4. Discussion
Even though olive polyphenols have many beneficial effects on animal and human health [
1,
2,
3,
5], only a few trials have investigated their use as antioxidants in pigs. Moreover, the potential of extracted olive polyphenols at different dietary levels has not been studied in detail in animals.
In the present study, oxidative stress was induced in Cont+, Vit-E and the three OLE-supplemented groups by including high linseed oil, which was exchanged for wheat starch in the diet fed to the Cont− group. Consequently, Cont− feed contained less energy than Cont+, and therefore a higher feed intake and a poorer feed conversion ratio were observed in animals fed Cont− than in the rest. Moreover, dietary supplementation with OLE did not affect the growth performance of the piglets compared to Cont+, contrarily to the results obtained in pigs fed dietary olive leaves [
3,
5]. The inclusion of OLE did not affect the energy content of the feed or antinutritive components, as might be the case with olive leaf supplementation. In spite of this, further studies under practical rearing conditions without feed restriction should be performed to confirm our hypothesis, since the inclusion of OLE could affect feed consumption, growth and feed conversion, as reported for some polyphenols [
12].
One of the most accepted markers of in vivo oxidative stress is urinary excretion of F
2-isoprostanes [
13]. We did not detect any differences in urinary excretion of F
2-isoprostanes between Cont− and Cont+, suggesting that oxidative stress caused by the high PUFA intake was not very pronounced. Nevertheless, dietary OLE did not have an effect on F
2-isoprostanes, which is in agreement with studies in humans [
2], although some trials showed lower excretion of isoprostanes in humans after the consumption of olive polyphenols [
14]. In the present study, the only reduction of F
2-isoprostanes was observed with the inclusion of vitamin E, which is in line with some trials in humans [
15]. F
2-isoprostanes could be a biased marker of oxidative stress, since the linseed oil-supplemented feeds had different n-6:n-3 PUFA ratios (0.54:1) in comparison to Cont− (5.3:1). The differences in the dietary linoleic fatty acid, which is elongated to arachidonic fatty acid, the precursor of F
2-isoprostanes, were reflected in the fatty acid composition of the erythrocytes. Animals in group Cont− had a higher proportion of arachidonic acid in the erythrocytes (Cont− = 4.93 wt %; n = 4) than the linseed oil-supplemented groups (Cont+ = 2.75 wt %; n = 4), and therefore higher F
2-isoprostane levels in Cont− were expected. Therefore, it is not clear if the differences were due to the difference in the level of arachidonic acid in the body or to the lower oxidative stress in animals fed Cont− than in those fed Cont+.
Prevention of the oxidation of LDL is an important effect of olive polyphenols in protection against cardiovascular diseases in humans. In the present study, the plasmatic level of oxLDL was not affected by any of the supplements used compared to both Cont− and Cont+. This does not comply with the results reported by Paiva-Martins et al. [
3], who observed a trend of dietary olive leaves to lower oxLDL in pigs, although the supplementation level of polyphenols was much higher than in the present trial. The authors related the outcome to the increased level of vitamin E in olive leaves, while in our study, dietary supplementation with vitamin E did not have an effect on oxLDL. Our results are also different than those reported in humans. For instance, EFSA [
2] substantiated the health claim that 5 mg of hydroxytyrosol and its derivatives per day protect LDL from oxidation, which is comparable to the concentration of polyphenols in group OLE-1. However, we observed a numerical difference between the OLE-supplemented and non-supplemented groups. The lack of statistical difference might have been attributed to the variability of oxLDL within the groups. In its trials, EFSA [
2,
16] supported the health claim because the difference in plasma oxLDL between the pre- and the post-intervention with polyphenol-rich olive oil was only 6.5%, while in our trial, OLE lowered the oxLDL level for approximately 20%, regardless of the concentration. The latter suggests that even the lowest concentration of OLE was sufficient to prevent LDL from oxidation. Nevertheless, a larger scale trial is suggested in order to confirm this hypothesis.
Dietary linseed oil (Cont+ vs Cont−) increased plasma MDA levels (
p < 0.05) and showed a tendency to increase urinary MDA excretion levels (
p = 0.115). This is in accordance with the previous findings of Frankič [
6,
17] and Frankič and Salobir [
11], who discovered that dietary linseed oil increased plasma and urinary MDA levels in pigs in comparison to a diet without linseed oil. In the present study, the dietary inclusion of OLE did not affect plasma and urinary MDA levels, similarly to the results reported by Andreadou et al. [
18] in rabbits. These authors observed that the dietary inclusion of oleuropein did not affect baseline MDA levels, but lowered plasma MDA levels when the oxidative stress was induced by ischemia and reperfusion. It is likely that ischemia and reperfusion had a stronger influence on the oxidative stress than the PUFA-rich diet in the present study, and therefore the effect of olive polyphenols could be different. Similarly, Al-Azzawie and Alhamdani [
19] reported that dietary oleuropein in alloxan-diabetic rabbits reduced plasma MDA levels to the level of non-diabetic animals. As in previous studies by Frankič et al. [
6,
17], supranutritional vitamin E did not affect plasma and urinary MDA levels in piglets fed a PUFA-rich diet. This was an interesting result, since vitamin E is known as one of the best lipid-soluble antioxidants.
Buckley et al. [
20] reported that plasmatic tocopherols firmly follow the concentration of dietary tocopherols. Similarly, in our study, there was a higher concentration of γ-tocopherol in the plasma of animals fed diets supplemented with linseed oil (rich in γ-tocopherol) than in those fed Cont−. Moreover, dietary α-tocopheryl acetate (group Vit-E) significantly raised the plasmatic concentration of α-tocopherol and lowered that of γ-tocopherol within the linseed oil supplemented groups (Cont+, Vit-E, OLE-1, OLE-2, OLE-3). This might be related to the preferential transfer of α-tocopherol to lipoproteins and of γ-tocopherol to the bile by the liver, as observed in humans [
21]. Furthermore, OLE did not have an effect on plasmatic α- and γ-tocopherols, unlike in the study by Paiva-Martins et al. [
3], who reported that the dietary supplementation of olive leaves increased the content of α-tocopherol in muscle in pigs. This could be related to the higher level of α-tocopherol in the leaves than in OLE, and/or to the higher level of the supplementation compared to our study.
In the present study, we included antioxidants with different polarities expecting different in vivo ACW and ACL. However, although some trials reported a positive change in plasmatic ACW and ACL in animals with dietary supplementation of antioxidants [
22,
23], we could not detect any differences in serum ACW and ACL between the groups. It is possible that the endogenous antioxidants were counteracting the presence of the exogenous antioxidants in order to retain the equilibrium of the total antioxidative capacity [
24]. The unresponsiveness could also be attributed to a low absorption, strong homeostasis, fast metabolism and/or rapid excretion of polyphenols [
12].
Glutathione peroxidase and SOD are among the most important enzymes involved in the antioxidant defense system [
25]. In our study, neither dietary linseed oil nor OLE or vitamin E affected their activities, which is contrary to the positive effects of olive polyphenols on the expression of GPx [
25,
26] and SOD [
27] reported by some in vitro studies. This could show a discrepancy between the in vivo and the in vitro trials, since the concentrations of the active compounds and their activities often differ between the two. Some authors reported an increase on the activities of GPx and SOD caused by dietary olive polyphenols in rats [
28] and in humans [
29,
30]. However, the animals in our trial might have reacted differently, since we included high amounts of PUFA that increased the oxidative load in comparison to the animals and humans in other trials.
The oxidative products of n-3 PUFA can form adducts with DNA, suggesting their involvement in the pathogenesis of different diseases [
31]. In our study, the inclusion of linseed oil (Cont+) caused higher DNA damage measured as DNA tail % in comparison to Cont−. This complies with previous results in a study on piglets, in which an increase in DNA damage was observed with the dietary inclusion of linseed oil [
6,
11,
17]. In our trial, diet OLE-1 showed some protective effects on DNA measured as tail DNA % in comparison to Cont+ (
p = 0.072), although the effect was not present in higher concentrations of OLE (OLE-2 and OLE-3). This partly agrees with some in vitro [
32]) and in vivo [
30] studies showing the protective effects of olive polyphenols against DNA damage. Interestingly, vitamin E presented prooxidative effects, as we detected higher OTM in the blood of animals fed Vit-E than in those fed Cont+, which does not comply with the previous findings of Frankič and Salobir [
11]. These authors reported that dietary vitamin E completely mitigated the negative effects of dietary linseed oil on DNA damage in piglets, although the level of supplementation with vitamin E was lower than in our study.
The urinary level of 8-OHdG is a common marker of DNA damage or its repair [
13]. In the present study, piglets fed Vit-E and OLE-1 had a similar excretion of 8-OHdG as piglets fed Cont−, which may suggest that the extent of DNA repair and/or damage was not altered in these groups. Contrary to our results, studies concerning DNA damage reported a reduction of 8-OHdG with dietary supplementation of olive polyphenols in humans [
30]. The latter suggests that olive polyphenols might not be efficient in preventing DNA damage caused by PUFA-induced oxidative stress, or that the concentration of polyphenols might not have been sufficient to protect against DNA damage.
Lipids are primarily metabolized by the liver, and therefore its enzymes are an important marker of oxidative stress caused by n-3 PUFA. In our study, the inclusion of linseed oil (Cont+ vs. Cont−) increased the GGT activity in serum, suggesting an effect of the high intake of PUFA on liver function. Animals fed OLE-1 and Vit-E had lower GGT activity (
p < 0.05 and
p = 0.078, respectively) than those fed Cont−. This could indicate protective effects of OLE-1 and Vit-E, as demonstrated for other dietary antioxidants [
33]. However, the differences between the latter and Cont+ were not significant (
p > 0.05). In the present study, the activities of AST and ALT were not influenced by any of the supplements used, which is in agreement with the study by Bock et al. [
34]. These authors reported no effects of olive polyphenols in plasmatic AST, ALT and GGT in humans. The results suggested that olive polyphenols do not have an apparent effect on the liver function, even in the case of high n-3 PUFA intake.
Antioxidants commonly have a concentration-dependent effect on the prevention of oxidative damage. Thus, we hypothesized that this could also be the case for OLE. Even though there are some reports on the concentration-dependent effects of olive polyphenols in pigs [
3], turkeys [
35], humans [
16] and human erythrocytes [
36], we did not obtain similar results. The lack of a clear dose-dependent antioxidative effect could be attributed to numerous nutritional and environmental factors. For instance, the oxidative stress caused by the high dietary intake of PUFA might have been overestimated, and the additional dietary antioxidant supplementation might have been unable to have an apparent effect even when included in high concentrations. Oxidative stress is also influenced by the duration of the stress: long stress periods could influence oxidative-induced damages in a substantially different way than short-term stress. Data on this are scarce, although some trials reported that a high intake of PUFA caused a rapid increase in the markers of oxidative stress in pigs [
37]. Another important factor involved in the response to oxidative stress is the age of the animals. In our study, the animals were very young and could have had antioxidative defense systems with higher adaptability, antioxidative capacity and lower basal oxidative stress-induced damages, as is the case in humans [
24,
38]. Furthermore, the basal supplementation level of OLE was that recommended to prevent the oxidation of LDL in humans [
2]. Hence, the response of humans and pigs could be different due to differences in metabolism, nutrition, age and genetic predispositions [
39]. Previous studies in animals used higher amounts of olive polyphenols than in our study.