Effect of soya bean and fish oil inclusion in diets on milk and plasma enzymes from sheep and goat related to oxidation
Summary
This study investigated the effects of dietary inclusion of soya bean oil combined with fish oil (SFO) on the activities of a) superoxide dismutase (SOD), glutathione reductase (GR), catalase (CAT) and glutathione transferase (GST) in blood plasma and b) SOD, GR, CAT and lactoperoxidase (LPO) in the milk of sheep and goats. Furthermore, the oxidative stress indicators for measuring total antioxidant activity and free radical scavenging activity [ferric reducing ability of plasma (FRAP) and 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) assays] and oxidative stress biomarkers [malondialdehyde (MDA) and protein carbonyl (PC)] were also determined in the blood plasma and milk of the animals. For this purpose, twelve dairy sheep and twelve dairy goats were assigned each to two homogenous subgroups. Treatments in both animal species involved a control diet without added oil and a diet supplemented with 5% soya bean oil and 1% fish oil. The results showed that the inclusion of SFO in the diets of sheep and goats increased significantly the activities of CAT and GR in their blood plasma. The same effect was observed for the activities of GST and FRAP in the blood plasma of goats. Moreover, the fact that the goats had significantly higher average daily PUFA intake (3.62 g/kg BW0.75) compared to sheep (2.51 g/kg BW0.75) resulted in an enhancement in the MDA content in their plasma. A significant increase in CAT activity in the milk in both animal species fed with SFO diets was also found. Finally, due to the higher apparent transfer rate of n-3 FA from the diet to the milk in sheep, the PC concentrations were found to be enhanced in their plasma and milk. In conclusion, the impact of dietary SFO supplementation on the oxidative status of body and/or on the milk of small ruminants depends not only on the daily PUFA intake, but also on the amount of n-3 FA that reach their milk.
Introduction
Supplementation of ruminant diets with vegetable oils, rich in linoleic acid, induces an increase in conjugated linoleic acid (CLA) content in the milk (Martínez Marín et al., 2012), while the inclusion of marine lipids, such as fish oil, enhances the n-3 polyunsaturated fatty acid (PUFA) content in milk fat (Capper et al., 2007), which is very important because the contribution of n-3 PUFAs to milk and dairy products in order to meet the human nutritional requirements is low in a balanced diet (Shingfield et al., 2008).
Nevertheless, this approach either could increase the risk of plasma lipid peroxidation with deleterious consequences on animal health or milk spoilage (Grandelli et al., 1998), or could cause unpleasant oxidation (Palmquist et al., 1993), because fatty acids, especially PUFAs, are easily oxidized. As a result, the inclusion of PUFA in ruminant diets, in most cases, is accompanied by the addition of antioxidant compounds (Gobert et al., 2009; Santos et al., 2014).
Recently, it has been proven that some feedstuffs, such as flax hulls, are not only rich in PUFA, but also rich in antioxidant compounds that improve the oxidative status of cows (Côrtes et al., 2012; Schogor et al., 2013) without an extra administration of an antioxidant source. The soya bean oil could be another option because it has the highest antioxidant capacity compared to other vegetable oils, such as extra virgin olive, corn and sunflower oil (Pellegrini et al., 2003), and it also contains high tocopherol content (Castelo-Branco and Torres, 2012). In addition, the use of soya bean oil as a possible dietary source to improve the oxidative status of ruminants has been proposed by Scislowski et al. (2005a). Positive impact on the antioxidant defence system may also be observed with the fish oil too, because its inclusion in the diets of rabbits (Hsu et al., 2001) and rats/hamsters (Erdogan et al., 2004; Muga and Chao, 2014) inhibited the oxidative stress and prevented the formation of free radicals. Although fish oil is not rich in antioxidants per se, generally it is supplied with natural antioxidants, such as tocopherols, by the food industry to prevent oxidative deterioration due to its high content of unsaturated fatty acids (Rossell, 2009; Yi et al., 2011).
Oxidative stress in ruminants occurs not only during periparturient period or heat stress but also when they receive diets rich in PUFA (Scislowski et al., 2005a,b). Although dietary PUFAs are subjected to ruminal biohydrogenation, some of them escape from this process. Thus, before their absorption by various tissues, including in the milk, dietary PUFAs are absorbed by small intestine and recycled in blood mainly as TG-rich lipoprotein particles. At this stage, PUFA becomes preferential targets for the action of free radicals that induce an oxidative stress. The oxidative stress is faced normally by the body with a wide range of antioxidant mechanisms that can be divided into enzymatic and non-enzymatic (e.g. metabolites) (Ye et al., 2015). This type of stress is facilitated if an imbalance occurs between the respective amounts of PUFA (which can be attacked by free radicals) and antioxidant systems (involved in PUFA protection against free radicals). Several endogenous enzymes, such as superoxide dismutase (SOD), glutathione reductase (GR), catalase (CAT) and glutathione transferase (GST) (Miller et al., 1993; Board and Menon, 2013), found both in the blood and in the milk represent the main intracellular antioxidant defence system that regulates reactive oxygen species accumulation within the tissues (Celi, 2010; Sordillo, 2013) (Fig. 1). Furthermore, the enzyme lactoperoxidase (LPO) is related to the oxidation of milk lipids (O'Connor and O'Brien, 2006). Thus, taking into account all the aforementioned parameters, the present study investigated the effects of dietary inclusion of soya bean oil combined with fish oil (SFO) on the activities of (i) SOD, GR, CAT and GST in the blood plasma and (ii) SOD, GR, CAT and LPO in the milk of sheep and goats.
Materials and methods
Twelve 3-year-old Friesian cross-bred dairy sheep and twelve 3-year-old Alpine cross-bred dairy goats, at 90 ± 2 days of milking, were maintained at the Agricultural University of Athens. Housing and care of animals conformed to Ethical Committee Guidelines of the Faculty of Animal Science. Both animal species were assigned to two homogeneous subgroups, each (n = 6) balanced by their body weight (BW) and milk yield. The average initial BW and milk yield was 63 ± 2.1 and 1.1 ± 0.46 kg, respectively, for sheep and 44.9 ± 1.1 and 2.1 ± 0.64 kg for goats. Throughout the experimental period, each animal of each group was fed individually according to its daily individual energy and crude protein requirements (National Academic Press, 1981; Zervas, 2007). The sheep and goats of both groups were fed after 2-week adaptation period with a ration consisting of alfalfa hay and concentrates with a forage-to-concentrate ratio (F/C) of 53:47 and 50:50 respectively. The concentrate diet of the control group had no added oil, while that of the treated group was supplemented with 5% (w/w) soya bean oil and 1% (w/w) fish oil (SFO). In order to have isoenergetic and isoproteic concentrate diets among the groups, some different raw ingredients along with the oil inclusion in the concentrate diet of the treated groups were used. More specifically, the concentrate diet (g/kg) of the control groups consisted of corn grain, 670; soya bean meal, 200; wheat middlings, 100; mineral and vitamin pre-mix, 30, whereas that of the treated group consisted of corn grain, 300; sunflower meal, 280; sugar beet pulp, 70; wheat middlings, 260; soya bean oil, 50; fish oil, 10; mineral and vitamin pre-mix, 30. The mineral and vitamin pre-mix of both dietary groups contained (per kg as mixed) 150 g Ca, 100 g P, 100 g Na, 100 mg Co, 300 mg I, 5000 mg Fe, 10 000 mg Mn, 20 000 mg Zn, 100 000 mg Se, 5 000 000 IU retinol, 500 000 IU cholecalciferol and 15 000 mg α-tocopherol. The diets were offered to the animals twice a day (two equal parts at 0800 and 1600 h). The concentrate diets were prepared every week for both animal species groups. The quantities of food offered to the animals were adjusted weekly according to their individual requirements (maintenance and lactation) based on their BW and fat-corrected milk yield. The average daily dry matter intake throughout the experimental period for the control group and the treated group was 1.81 and 1.85 kg, respectively, for the sheep and 2.09 and 2.15 kg, respectively, for the goats. The whole experimental period lasted 42 days. All animals had free access to fresh water.
Samples collection
All animals were milked twice a day at 8 am and 6 pm by a milking machine. At 28th and 42nd experimental day, respectively, 5% from the morning milk yield and 5% from the evening milk yield from each animal species were pooled together as one sample and subjected to enzyme analysis. At day 28 and at day 42, blood samples were also collected from the jugular vein into EDTA-containing tubes and subsequently centrifuged at 2700 g for 15 min to separate plasma from the cells, in order to determine the activity of enzymes. Both milk and blood samples were stored at −80 °C, prior to analysis.
Enzyme assays in both blood plasma and milk
Glutathione transferase activity was measured using 1-chloro-2, 4-dinitrobenzene (97%, Aldrich) as substrate by monitoring the formation of the conjugate of each substrate and reduced glutathione (GSH), according to the method described by Labrou et al. (2001). Glutathione reductase (GR) activity was measured according to the method described by Mavis and Stellwagen (1968). Catalase (CAT) activity was assessed using continuous spectrophotometric rate for the determination of H2O2 at 240 nm, based on the method described by Beers and Sizer (1952) and Stern (1937). Lactoperoxidase activity was measured using ABTS (2, 2′-azino-bis(3 ethylbenzothiazoline-6-sul phonic acid)) as a substrate according to the method described by Keesey (1987) and Pütter and Becker (1983). SOD activity was assayed using a modified method of McCord and Fridovich (1969). One unit will inhibit the rate of reduction of cytochrome c by 50% in a coupled system, using xanthine and xanthine oxidase at pH 7.8. The xanthine oxidase concentration should produce an initial (uninhibited) ΔA550 nm of 0.025 ± 0.005 per minute.
Lipid peroxidation activity and protein carbonyl determination in both blood plasma and milk
Lipid peroxidation activity was assayed by measuring malondialdehyde (MDA) according to the method of Heath and Packer (1968). Protein carbonyl (PC) contents were determined based on a published method (Patsoukis et al., 2004). Moreover, protein concentration was determined by the Bradford assay using bovine serum albumin (fraction V) as standard (Bradford, 1976).
Antioxidant and free radical scavenging activities
Ferric reducing antioxidant power (FRAP) assay was used to measure total antioxidant potential according to the method described by Benzie and Strain (1996) in the blood plasma, while the 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) radical scavenging assay was based on the published methods (Pellegrini et al., 2003; Li et al., 2011). The same protocols were used for milk samples for the determination of FRAP and ABTS, but with some modifications. More specifically, for extraction procedure, one normal solution of HCl (1 N)/95% ethanol (v/v, 15/85) was prepared and used as an extraction solvent. Then, 1 ml of the fresh milk was added to 10 ml solvent in 50-ml brown bottles and was shaken for 1 h at 30 °C in a rotary shaker set at 300 rpm. The mixture of solvent and samples was then centrifuged at 7800 g at 5 °C for 15 min. The supernatant fluids were kept at −20 °C in the dark until further analysis of FRAP and ABTS.
Statistical analysis
where Yijklm is the dependent variable, μ the general mean, Si the effect of specie, Dj the effect of dietary treatment, Tk the effect of sampling time, Al the animal's random effect, S × Dij, S × Tik, D × Tjk and S × D × Tijk the first- and second-level interactions and eijkl the residual errors. Because the interactions were not statistically significant in most of the cases, they are not presented. Moreover, data for each animal species (sheep, goat) were analysed using a repeated-measures GLM considering the sampling time as a repeated measure, with fixed effects of diet, time and the interactions between them and with animal as random effect. Principal component analysis (PCA) was also applied to pooled data of the blood plasma or milk in order to establish those enzyme activities or lipid peroxidation or transfer rates of fatty acids capable of discriminating and classifying samples by the animal species, the dietary treatment and the sampling time. For all tests, a p-value of less than 0.05 was considered to be statistically significant. Statistical analysis was performed using the statistical packages spss (version 16.0, SPSS Inc., Chicago, IL, USA) and statgraphics centurion xv (Manugistics Inc., Rockville, MD, USA).
Results and discussion
The inclusion of SFO in the diets of sheep and goats significantly increased CAT activity and did not affect the SOD activity in their blood plasma (Table 1). SOD is the first enzyme involved in the conversion of oxygen radicals to peroxides, while CAT is involved in the second step of removing these peroxides and converting them into O2 (Yu, 1994) (Fig. 1). Increased levels of hydrogen peroxides, as a consequence of their increased production rate due to the presence of PUFA in the SFO diets, may serve as a factor for SOD inactivation. Besides, the inactivation of SOD by hydrogen peroxide has been described in several in vitro studies (Bray et al., 1974; Hodgson and Fridovich, 1975). In accordance with our findings, Shireen et al. (2008) observed a significant increase in the activity of CAT, while SOD activity remained unchanged in the liver of rats fed with soya bean oil in combination with the vitamins E and C. Moreover, in the blood plasma of sheep and goats fed with the SFO diets, significantly higher GR activity was found (Table 1). GR has also an important role in the antioxidant defence system because it catalyses the conversion of oxidized glutathione disulphide to the reduced form of glutathione, which is a critical molecule in resting oxidative stress (Fig. 1). A significant increase in GST activity and in the total antioxidant capacity (FRAP) in the blood plasma of goats (data not shown) was also observed when the animals consumed the SFO diet. GST is involved in the antioxidant defence system because it is responsible for the high-capacity metabolic inactivation of electrophilic compounds including lipid hydroperoxides and toxic substrates (Board and Menon, 2013; Labrou et al., 2015).
| Species (S) | Diets (D) | Time (T) | Effects | |||||||
|---|---|---|---|---|---|---|---|---|---|---|
| Sheep | Goat | Control | Treated | At 28th day | At 42nd day | RMSE | S | D | T | |
| SOD Units/ml | 10.24 | 9.75 | 9.92 | 10.08 | 10.51 | 9.48 | 0.480 | NS | NS | NS |
| SOD Units/mg of protein | 0.166a | 0.106b | 0.133 | 0.139 | 0.151 | 0.120 | 0.021 | b | NS | NS |
| GR Units/ml | 0.046 | 0.042 | 0.035A | 0.053B | 0.042 | 0.045 | 0.006 | NS | b | NS |
| GR Units/mg of protein | 0.0007a | 0.0005b | 0.0005A | 0.0007B | 0.0006 | 0.0006 | 0.00008 | a | b | NS |
| CAT Units/ml | 63.50 | 72.07 | 51.80A | 83.78B | 69.64 | 65.94 | 8.080 | NS | c | NS |
| CAT Units/mg of protein | 1.051 | 0.783 | 0.665A | 1.169B | 1.022 | 0.812 | 0.274 | NS | a | NS |
| GST Units/ml | 0.102 | 0.114 | 0.086A | 0.130B | 0.115 | 0.101 | 0.019 | NS | a | NS |
| GST Units/mg of protein | 0.002 | 0.001 | 0.001 | 0.002 | 0.002 | 0.001 | 0.0004 | NS | NS | NS |
| FRAP μmol ascorbic acid | 0.903a | 1.272b | 1.033 | 1.142 | 1.250x | 0.925y | 0.067 | c | NS | c |
| ABTS μm ascorbic acid | 92.54a | 50.63b | 70.09 | 73.08 | 71.06 | 72.12 | 2.856 | c | NS | NS |
- SOD, superoxide dismutase; GR, glutathione reductase; CAT, catalase; GST, glutathione transferase; FRAP, ferric reducing ability of plasma; ABTS, 2,2′-azino-di(3-ethylbenzothiazoline-6-sulphonic acid); RMSE, root mean square error.
- Means with different superscripts in each row [between the two animal species (a, b), between the two diets (A, B) and between the two sampling time (x, y)] for each parameter differ significantly (p ≤ 0.05).
- a p < 0.05.
- b p < 0.01.
- c p < 0.001.
The enhancement in the antioxidant status of both animal species fed with diets supplemented with SFO may be due to its antioxidants (although not determined in this study) and/or due to the PUFA-rich content in the diet because the organism in order to face the oxidative stress could enhance its antioxidant defence system. Moreover, fish oil derived from fish fed with microalgae may be enriched with antioxidant compounds such as phenols, flavonoids, carotenoids (Geetha et al., 2010; Goiris et al., 2012), which inhibit the oxidative stress (Muga and Chao, 2014). Additionally, although fish oil is not rich in antioxidant per se, the food industry supplied it with natural antioxidants in order to prevent the oxidative deterioration due to its high content of unsaturated fatty acids (Rossell, 2009). Thus, the food industry incorporates natural antioxidants into oils. Among these natural antioxidants, tocopherols are more effective in preventing the fish oil from oxidation (Rossell, 2009; Yi et al., 2011). Indeed, higher SOD and CAT activities have been reported in the liver of rats fed a diet enriched with fish oil (Ruiz-Gutiérrez et al., 1999). An enhancement in the antioxidant defence system was observed also in rats (Muga and Chao, 2014), rabbits (Hsu et al., 2001) and cows (Puppel et al., 2013) when fish oil was included in their diets. It should be pointed out here that in this study, soya bean oil and fish oil were included in the diet of sheep and goats at the same time, so it is difficult to assume whether the enhancement in some antioxidant enzymes in the blood plasma is due to either the inclusion of soya bean oil or the inclusion of fish oil (Table 1). It is possible that both soya bean oil and fish oil affected the activities of enzymes simultaneously, because it is well known that vegetable oils contain also natural antioxidant compounds such as tocopherols, tocotrienols, phenolic and possible sterols, which protect them from oxidation (Dauqan et al., 2011; Castelo-Branco and Torres, 2012). Indeed, the soya bean oil has high total tocopherol content (163.3–184.7 mg of total tocopherols/100 g) (Castelo-Branco and Torres, 2012) and contains small amounts of β- and γ-tocotrienols (Castelo-Branco and Torres, 2012) as well as phenolic compounds (1.48 ± 0.05 mg CAE/100 g oil) (Singer et al., 2008), which may also contribute to its high antioxidant capacity.
However, in the blood plasma of goats fed with SFO diet, besides the enhancement in the activities of antioxidant enzymes and/or in the total antioxidant capacity, a significant increase in the malondialdehyde (MDA) content was also observed (Table 2). MDA is one of the biomarkers of damage caused in the body by reactive oxygen and nitrogen substrates (Vasconcelos et al., 2007). Thus, the increase in MDA content in the blood plasma of goats only may be due to their significantly higher average daily PUFA intake (3.62 g/kg BW0.75) compared to sheep (2.51 g/kg BW0.75), because goat produced more milk. Besides, the PUFAs that found in the plasma become preferable targets for the action of free radicals, derived from the dietary PUFAs that escape from the rumen biohydrogenation process. Indeed, an enhancement in lipid peroxidation in the heart and liver of rats fed with a diet supplemented with 160 g fish oil blend/kg diet was observed by Yuan and Kitts (2003), while the opposite was found by the same researchers with the inclusion of 80 g fish oil blend/kg diet (Yuan and Kitts, 2002). It has been suggested that the daily intake of fat supplement may influence the oxidative status of target tissues through the effects on membrane lipid composition and through the ability of reducing equivalents (i.e. reduced NADPH) for the activities of antioxidant enzymes. Rats fed with 20% soya bean oil diet showed a reduced activity of glutathione peroxide (GPx) compared to those fed with 5% soya bean oil diet (Huang and Fwu, 1992). Both GPx and CAT are involved in H2O2 degradation, which is produced by the action of SOD (Fig. 1).
| Species (S) | Diets (D) | Time (T) | Effects | |||||||
|---|---|---|---|---|---|---|---|---|---|---|
| Sheep | Goat | Control | Treated | At 28th day | At 42nd day | RMSE | S | D | T | |
| MDA blood plasma μμ TMP | 0.197a | 0.241b | 0.201A | 0.236B | 0.277x | 0.161y | 0.013 | b | a | c |
| MDA milk μμ TMP | 0.251a | 0.199b | 0.223 | 0.227 | 0.184x | 0.266y | 0.024 | a | NS | c |
| PC blood plasma nmol/mg protein | 0.013 | 0.010 | 0.010 | 0.012 | 0.011 | 0.012 | 0.002 | NS | NS | NS |
| PC milk nmol/mg protein | 0.027a | 0.083b | 0.049 | 0.061 | 0.056 | 0.054 | 0.012 | c | NS | NS |
| Transfer rate | ||||||||||
| C18:3n-3 | 4.709 | 5.390 | 5.221 | 4.879 | 5.109 | 4.990 | 0.491 | NS | NS | NS |
| C18:2n-6 | 6.852a | 8.208b | 8.081A | 6.978B | 7.795x | 7.265y | 0.470 | a | a | a |
| C20:5n-3 | 1.716a | 1.445b | 0.000A | 3.160B | 1.619x | 1.542y | 0.259 | a | c | a |
| C22:6n-3 | 7.078a | 6.447b | 0.000A | 13.525B | 6.694 | 6.831 | 0.546 | a | c | NS |
- RMSE, root mean square error.
- Means with different superscripts in each row [between the two animal species (a, b), between the two diets (A, B) and between the two sampling time (x, y)] for each parameter differ significantly (p ≤ 0.05).
- a p < 0.05.
- b p < 0.01.
- c p < 0.001.
The dietary supplementation with a combination of SFO caused a significant increase in milk CAT enzyme activity in both animal species (Table 3). This enhancement in milk CAT enzyme activity may be again explained by the presence of antioxidant compounds (although not determined in this study) in the supplemented oils as previously described for blood. Indeed, the inclusion of different fat supplements, including fish oil, in the diets of cows caused a significant increase in milk α-retinol and α-tocopherol concentrations, which was accompanied by an improvement in its antioxidant capacity (Puppel et al., 2013). Moreover, Puppel et al. (2013) found that the dietary fat supplementation in cows also resulted in a significant reduction in the milk MDA content in the samples collected at the 21th day of supplementation period when compared with the samples collected at the 1st day. In this study, the SFO diet resulted in significantly higher FRAP values, in the milk of goats only (data not shown), in order to protect the milk from possible oxidation as a consequence of its enrichment with PUFA supplemented by the SFO diet. This assumption was further supported by the fact that the goats had significantly higher C18:2n-6 transfer rate compared with sheep (Table 2).
| Species (S) | Diets (D) | Time (T) | Effects | |||||||
|---|---|---|---|---|---|---|---|---|---|---|
| Sheep | Goat | Control | Treated | At 28th day | At 42nd day | RMSE | S | D | T | |
| SOD Units/ml | 93.10a | 59.53b | 77.64 | 74.99 | 77.70 | 74.92 | 5.336 | c | NS | NS |
| SOD Units/mg of protein | 1.446a | 1.666b | 1.519 | 1.593 | 1.589 | 1.523 | 0.101 | a | NS | NS |
| GR Units/ml | 0.295a | 0.098b | 0.229 | 0.163 | 0.220x | 0.173y | 0.052 | c | NS | b |
| GR Units/mg of protein | 0.005 | 0.003 | 0.004 | 0.003 | 0.004 | 0.003 | 0.001 | NS | NS | NS |
| CAT Units/ml | 113.85 | 120.97 | 95.01A | 139.81B | 102.43x | 132.39y | 13.395 | NS | b | a |
| CAT Units/mg of protein | 1.806a | 3.422b | 1.961A | 3.268B | 2.230x | 2.999y | 0.404 | c | b | b |
| LPO† Units/ml | 0.152 | 0.098 | 0.120 | 0.130 | 0.135 | 0.115 | 0.047 | NS | NS | NS |
| LPO† Units/mg of protein | 0.002 | 0.003 | 0.002 | 0.003 | 0.003 | 0.002 | 0.001 | NS | NS | NS |
| FRAP μmol ascorbic acid | 2.024a | 1.628b | 1.766 | 1.886 | 1.753 | 1.899 | 0.159 | a | NS | NS |
| ABTS μm ascorbic acid | 109.67a | 59.15b | 85.83 | 82.99 | 85.20 | 83.62 | 2.856 | c | NS | NS |
- SOD, superoxide dismutase; GR, glutathione reductase; CAT, catalase; LPO, lactoperoxidase; FRAP, ferric reducing ability of plasma; ABTS, 2,2′-azino-di(3-ethylbenzothiazoline-6-sulphonic acid); RMSE, root mean square error.
- Means with different superscripts in each row [between the two animal species (a, b), between the two diets (A, B) and between the two sampling time (x, y)] for each parameter differ significantly (p ≤ 0.05).
- a p < 0.05.
- b p < 0.01.
- c p < 0.001.
Protein carbonyl (PC) contents are widely analysed as a measure of protein oxidation. Protein carbonylation can be achieved either through a variety of reactions or from an indirect mechanism involving the hydroxyl radical-mediated oxidation of lipids (Grimsrud et al., 2008). It has been suggested that protein carbonyls formed from lipid-derived aldehydes are more prevalent than those formed via direct amino acid side chain oxidation (Yuan et al., 2007). In this study, in both the milk and plasma of sheep fed with SFO diet, significantly higher PC concentrations were observed (data not shown). This may occur because the apparent transfer rate of C20:5n-3 and C22:6n-3 FA from the diet to milk was significantly higher in sheep than that in goats, while the opposite occurred for the apparent transfer rate of C18:2n-6 FA (Table 2). It has been suggested that the n-3 PUFAs are easily oxidized due to the presence of its numerous double bonds (Nielsen et al., 2000; Nagyova et al., 2001). Indeed, an increase in free radical production in prostate cells, supplemented with C18:3n-3 FA, compared with those supplemented with C18:2n-6 in an in vitro study, was found by Meng et al. (2013). Accordingly, Scislowski et al. (2005a) observed that the production of conjugated diene, derived from PUFA peroxidation, was higher in the plasma of steers given an oil rich in C18:3n-3 FA than in the plasma of those given an oil rich in C18:2n-6 FA.
Principal component analysis (PCA) was applied to pooled data of enzyme activities (SOD, GR, CAT, GST), oxidative stress indicators (FRAP, ABTS) and oxidative stress biomarkers (MDA, PC) in the blood plasma in order to investigate the relationships between the variables and detect those enzyme activities or lipid peroxidation capable of distinguishing samples by the animal species, the dietary treatment and the sampling time. PCA resulted in four principal components, which accounted for 73.58% of the total variability. Enzyme activities and oxidative stress indicators are represented in Fig. 2 as a function of both first and second principal components. The first principal component explained 26.46% of the total variability and was defined by FRAP, MDA and ABTS. These indicators were located away from the axis origin, suggesting that they were well represented by the first PC. FRAP and MDA were placed closed together on the positive side of the horizontal axis, indicating a strong positive correlation which showed that the organism, in order to face the increase in MDA content, activates some antioxidant factors that are determined using FRAP. On the other hand, these oxidative stress indicators were lying opposite to ABTS, and therefore, they were found to be negatively correlated with this. The first PC could be considered as a representative of the animal species, because most of the samples collected from sheep were placed on the negative side of PC1, near ABTS, and therefore, they had higher contents of ABTS, compared to the samples from goat; the samples that were placed on the positive side of PC1, near MDA and FRAP, had the highest contents of these oxidative stress indicators. The second principal component explained another 18.12% of the total variability and was defined by CAT, GR and PC. CAT and GR were located closed together on the positive side of PC2, indicating that they were found to be positively correlated with each other (Fig. 2). Samples from animals treated with SFO diet were also placed near them, and therefore, these samples had higher contents of CAT and GR than the samples from animals that treated with control diet: these samples were placed on the negative side of PC2, opposite to CAT and GR and near PC, and had the highest contents of PC. Consequently, the second principal component could be considered as a representative of dietary treatment. Samples from animals fed with SFO diet were thoroughly distinguished from the samples from the animals treated with the control diet. The third principal component explained another 15.53% of the total variability and was defined by GST. Samples from goats treated with SFO diet were clustered near this enzyme and had higher contents of GST than samples that represented the opposite characteristics. The fourth principal component explained another 13.46% of the total variability and was defined by SOD. This enzyme was placed near the axis origin, indicating that it remained unchanged by SFO diet. However, the discrimination of samples according to the sampling time was not attained, because many misclassifications were observed.
Principal component analysis (PCA) was also applied to pooled data of enzyme activities (SOD, GR, CAT, LPO), oxidative stress indicators (FRAP, ABTS), oxidative stress biomarkers (MDA, PC) and transfer rates of C18:2n-6, C18:3n-3, C20:5n-3 and C22:6n-3 in the milk in order to investigate the relationships between the variables and detect those enzyme activities or lipid peroxidation or transfer rates of fatty acids capable of distinguishing samples by the animal species, the dietary treatment and the sampling time. PCA resulted in three principal components, which accounted for 70.81% of the total variability. Enzyme activities, oxidative stress indicators and transfer rates of fatty acids are represented in Fig. 3 as a function of both first and second principal components. The first principal component explained 35.99% of the total variability and was defined by GR, SOD, ABTS, PC and transfer rates of C18:2n-6 and C18:3n-3. GR, SOD and ABTS were placed closed together on the positive side of the PC1, indicating that they were found to be positively correlated with each other and negatively correlated with PC and transfer rates of C18:2n-6 and C18:3n-3. The first principal component could be considered as a representative of the animal species, because all samples collected from the sheep were placed on the positive side of PC1, near GR, SOD and ABTS, and had higher contents of them than the samples from the goat; these samples were located on the negative side of PC1, near PC and transfer rates of C18:2n-6 and C18:3n-3. The second principal component explained another 23.21% of the total variability and was defined by CAT and transfer rates of C20:5n-3 and C22:6n-3. All of them were clustered on the positive side of PC2, indicating a strong positive correlation which might show that there was an increase in CAT activity in the milk in order to prevent possible oxidation of C20:5n-3 and C22:6n-3 FA. Samples from the animals treated with SFO diet were also placed on the positive side of PC2, which were thoroughly distinguished from the samples from the animals treated with control diet. Consequently, the second principal component could be considered as a representative of dietary treatment. The third principal component explained another 11.60% of the total variability and was defined by LPO, MDA and FRAP. Samples collected from the sheep were placed near these indicators and had higher contents of them, and samples from the animals treated with SFO diet also had higher contents of LPO than the samples that represented the opposite characteristics. As in the case of samples from the blood plasma, the discrimination of samples according to the sampling time was not attained.
Conclusions
The SFO diet significantly increased the activities of CAT and GR in the blood plasma of both animal species and the GST and FRAP values in goats only. The enhancement in the antioxidant defence system of animals may be due to the antioxidant compounds present in soya bean oil and fish oil, although they were not determined in this study. However, the fact that the SFO diet resulted in a significant increase in the MDA and PC contents in the blood plasma of goats and sheep, respectively, indicates that the enhancement in the antioxidant enzymes maybe is, also, a reaction of the organism to face the oxidative stress. Moreover, in the milk of sheep and goats fed with the SFO diet, a significantly increased CAT activity was found, which in the case of sheep was accompanied by higher PC content possibly due to its higher transfer rate of C20:5n-3 and C22:6n-3 FA from the diet to the milk. Finally, great species differences exist among the two animal species (sheep vs. goats) with respect to activities of the antioxidant enzymes: sheep fed diet with SFO seem to be more sensitive to protein oxidation when compared with goats fed diet with SFO.
