 13.7% FM [
100]. Additionally, GPx1 and GPx3, two isoforms of GPx, were not impacted with feeding varying amounts of FM [
98,
99,
100]. However, GPx1 and GPx3 were up- and downregulated, respectively, in dairy cows fed 9.88% FH compared with those fed the control diet [
56]. These contradictory effects on GPx1 and GPx3 mRNA abundance with feeding 9.88% FH may be associated with different functions of GPx genes [
101]. In addition to CAT and GPx, the mRNA abundance of three isoforms of SOD genes including SOD1, SOD2, and SOD3 were quantified. Both De Marchi et al. [
100] and Schogor et al. [
99] showed that the mRNA abundance of SOD genes was not modified by FM supplementation to lactating dairy cows. In contrast, an increase in the mRNA abundance of SOD1 and decreases in that of SOD2 and SOD3 were detected in dairy cows fed 9.88% FH [
56]. The promoter region of SOD1 contains an antioxidant response element not found in SOD2 and SOD3, thereby consistent with the variable responses of SOD genes to FH supplementation [
102]. Collectively, the effects of flaxseed products on modifying antioxidant enzymes or their expression in mammary or hepatic tissues were limited.
The nuclear factor (erythroid-derived 2)-like 2 (
NFE2L2) relative mRNA abundance in mammary tissues increased linearly in cows fed incremental amounts of FM [
99] (see
Table 4). The
NFE2L2 gene encodes for a transcription factor involved in activating the expression of a series of genes that are transcribed and translated into antioxidant proteins [
103,
104]. It is noteworthy that increased
NFE2L2 [
99] did not coincide with changes in mRNA abundance of most antioxidant enzymes as discussed above. A trend was observed for decreased relative mRNA abundance of the nuclear factor kappa-light-chain-enhancer of activated B cells subunit 1 (NF-κB1) gene with feeding 13.7% FM to lactating dairy cows [
100]; however, two other studies [
98,
99] did not detect changes in mRNA abundances of NF-κB and NF-κB1, respectively, when similar amounts of FM were fed. The NF-κB1 gene is one of the five members of the NF-κB family, which regulates numerous genes involved in inflammatory and immune responses, apoptosis, and tumor progression [
105,
106,
107]. The polyphenolic compound quercetin protected interstitial Leydig cells against atrazine-induced toxicity by decreasing the expression of NF-κB and preventing oxidative stress [
107]. As shown in
Table 2, FM is the richest source of the lignan SDG, a polyphenolic compound like quercetin, thus in line with the reduced expression of NF-κB1 gene [
100]. These results suggest that FM supplementation has potential to decrease inflammation and cell death in mammary tissues [
100]. Interestingly, decreased NF-κB1 was not associated with changes in the relative mRNA abundance of antioxidant enzymes [
100], possibly because FM supplementation did not affect the nuclear factor erythroid 2–related factor 2 (
NRF2) mRNA abundance, which agrees with previous work [
98]. As known,
NRF2 is a transcription factor that activates the expression of multiple genes holding an antioxidant response element in their promoters for codifying antioxidant proteins and phase 2 detoxifying enzymes [
105]. Future research is needed to better understand how the relationship between flaxseed supplementation and expression of antioxidant enzyme genes may interact to modulate inflammatory, immunological, and health responses in dairy cows experiencing oxidative stress.
Thiobarbituric acid-reactive substances (TBARS) are markers of oxidative status and mainly used to estimate oxidative damage to lipids or lipoperoxidation [
109]. Lipoperoxidation can cause damages to cell membranes and membrane-bound enzymes [
110]. The impact of flaxseed products supplementation on TBARS concentration in milk, plasma, and ruminal fluid are summarized in
Table 5. Quadratic and cubic responses for milk TBARS production were observed in cows fed incremental amounts of FM, with 5% FM and 10% FM resulting in the lowest values [
99]. They [
99] also reported a significant treatment × sampling time interaction for ruminal TBARS concentration; a linear decrease in TBARS was found with increasing FM supplementation at 2 h after feeding, but no changes were detected at 0 (pre-feeding), 4, and 6 h post-feeding. It was hypothesized that the defense of FM-lignans against oxidation in the rumen is a time-dependent process, with protection being more effective within the first hours after feeding and weakening over time [
99]. However, another study [
111] reported no changes in ruminal TBARS concentration at 0 (pre-feeding) and 2 h post-feeding but decreased thereafter (4 and 6 h) with feeding 12.4% FM. A third experiment [
112] showed a significant decrease in ruminal TBARS concentration in dairy cows fed 13.7% FM despite no treatment × sampling time interaction effect. None of the studies listed in
Table 5 (i.e., [
99,
111,
112]) reported effects of FM on plasma TBARS concentration. Similarly, no effects of FM supplementation were observed for the plasma peroxidizability index and total antioxidant capacity [
111,
112]. As pointed out earlier, research using dairy cattle during stages of life (e.g., transition period, neonatal phase, weaning) more conducive of oxidative stress is needed to better assess the role of flaxseed lignans on animal oxidative status and overall health.