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Effect of PUFAs-ω3 and ω6 on oxidative stress of sheep erythrocytes

BMC Veterinary Research volume 21, Article number: 334 (2025) Cite this article

Abstract

Background

In recent years, the use of long-chain polyunsaturated fatty acids (PUFA) ω3 and ω6, as food supplements in livestock has increased due to their beneficial properties related to their antioxidant activity. It has been demonstrated however that a high intake of these substances has prooxidant and cell-damaging effects, especially if their circulating concentrations are unbalanced. Starting from these premises, and taking advantage of previous findings, the present study aimed at defining the optimal circulating concentrations and PUFAs ω3/ω6 ratio, to ensure the antioxidant/oxidant balance in sheep RBCs.

Results

All tested concentrations (25–300 µg/mL in PBS) of PUFAs-ω3 after 4 h of treatment on sheep RBCs, showed antioxidant properties with a significant decrease in reactive oxygen species (ROS) versus the control group (CTRL) (p < 0.05). Furthermore, ω6 showed an antioxidant effect at low concentrations (25–200 g/mL) but a pro-oxidant effect at the highest concentrations (250 and 300 µg/mL) with a significant increase in ROS production (123.6 ± 2.1 and 131.4 ± 6.5% sloope RFU of CTRL respectively p < 0,001), malondialdehyde (MDA) (p < 0.01), and haemolysis (p < 0.01) versus CTRL group (1.1 ± 0.1%), and, also with a decrease of Trolox equivalent antioxidant capacity (TEAC) (p < 0,05). The ratio ω3/ω6 of 1:10 (25/250 µg/mL) and 1:4 (25/100 µg/mL) showed an intracellular ROS level like the CTRL group whereas, the ratio 1:2 (100/200 µg/mL) resulted in a significant decrease in ROS production (62.71 ± 2.31% slope RFU of CTRL, p < 0.001) and MDA (p < 0.001), with an increase in TEAC (p < 0.05), and a decrease haemolysis versus the control group (p < 0,01).

Conclusions

Our results showed that a beneficial effect on the oxidative state of sheep RBCs was obtained with in vitro administration of low concentrations of ω6 and with all tested concentrations of ω3. The addition of ω6 at high concentrations leads to an imbalance in the PUFA ω3/ω6 ratio, compromising the oxidative state and viability of the RBCs. The maximum antioxidative effect was found at ω3/ω6 ratio 1:2).

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Introduction

Long-chain polyunsaturated fatty acids omega 3 (PUFAs-ω3) and 6 (PUFAs-ω6) represent one of the most widely used antioxidants. In mammals, the body cannot synthesise them but they must be introduced through diet [1, 2]. PUFA-ω3 and -ω6 differ in the location of the first double bond, starting from the methyl end of the molecule. Linoleic acid (LA) is the predominant PUFA-ω6 in nature and it is found in almost all plants except coconut, cocoa, and palm. The most important PUFA-ω3, alpha-linolenic acid (ALA), is found in green leafy vegetables, seeds of flax, rape, chia, perilla, and walnuts [2]. The main biological processes in which they are involved are vision, growth, brain development and reproduction [1, 2, 6].

PUFAs-ω3 and -ω6 are considered the most susceptible reactive oxygen species (ROS) substrates during oxidative stress (OS) conditions [5]. Nutritional epidemiology studies and clinical trials confirm the beneficial effects of food consumption in -ω3 and -ω6 on preventing coronary artery diseases, stroke and dementia in humans [4]. Moreover, a diet enriched in PUFAs-ω3 can prevent vascular dysfunction via multiple mechanisms, including an antioxidant action through the increased activity of endogenous antioxidant response [6, 7]. PUFA-ω3 has also been shown to positively affect the reproductive system in dairy cattle [8]. In a previous work in Sarda sheep [9] we showed that a diet integrated with by-pass linseed oil (LO) rich in PUFAs-ω3 provided beneficial effects on the function of the ovarian corpus luteum. Moreover, it proved to have a positive impact on antioxidative defences on maternal structures during the embryo-maternal recognition period in ewes particularly on the luteal tissues, with an increase in total antioxidant capacity (TEAC), in the enzymatic activity of antioxidant defences and total thiols [9].

The interaction between -ω3 and -ω6 metabolism in the context of inflammation is not fully understood, but it is thought that PUFAs-ω3 and -ω6 likely work synergistically to produce anti-inflammatory effects [10, 11]. However, it is also necessary to underline that a high intake of PUFAs-ω6 in the diet can shift the physiological state to a proinflammatory and prothrombotic one [12]. The metabolism of ω6 specifically produces eicosanoids such as prostaglandins, thromboxanes, leukotrienes, hydroxy fatty acids, and lipoxins in greater quantity than those derived from PUFAs-ω3 [13]. These products, already biologically active in small amounts, contribute to inflammatory disorders, thrombus formation and allergic processes, particularly in susceptible subjects [14]. These inflammation processes are associated with oxidation phenomena and are implicated in initiating atherosclerosis and endothelial and blood cell dysfunction [11].

In non-human primates and rats, dietary supplementation rich in -ω3 did not modify the ratio of unsaturated/saturated fatty acids in the erythrocyte membranes but only the concentrations of unsaturated fatty acids. In fact, after 12 weeks of -ω3 supplementation, in the RBCs membrane, PUFAs-ω3 increased and PUFAs-ω6 decreased, while concentrations of saturated fatty acids remained unchanged. This alteration of the fatty acid profile of the RBC membranes could have a potential biological impact on the inflammation process, lipid metabolism, and overall health state [15, 16].

Therefore, the balance of PUFAs-ω3 and -ω6 in the diet is crucial because they can have opposing physiological effects. Considering that the mammalian cell cannot convert PUFAs-ω3 into -ω6, due to the lack of the enzyme ω3 desaturase, it is necessary to supply the body with a balanced quantity of these substances [17]. For this reason, many studies investigated the effects of different PUFA ratios in order to identify the optimal dietary intake [18,19,20,21,22].

For example, Ghazali et al. reported that in hepatic cells low ω3/ω6 ratios (1:15, 1:25) damaged mitochondrial functions, increasing ROS production, and reducing mitochondrial respiratory chain and ATP production; on the contrary, at higher ratios (1:1 and 1:4) hepatic cells showed the same mitochondrial functionality as the control [22]. Some researchers have suggested reducing PUFAs-ω6 consumption [18, 23, 24], or increasing ω3 intake [18, 25] to improve the ω3/ω6 ratio and reduce cardiovascular diseases. Furthermore, several authors agree on the ideal ω3/ω6 ratio of 1:1 or 1:2 for treating cancer [18, 26].

Based on these findings, the present study was designed to determine the concentrations of PUFAs ω3, -ω6 and their ratio that would ensure the optimal antioxidant/oxidant balance in sheep red blood cells (RBCs). Oxidative stress in cells results from an imbalance between the level of reactive oxygen species (ROS) and antioxidant defence systems [27, 28]. Since RBCs are naturally exposed to circulating concentrations of PUFAs ω3/ω6 that can vary with the animal’s diet, we investigated the effects of different ratios of PUFAs ω3/ω6 on the redox state of sheep erythrocytes in vitro.

Results

All tested concentrations of PUFAs-ω3 showed antioxidant properties with a significant (p < 0.05) decrease in ROS production versus CTRL group (Fig. 1). In contrast, -ω6 showed an antioxidant effect at concentrations ≤ 200 µg/mL, and a pro-oxidant effect at the highest concentrations (250 and 300 µg/mL), with a significant (p < 0.001) increase of ROS production versus CTRL group (123.61 ± 2.08 and 131.45 ± 6.58% slope RFU of CTRL respectively) (Fig. 1).

Fig. 1
figure 1

Intracellular ROS production in sheep RBCs using H2DCF-DA and measuring RFU (RFU measurement RFU = Relative Fluorescent Units) production every 10 min for 4 h. RBCs were treated in vitro at 37 °C with PUFAs-ω3 and PUFAs-ω6 at different concentrations individually (25, 50, 100, 150, 200, 250, and 300 µg/mL), and using the ratio ω3/ω6 of 1:2 (100/200), 1:4 (25/100 µg/mL), 1:10 (25/250 µg/mL). Untreated RBCs at 37 °C for 4 h represent the control group (CTRL). Uppercase letters indicate significant statistical differences between groups of RBCs supplemented with different concentrations of PUFAs (red bars indicate groups with added ω3. Blue bars indicate groups with added ω6. Blue/red bars indicate groups with added ω3/ω6 ratio. Black bar indicated CTRL): p < 0.05 (one-way ANOVA)

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The combination of PUFAs-ω3/-ω6 at a 1:2 ratio, resulted in a significant decrease in ROS production (62.71 ± 2.31% slope RFU of CTRL) in sheep erythrocytes (p < 0.01) vs. CTRL, while lower ratios were not effective in this regard (Fig. 1).

The concentrations of total thiols did not change significantly when compared to the CTRL group for any investigated PUFA concentrations and ω3/ω6 ratios (Fig. 2A). TEAC significantly decreased (p < 0.05) with ω6 at 250 and 300 µg/mL (5.78 ± 0.05 and 5.87 ± 0.27 nmol equivalent Trolox/106 RBCs respectively), whereas increased (p < 0.05) in the presence of the ω3/ω6 ratio 1:2 (8.31 ± 0.15 nmol equivalent Trolox/106 RBCs) when compared to CTRL (7.02 ± 0.39 nmol equivalent Trolox/106 RBCs) (Fig. 2B). Moreover, MDA levels significantly increased with ω6 at 250 and 300 µg/mL (p < 0.01) (3.23 ± 0.25 and 3.46 ± 0.22 nmol MDA/106RBCs respectively) and decreased with the ratio 1:2 (p < 0.01) (2.18 ± 0.07 nmol MDA/106RBCs), when compared to CTRL group (2.67 ± 0.09 nmol MDA/106RBCs) (Fig. 2C). For all the other tested concentrations and ratios, no significant changes versus CTRL groups in TEAC and MDA levels were found (Fig. 2B and C).

Fig. 2
figure 2

Total thiols (A), TEAC (B), and MDA (C) assay in sheep RBCs treated for 4 h in vitro at 37 °C with PUFAs-ω3 and PUFAs-ω6 at different concentrations individually (25, 100, 200, 250 and 300 µg/mL), and using the ratio ω3/ω6 of 1:2 (100/200 µg/mL), 1:4 (25/100 µg/mL), 1:10 (25/250 µg/mL). Untreated RBCs after 4 h at 37 °C represent the control group (CTRL). Uppercase letters indicate significant statistical differences between groups of RBCs supplemented with different concentrations of PUFAs (red bars indicate groups with added ω3. Blue bars indicate groups with added ω6. Blue/red bars indicate groups with added ω3/ω6 ratio. Black bar indicated CTRL): p < 0.05 (one-way ANOVA)

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Given the intracellular ROS change, both RBCs’ morphological characteristics (Fig. 3) and haemolytic status (Fig. 4) showed significant differences among treated groups.

Fig. 3
figure 3

Morphological evaluation of sheep RBCs treated for 4 h in vitro at 37 °C with different concentrations of PUFAs-ω3 and PUFAs-ω6. In Fig. 3A the % damaged RBCs are reported for the CTRL, and the highest concentrations of PUFAs-ω3 and PUFAs-ω6 (250 and 300 µg/mL), and the ratio ω3/ω6 of 1:2 (100/200 µg/mL) and 1:4 (25/100 µg/mL). Uppercase letters indicate significant differences, between groups: p < 0.001 (one-way ANOVA). Figure 3B and C show microscopic images of healthy and damaged RBCs, respectively. White arrows indicate examples of damaged erythrocytes (Fig. 3C)

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Fig. 4
figure 4

Effect of the different treatments on haemolysis. Relative haemoglobin percentage of sheep RBCs treated for 4 h in vitro at 37 °C with different concentrations of PUFAs-ω3 and PUFAs-ω6 (25, 100, 200, and 250 µg/mL), and using the ratio ω3/ω6 of 1:2 (100/200 µg/mL), 1:4 (25/100 µg/mL), 1:10 (25/250 µg/mL). Untreated RBCs after 4 h at 37 °C represent the control group (CTRL). Uppercase letters indicate significant differences, between groups of RBCs supplemented with different concentrations of PUFAs Uppercase letters indicate significant statistical differences between groups of RBCs supplemented with different concentrations of PUFAs (red bars indicate groups with added ω3. Blue bars indicate groups with added ω6. Blue/red bars indicate groups with added ω3/ω6 ratio. Black bar indicated CTRL): p < 0.05 (one-way ANOVA)

Full size image

As shown in Fig. 3, the sheep RBCs treated with the highest concentrations of ω3 (250 and 300 µg/mL) had an aspect similar to the control (% damaged RBCs 1.09 ± 0.08 and 1.25 ± 0.05%, respectively, versus CTRL 1.12 ± 0.03%). In contrast, the corresponding concentrations of ω6 showed a damaged status (% damaged RBCs of 17.2 ± 0.70 and 15.9 ± 0.86%, respectively) when compared to the CTRL (p < 0.001). However, when ω6 was combined with ω3 the erythrocytes’ aspect returned like the control (% damaged RBCs of 0.99 and 1.3% for 1:2 and 1:4 ratios, respectively). Moreover, the relative haemoglobin percentage was significantly (p < 0.01) lower than CTRL for all ω3 concentrations (50 and 150 µg/mL were not shown), for the lowest ω6 concentrations (from 25 to 150 µg/mL) and ω3/ω6 ratio 1:2. The haemolysis percentage for ω6 200 µg/mL and for ω3/ω6 ratios 1:4 and 1:10 was comparable to the CTRL group (Fig. 4). Instead, the highest ω6 concentrations (250 and 300 µg/mL) showed a significant increase (p < 0.01) in the % RBCs haemolysis when compared to the control (Fig. 4).

Discussion

In recent years, dietary supplementation with ω3 and ω6 has become highly popular among humans and animals [9, 29, 30]. PUFAs-ω3 and -ω6 have demonstrated antioxidant and anti-inflammatory properties [31]. However, several studies have indicated that a diet high in PUFAs-ω6 may lead to a shift from a physiological to prothrombotic and pro-aggregatory state, resulting in increased blood viscosity, vasospasm, and vasoconstriction, and a decreased bleeding time [32, 33]. Furthermore, increased dietary intake of ω6 leads to oxidation of low-density lipoprotein, platelet aggregation, and interferes with the incorporation of essential fatty acids in cell membrane phospholipids [34].

Although in recent years, the consumption of antioxidant substances as supplements to the normal diet has been a diffused trend to reduce the negative effects of OS, a surplus could make them pro-oxidants, generating cell-damaging effects [35,36,37,38,39,40,41]. Therefore, it is important to maintain the right ω3/ω6 PUFA ratio in the bloodstream [18, 42, 43].

In a recent paper, a positive antioxidant effect of PUFAs-ω3 on plasma and reproductive tissues (uterus and corpus luteum), after supplementation with linseed oil (rich in PUFAs-ω3), during maternal recognition of the pregnancy period in ewes, has been reported [9]. After this supplementation, the ω3/ω6 ratio in sheep blood plasma was 1:2, versus 1:5 for non-supplemented sheep [9]. The authors indicated a protective effect of LO from OS on maternal structures, guaranteeing embryo survival.

Considering that erythrocytes are naturally exposed to circulating concentrations of PUFAs ω3/ω6 and that these concentrations change according to the animal’s diet, we investigated in vitro the effects of different concentrations and different ratios of PUFAs ω3/ω6 on the redox status of sheep erythrocytes.

The results of our work showed that for all studied concentrations of ω3, after 4 h of treatment, there was a decrease in ROS production compared to the control; this is in agreement with what was found in “in vitro” cultures for other cell lines [44,45,46]. In addition, with the same PUFA-ω3 concentrations, TEAC, total Thiols, MDA and RBCs morphology did not change compared to the control. The observed decrease in ROS, with the non-change in the other assayed parameters on our cell model, could suggest a capacity of ω3 to act as an ROS scavenger without changing the concentration of cellular antioxidants, as also reported by Doriane et al. [47] for human aortic endothelial cells.

Moreover, we observed a decrease in haemolysis percentage versus the control with all tested concentrations of PUFA-ω3. The literature reported that by adding fatty acids, the biophysical parameters of cell membranes changed, including their fluidity, and deformability [43, 48]. In particular, Bazzano et al. [49] reported that the supplementation with PUFA-ω3 in horses showed reduced osmotic fragility of erythrocytes and, therefore, greater resistance to haemolysis than horses without supplementation in their diet after show jumping courses. Also, other researchers reported the same effect of PUFA-ω3 on decreased haemolysis in rabbit RBCs [50]. This evidence supports our results for sheep RBCs treated with ω3. However, our results, compared to the literature studies, provide additional information about the different concentrations of ω3 used.

Unlike PUFA-ω3, the -ω6, at the highest concentrations (250 and 300 µg/mL) tested, showed a prooxidant effect on RBCs with a significant increase of ROS production and MDA levels, and a decrease in TEAC versus CTRL. This framework suggests the presence of OS with decreased antioxidant defences, increased ROS levels and lipid peroxidation with consequent membrane damage, confirmed by increased haemolysis and alterations in the form and aspect of erythrocytes.

To our knowledge, the effect of ω6 on erythrocytes has not yet been reported. Despite the relationship between PUFAs-ω6 and ROS being poorly investigated in animal cells [51], in humans it has been observed that LA induces ROS production in hepatic cell lines [52, 53] and causes apoptosis, increasing lipid peroxidation in carcinoma cells [52, 54]. Furthermore, Doriane R. et al. [47] reported that supplementation of human aortic endothelial cells with PUFAs-ω3 resulted in a lower production of ROS compared to cells supplemented with PUFAs-ω6.

This trend would confirm what we found in the RBCs and explain the increased haemolysis at high concentrations of -ω6, which acted as a pro-oxidant. Under physiological conditions, the structural alterations linked to oxidative damage lead to the removal of senescent erythrocytes from circulation through an eryptosis process [27]. Under in vitro conditions, as reported by Baralla et al. [55], sheep RBCs, damaged by treatment with pro-oxidant molecules, undergo haemolysis due to membrane damage since they cannot be eliminated by eryptosis.

Several studies also focused on the effect of ω3/ω6 ratio. For example, a decrease of ω3/ω6 ratio was linked to inflammation and chronic diseases [56]. On the contrary, a meta-analysis study indicated that the intake of PUFA-ω3, but not of -ω6 (resulting in a higher ω3/ω6 ratio in the blood), was associated with a lower risk of metabolic syndrome [57]. Also, Park et al. reported the beneficial effect of a higher ω3/ω6 ratio on metabolic dysfunction in mice, inhibiting hepatic triglyceride accumulation and inflammation [58]. Contreras-Solis et al. also report a beneficial effect of a high ratio ω3/ω6 in sheep pregnancy [9].

The results of our study about different ratios ω3/ω6 in sheep erythrocytes confirmed this trend. A protective effect on OS, with a significant decrease in ROS production and MDA and with an increase in TEAC, was found at the 1:2 ratio used. These data were confirmed by a decrease in haemolysis and by RBCs’ morphological aspect.

Conclusion

The results of our in vitro investigation with sheep RBCs, demonstrated the antioxidant properties of PUFA-ω3 and the prooxidant effect of PUFA-ω6 at high concentrations. However, when ω3 and ω6 were combined in different ratios, the prooxidant effect of ω6 was neutralised, emphasising the importance of monitoring the ω3/ω6 ratio for erythrocyte viability. Our findings confirmed that the in vitro supplementation of ω3/ω6 at a 1:2 ratio, improved the antioxidative/oxidative status of sheep RBCs compared to lower ratios (1:4 and 1:10).

Materials and methods

Reagents and animals

All reagents used were purchased from Merck Sigma Aldrich: Fructose (cod: 729051), H2DCF-DA (cod: D6883), Linoleic acid (LA) (cod: L8134), alpha-linolenic acid (ALA) (cod: L2376).

All procedures involving animals in this study were approved by the Local Animal Care and Use Committee of the University of Sassari (Authorization code: 2899 of 17/01/2018). Ewes were all owned by the Department of Veterinary Medicine, and they were confined outdoors with access to a sheltered area, at the experimental facilities of the Department of Veterinary Medicine at the University of Sassari, Italy (40°43′40.33″N, 8°33′1.33″E). The experiments were repeated 4 times and each time, blood samples, from four ewes (different ewes each time, aged between 2 and 4 years, average body weight 40–50 kg), were collected at fasting (07:00 a.m.) from the jugular vein using 9 mL vacuum collection tubes containing EDTA K2 (Vacutainer Systems Europe; Becton Dickinson, Meylan Cedex, France). The mean sheep erythrocyte concentration was 12 × 109/mL. The four blood samples were pooled before treatment and analyses.

Protocol design

Sheep whole blood (pooled blood) was aliquoted and treated with ALA (group ω3, as ALA is the most important PUFA-ω3) and LA (group ω6; as LA is the most important PUFA-ω6) at different concentrations individually (25, 50, 100, 150, 200, 250 and 300 µg/mL), and using the ratio ω3/ω6 of 1:2 (100/200 µg/mL), 1:4 (25/100 µg/mL), 1:10 (25/250 µg/mL) to obtain different treated groups. Every aliquot was incubated at 37 °C for 4 h and used to assay intracellular ROS, total Thiols, Malonyl di Aldehyde (MDA), Trolox Equivalent Antioxidant Capacity (TEAC) and percentage haemolysis as described below; also, the RBCs morphology after treatment was observed. All the assays were performed in quadruplicate. A control group (CTRL) without treatment was incubated for 4 h at 37° C for all experiments.

The concentrations used were chosen based on the PUFAs-ω3 and PUFAs-ω6 circulating concentrations found in sheep after LO administration as a dietary supplement, as described by Contreras-Solís et al. (CTRL PUFAs-ω3 = 0.034 ± 0.014 mg/mL and PUFAs-ω6 = 0.170 ± 0.046 mg/mL PUFAs-ω3/ω6 = 1:5; LO supplemented group PUFAs-ω3 = 0.096 ± 0.034 mg/mL and PUFAs-ω6 = 0.214 ± 0.064 mg/mL PUFAs-ω3/ω6 = 1:2) [9]. In this study, twenty ewes (4.23 ± 0.10 years old Sarda ewes, average body weight 50–52 kg), were fed a diet enriched with microencapsulated LO (18% ALA; SILA™; Verona, Italy), while a separate control group of twenty ewes was fed with an LO-free diet. The experimental period lasted 36 days and concentrations of PUFAs-ω3 and PUFAs-ω6 were determined on day 15 of the experimental phase using Fatty Acids Methyl-Esters (FAMES) analyses [9].

ROS assay

Blood samples from each treatment were further diluted to 7 × 106 RBCs/mL using PBS plus (PBS with 0,1% fructose) to measure ROS production in erythrocytes. To each treated sample, 2′,7′-dichlorodihydrofluorescein diacetate (H2DCF-DA) was added at the final concentration of 3µM. Within the cell, the esterase cleaves the acetate groups on H2DCF-DA, thus trapping the reduced form of the probe 2′,7′-dichlorodihydrofluorescein (H2DCF). Intracellular ROS oxidise H2DCF, yielding the fluorescent product, DCF. After 30 min of incubation at 37 °C, fluorescence was measured using a FLUOstar Omega microplate reader (BMG LABTECH) every 10 min for 4 h (kinetic from t0 to t4). Excitation and emission wavelengths used for fluorescence quantification were 485 and 535 nm, respectively. All fluorescence measurements (RFU = Relative Fluorescence Unit) were corrected for background fluorescence and replicated four times. Kinetic data were expressed as % slope RFU mean ± SD vs. CTRL [55, 59].

Total thiols, TEAC, and MDA assays

Blood cells were treated with PBS containing TritonX100 to 0.1% to obtain the cell extract samples for TEAC, MDA, and total thiols assays. These assays were performed as reported in the previous manuscript [27, 55, 60]. All data were normalised for total cells and were assayed using an automatic cell counter instrument (Haematology analyser Alcyon Mindray BC5000, Shenzhen, China).

Total thiols

Total Thiols were assayed using the Ellman’s Reagent 5,5-dithiol-bis-(2-nitrobenzoic acid) (DTNB) solved in PBS. Thiols were quantified using a spectrophotometer by measuring the absorbance of visible light at 412 nm, using an extinction coefficient of 14,150 M− 1 cm− 1 for dilute buffer solutions [35]. The values of Thiols in the samples were expressed in nmol/106 erythrocytes.

TEAC assay

TEAC was determined using the method described by Reetal. And modified by Lewinska et al. [61]. Briefly, a fresh solution was prepared by dissolving 19.5 mg 2,20-azinobis (3- ethylbenzthiazoline-6-sulphonic acid [ABTS]) and 3.3 mg potassium persulphate in 7 mL of 0.1 mol/L phosphate buffer, pH 7.4. This solution was stored in the dark for 12 h for the completion of the reaction. ABTS solution was diluted (usually approximately 1:80) in 0.1 mol/L phosphate buffer, pH 7.4, to give an absorbance reading at 834 nm of 1.0 and mixed thoroughly. The antioxidant capacity was expressed as the concentration of Trolox producing the same effect as the sample studied, (TEAC). The values of TEAC in the samples were calculated using a standard curve (5–20 mM Trolox in a total volume of 550 mL) and were expressed as nmoles of Trolox equivalent / Cell extract (nmol TEAC/106 erythrocytes).

MDA assay

MDA, one of the several low-molecular-weight end-products of lipid peroxidation, was evaluated by the TBARS assay using Thiobarbituric acid and a spectrophotometric method according to the TBA test described by Spanier and Traylor [62]. The values of MDA in the samples were calculated using a standard curve (2–100 µM) and expressed in nmol/106 erythrocytes.

RBCs morphological evaluation

After incubation (4 h at 37 °C), blood samples were used to prepare blood smears. A slide for each concentration of PUFAs-ω3 and PUFAs-ω6 and their ratios was viewed at high magnification using an inverted system microscope (Olympus X71 model TH4200). An untreated blood smear was used as the control [55, 60]. RBCs with a well-rounded shape and an intact membrane were considered healthy, while those with damaged membranes and irregular shapes were deemed defective. Morphological evaluation was performed using the Cell* Imaging Software for Life Sciences Microscopy, and the damage was expressed as a percentage of damaged RBCs on a total of 500 RBCs for each treatment group.

Haemolysis test

After incubation at 37 °C for 4 h with PUFAs-ω3 and -ω6, blood samples were gently centrifuged (100 g for 3 min), and the supernatant was used for the haemolysis test. The test measured the % haemoglobin released from damaged RBCs in the supernatant, reading the absorbance at 405 nm. Data were expressed as means of % relative haemoglobin ± SD. The absorbance value in the supernatant of sheep RBCs lysed in H2O was considered 100% haemolysis [55, 60, 63].

Statistical analysis

Data are expressed as mean ± SEM of at least four replicates. After checking the normality and homogeneity of variances assumptions, differences between experimental groups were analysed by a one-way ANOVA (SigmaPlot 15.0). Statistical significance was accepted at p < 0.05.

Data availability

The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

References

  1. Wathes DC, Abayasekara DRE, Aitken RJ. Polyunsaturated fatty acids in male and female reproduction. Biol Reprod. 2007;77:190–201.

    Article  CAS  PubMed  Google Scholar 

  2. Kaur N, Chugh V, Gupta AK. Essential fatty acids as functional components of foods- a review. J Food Sci Technol. 2014;51:2289–303.

    Article  CAS  PubMed  Google Scholar 

  3. Moallem U. Invited review: roles of dietary n-3 fatty acids in performance, milk fat composition, and reproductive and immune systems in dairy cattle. J Dairy Sci. 2018;101:8641–61.

    Article  CAS  PubMed  Google Scholar 

  4. Román GC, Jackson RE, Gadhia R, Román AN, Reis J. Mediterranean diet: the role of long-chain ω-3 fatty acids in fish; polyphenols in fruits, vegetables, cereals, coffee, tea, Cacao and wine; probiotics and vitamins in prevention of stroke, age-related cognitive decline, and alzheimer disease. Rev Neurol (Paris). 2019;175:724–41. https://doiorg.publicaciones.saludcastillayleon.es/10.1016/j.neurol.2019.08.005.

    Article  PubMed  Google Scholar 

  5. Mavangira V, Sordillo LM. Role of lipid mediators in the regulation of oxidative stress and inflammatory responses in dairy cattle. Res Vet Sci. 2018;116:4–14.

    Article  CAS  PubMed  Google Scholar 

  6. Didara M, Poljičak-Milas N, Milinković-Tur S, Mašek T, Šuran J, Pavić M, et al. Immune and oxidative response to linseed in the diet of periparturient Holstein cows. Animal. 2015;9:1349–54.

    Article  CAS  PubMed  Google Scholar 

  7. Heshmati J, Morvaridzadeh M, Maroufizadeh S, Akbari A, Yavari M, Amirinejad A et al. Omega-3 fatty acids supplementation and oxidative stress parameters: A systematic review and meta-analysis of clinical trials. Pharmacol Res. 2019;149 June.

  8. Gulliver CE, Friend MA, King BJ, Clayton EH. The role of omega-3 polyunsaturated fatty acids in reproduction of sheep and cattle. Anim Reprod Sci. 2012;131:9–22.

    Article  CAS  PubMed  Google Scholar 

  9. Contreras-Solís I, Pasciu V, Porcu C, Sotgiu FD, Todorova N, Baralla E, Mara L, Gallus M, Cabiddu A, Dattena M, Abecia JA, Berlinguer F. The effect of By-Pass linseed oil supplementation on the maternal antioxidant system during the Embryo-Maternal recognition period in Ewes. Anim (Basel). 2023;13:2565. https://doiorg.publicaciones.saludcastillayleon.es/10.3390/ani13162565.

    Article  Google Scholar 

  10. Shearer GC, Walker RE. An overview of the biologic effects of omega-6 Oxylipins in humans. Prostaglandins Leukot Essent Fat Acids. 2018;137:26–38. 10.1016/ j.plefa.2018.06.005.

    Article  CAS  Google Scholar 

  11. Nayda NC, Thomas JM, Delaney CL, Miller MD. The effect of omega-3 polyunsaturated fatty acid intake on blood levels of omega-3s in people with chronic atherosclerotic disease: a systematic review. Nutr Rev. 2023;81:1447–61. https://doiorg.publicaciones.saludcastillayleon.es/10.1093/nutrit/nuad020.

    Article  PubMed  PubMed Central  Google Scholar 

  12. Simopoulos AP. An increase in the Omega-6/Omega-3 fatty acid ratio increases the risk for obesity. Nutrients. 2016;8:128. https://doiorg.publicaciones.saludcastillayleon.es/10.3390/nu8030128.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Mariamenatu AH, Abdu EM. Overconsumption of Omega-6 polyunsaturated fatty acids (PUFAs) versus deficiency of Omega-3 PUFAs in Modern-Day diets: the disturbing factor for their balanced antagonistic metabolic functions in the human body. J Lipids. 2021;17:8848161. https://doiorg.publicaciones.saludcastillayleon.es/10.1155/2021/8848161.

    Article  CAS  Google Scholar 

  14. Simopoulos AP. The importance of the omega-6/omega-3 fatty acid ratio in cardiovascular disease and other chronic diseases. Exp Biol Med. 2008;233:674–88. https://doiorg.publicaciones.saludcastillayleon.es/10.3181/0711-MR-311.

    Article  CAS  Google Scholar 

  15. Hals PA, Wang X, Piscitelli FDMV, Xiao Y-F. The time course of erythrocyte membrane fatty acid concentrations during and after treatment of non-human primates with increasing doses of an omega-3 rich phospholipid Preparation derived from krill-oil. Lipids Heal Dis (BioMed Cent. 2017;16:16.

    Article  Google Scholar 

  16. Shen Y, Sen A, Turgeon DK, Ren J, Graifman G, Ruffin MT, Smith WLB, DE, Djuric Z. Changes in serum, red blood cell, and colonic fatty acids in a personalized Omega-3 fatty acid supplementation trial. Nutr Cancer. 2021;:1–14.

  17. Lee JM, Lee H, Kang S, Park WJ. Fatty acid desaturases, polyunsaturated fatty acid regulation, and biotechnological advances. Nutrients. 2016;8:23. https://doiorg.publicaciones.saludcastillayleon.es/10.3390/nu8010023.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Gómez Candela C, Bermejo López LM, Loria Kohen V. Importance of a balanced Omega 6/omega 3 ratio for the maintenance of health: nutritional recommendations. Nutr Hosp. 2011;26:323–9. https://doiorg.publicaciones.saludcastillayleon.es/10.1590/S0212-16112011000200013.

    Article  PubMed  Google Scholar 

  19. Attia YAA-HM, Al-Sagan AA, Alqurashi AD, Korish MAAN, Olal MJ, Bovera FD. Dietary supplementation with different ω-6 to ω-3 fatty acid ratios affects the sustainability of performance, egg quality, fatty acid profile, immunity and egg health indices of laying hens. Agric ·. 2022;12:1712. https://doiorg.publicaciones.saludcastillayleon.es/10.3390/agriculture12101712.

    Article  CAS  Google Scholar 

  20. Liput KP, Lepczyński A, Ogłuszka M, Nawrocka A, Poławska E, Grzesiak A, Ślaska B, Pareek CS, Czarnik U, Pierzchała M. Effects of dietary n-3 and n-6 polyunsaturated fatty acids in inflammation and cancerogenesis. Int J Mol Sci. 2021;22:6965. https://doiorg.publicaciones.saludcastillayleon.es/10.3390/ijms22136965.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Horman T, Fernandes MF, Tache MC, Hucik B, Mutch DM, Leri F. Dietary n-6/n-3 ratio influences brain fatty acid composition in adult rats. Nutrients. 2020;12:1847. https://doiorg.publicaciones.saludcastillayleon.es/10.3390/nu12061847.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Ghazali R, Mehta KJ, Bligh SA, Tewfik I, Clemens D, Patel VB. High Omega arachidonic acid/docosahexaenoic acid ratio induces mitochondrial dysfunction and altered lipid metabolism in human hepatoma cells. World J Hepatol. 2020;12:84–98. https://doiorg.publicaciones.saludcastillayleon.es/10.4254/wjh.v12.i3.84.

    Article  PubMed  PubMed Central  Google Scholar 

  23. Harris WS, Mozaffarian D, Rimm E, Kris-Etherton P, Rudel LL, Appel LJ, Engler MM, Engler MB, Sacks F. Omega-6 fatty acids and risk for cardiovascular disease: a science advisory from the American heart association nutrition subcommittee of the Council on nutrition, physical activity, and metabolism; Council on cardiovascular nursing; and Council on epidem. Circulation. 2009;119:902–7. https://doiorg.publicaciones.saludcastillayleon.es/10.1161/CIRCULATIONAHA.108.191627.

    Article  PubMed  Google Scholar 

  24. Willett WC. The role of dietary n-6 fatty acids in the prevention of cardiovascular disease. J Cardiovasc Med (Hagerstown). suppl 2007;1:S42–5. https://doiorg.publicaciones.saludcastillayleon.es/10.2459/01.JCM.0000289275.72556.13.

    Article  Google Scholar 

  25. BA. G. How relevant is the ratio of dietary n-6 to n-3 polyunsaturated fatty acids to cardiovascular disease risk? Evidence from the OPTILIP study. Curr Opin Lipidol. 2008;19:57–62. https://doiorg.publicaciones.saludcastillayleon.es/10.1097/MOL.0b013e3282f2e2a8

  26. Granados S, Quiles JL, Gil A, Ramírez-Tortosa MC. Lípidos de La Dieta y cáncer. Nutr Hosp. 2006;21:44–54.

    Google Scholar 

  27. Pasciu V, Nieddu M, Sotgiu FD, Baralla E, Berlinguer F. An overview on assay methods to quantify ROS and enzymatic antioxidants in erythrocytes and spermatozoa of small domestic ruminants. Anim. 2023;13:2300. https://doiorg.publicaciones.saludcastillayleon.es/10.3390/ani13142300.

    Article  Google Scholar 

  28. Bo L, Jiang S, Xie Y, Kan H, Song W, Zhao J. Effect of vitamin E and Omega-3 fatty acids on protecting ambient PM2.5-Induced inflammatory response and oxidative stress in vascular endothelial cells. PLoS ONE. 2016;23(11):e0152216. https://doiorg.publicaciones.saludcastillayleon.es/10.1371/journal.pone.0152216.

    Article  CAS  Google Scholar 

  29. Zhang Q, Xu Q, Tian H, Chu Y, Qiu J, Sun M. Serum and diet long-chain omega-3 fatty acid nutritional status in Chinese elite athletes. Lipids. 2023;58:33–40. https://doiorg.publicaciones.saludcastillayleon.es/10.1002/lipd.12362.

    Article  CAS  PubMed  Google Scholar 

  30. Contreras-Solís I, Porcu C, Sotgiu FD, Chessa F, Pasciu V, Dattena M, et al. Effect of strategic supplementation of dietary By-Pass linseed oil on fertility and milk quality in Sarda Ewes. Animals. 2023;13:1–13.

    Article  Google Scholar 

  31. Oppedisano F, Macrì R, Gliozzi M, Musolino V, Carresi C, Maiuolo J, Bosco F, Nucera S, Caterina Zito M, Guarnieri L, Scarano F, Nicita C, Coppoletta AR, Ruga S, Scicchitano M, Mollace R, Palma E, Mollace V. The anti-inflammatory and antioxidant properties of n-3 PUFAs: their role in cardiovascular protection. Biomedicines. 2020;8:306. https://doiorg.publicaciones.saludcastillayleon.es/10.3390/biomedicines8090306.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Simopoulos AP. The omega-6/omega-3 fatty acid ratio, genetic variation, and cardiovascular disease. Asia Pac J Clin Nutr. 2008;17:131–4.

    CAS  PubMed  Google Scholar 

  33. Nguyen TX, Agazzi A, Comi M, Bontempo V, Guido I, Panseri S, Sauerwein H, Eckersall PD, Burchmore R, Savoini G. Effects of low Ω6:ω3 ratio in Sow diet and seaweed supplement in piglet diet on performance, colostrum and milk fatty acid profiles, and oxidative status. Animals. 2020;10:2049. https://doiorg.publicaciones.saludcastillayleon.es/10.3390/ani10112049.

    Article  PubMed  PubMed Central  Google Scholar 

  34. Simopoulos AP. Evolutionary aspects of diet, the omega-6/omega-3 ratio and genetic variation: nutritional implications for chronic diseases author links open overlay panel. Biomed Pharmacother. 2006;60 P:502–7.

    Article  Google Scholar 

  35. Sotler R, Poljšak B, Dahmane R, Jukić T, Pavan Jukić D, Rotim C, Trebše P, Starc A. Prooxidant activities of antioxidants and their impact on health. Acta Clin Croat. 2019;58:726–36. https://doiorg.publicaciones.saludcastillayleon.es/10.20471/acc.2019.58.04.20.

    Article  PubMed  PubMed Central  Google Scholar 

  36. Nowak M, Tryniszewski W, Sarniak A, Wlodarczyk A, Nowak PJ, Nowak D. Concentration dependence of Anti- and Pro-Oxidant activity of polyphenols as evaluated with a Light-Emitting Fe2+-Egta-H2O2 system. Molecules. 2022;27:3453. https://doiorg.publicaciones.saludcastillayleon.es/10.3390/molecules27113453.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Vahid F, Wagener L, Leners B, Bohn T. Pro- and antioxidant effect of food items and matrices during simulated in vitro digestion. Foods. 2023;12:1719. https://doiorg.publicaciones.saludcastillayleon.es/10.3390/foods12081719.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Taherkhani S, Suzuki K, Castell L. A short overview of changes in inflammatory cytokines and oxidative stress in response to physical activity and antioxidant supplementation. Antioxid (Basel). 2020;18:886. https://doiorg.publicaciones.saludcastillayleon.es/10.3390/antiox9090886.

    Article  CAS  Google Scholar 

  39. Chen M, Wang Y, Deng S, Lian Z, Yu K. Skeletal muscle oxidative stress and inflammation in aging: focus on antioxidant and anti-inflammatory therapy. Front Cell Dev Biol. 2022;10:964130. https://doiorg.publicaciones.saludcastillayleon.es/10.3389/fcell.2022.964130.

    Article  PubMed  PubMed Central  Google Scholar 

  40. Manaig YJY, Mármol-Sánchez E, Castelló A, Esteve-Codina A, Sandrini S, Savoini G, Agazzi, Armand Sánchez A, Folch JM. Exon-intron split analysis reveals posttranscriptional regulatory signals induced by high and low n-6/n-3 polyunsaturated fatty acid ratio diets in piglets Yron Joseph Yabut Manaig, Emilio Mármol-Sánchez, Anna Castelló, Anna Esteve-Codina, Silvia Sandrini. J Anim Sci. 2023;3(101):skad271. https://doiorg.publicaciones.saludcastillayleon.es/10.1093/jas/skad271.

    Article  Google Scholar 

  41. Patterson E, Wall R, Fitzgerald GF, Ross RP, Stanton C. Health implications of high dietary Omega-6 polyunsaturated fatty acids. J Nutr Metab. 2012;539426. https://doiorg.publicaciones.saludcastillayleon.es/10.1155/2012/539426.

  42. Di Nunzio M, Valli V, Bordoni A. Pro- and anti-oxidant effects of polyunsaturated fatty acid supplementation in HepG2 cells author links open overlay panel. Prostaglandins Leukot Essent Fat Acids. 2011;85:121–7. https://doiorg.publicaciones.saludcastillayleon.es/10.1016/j.plefa.2011.07.005.

    Article  CAS  Google Scholar 

  43. Aiello A, Medoro A, Accardi G, Calabrò A, Carru C, Cannavo A, Caruso C, Candore G, Scapagnini G, Corbi G, Ali S, Davinelli S. Polyunsaturated fatty acid status and markers of oxidative stress and inflammation across the lifespan: A cross-sectional study in a cohort with long-lived individuals. Exp Gerontol. 2024;195:112531. https://doiorg.publicaciones.saludcastillayleon.es/10.1016/j.exger.2024.112531.

    Article  CAS  PubMed  Google Scholar 

  44. Elsafty M, Abdeen A, Aboubakr M, Allicin. Omega-3 fatty acids attenuates acetaminophen mediated renal toxicity and modulates oxidative stress, and cell apoptosis in rats. Naunyn Schmiedebergs Arch Pharmacol. 2024;397:317–28. https://doiorg.publicaciones.saludcastillayleon.es/10.1007/s00210-023-02609-z. Epub 202.

    Article  CAS  PubMed  Google Scholar 

  45. Khan SA, Damanhouri GA, Ahmed TJ, Halawani SH, Ali A, Makki A, Khan SA. Omega 3 fatty acids - Potential modulators for oxidative stress and inflammation in the management of sickle cell disease. J Pediatr (Rio J). 2022;98:513–8. https://doiorg.publicaciones.saludcastillayleon.es/10.1016/j.jped.2022.01.001.

    Article  PubMed  Google Scholar 

  46. Østbye TK, Haugen JE, Wetterhus EM, Bergum SK, Nilsson A. Oxidized dietary oil, high in Omega-3 and Omega-6 polyunsaturated fatty acids, induces antioxidant responses in a human intestinal HT29 cell line. Nutrients. 2022;14:5341. https://doiorg.publicaciones.saludcastillayleon.es/10.3390/nu14245341.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Doriane R, Kaouthar K, Ullah B, Pedro B, Francesco V. Polyunsaturated fatty acids as antioxidants. Pharmacol Res. 2008;57:451–5. https://doiorg.publicaciones.saludcastillayleon.es/10.1016/j.phrs.2008.05.002.

    Article  CAS  Google Scholar 

  48. Ibarguren M, López DJ, Escribá, Pablo V. The effect of natural and synthetic fatty acids on membrane structure, microdomain organization, cellular functions and human health. Biochim Biophys Acta - Biomembr. 2014;1838:1518–28.

    Article  CAS  Google Scholar 

  49. Bazzano M, Rizzo M, Arfuso F, Giannetto C, Fazio F, Piccione G. Increase in erythrocyte osmotic resistance following polyunsaturated fatty acids (PUFA) supplementation in show jumper horses. Livest Sci. 2015;1–6. https://doiorg.publicaciones.saludcastillayleon.es/10.1016/j.livsci.2015.08.011.

  50. van den Berg JJ, de Fouw NJ, Kuypers FA, Roelofsen B, Houtsmuller UM, Op den Kamp JA. Increased n-3 polyunsaturated fatty acid content of red blood cells from fish oil-fed rabbits increases in vitro lipid peroxidation, but decreases hemolysis. Free Radic Biol Med. 1991;11:393–9. https://doiorg.publicaciones.saludcastillayleon.es/10.1016/0891-5849(91)90156-w.

    Article  PubMed  Google Scholar 

  51. Suzuki N, Sawada K, Takahashi I, Matsuda M, Fukui S, Tokuyasu H, Shimizu H, Yokoyama J, Akaike A, Nakaji S. Association between polyunsaturated fatty acid and reactive oxygen species production of neutrophils in the general population. Nutrients. 2020;12:3222. https://doiorg.publicaciones.saludcastillayleon.es/10.3390/nu12113222.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. Montecillo-Aguado M, Tirado-Rodriguez B, Huerta-Yepez S. The involvement of polyunsaturated fatty acids in apoptosis mechanisms and their implications in Cancer. Int J Mol Sci. 2023;24:11691. https://doiorg.publicaciones.saludcastillayleon.es/10.3390/ijms241411691.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. Teixeira FS, Pimentel LL, Vidigal SSMP, Azevedo-Silva J, Pintado ME, Rodríguez-Alcalá LM. Differential lipid accumulation on HepG2 cells triggered by palmitic and Linoleic fatty acids exposure. Molecules. 2023;28:2367. https://doiorg.publicaciones.saludcastillayleon.es/10.3390/molecules28052367.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. Dai J, Shen J, Pan W, Shen S, Das UN. Effects of polyunsaturated fatty acids on the growth of gastric cancer cells in vitro. Lipids Heal Dis. 2013;12:71. https://doiorg.publicaciones.saludcastillayleon.es/10.1186/1476-511X-12-71.

    Article  CAS  Google Scholar 

  55. Baralla E, Demontis MP, Varoni MV, Pasciu V. Bisphenol A, bisphenol S oxidative effects in sheep red blood cells: an in vitro study. BioMed Res Int. 2021. 6621264.

  56. Masetto Antunes M, Godoy G, Curi R, Vergílio Visentainer J, Barbosa Bazotte R. The myristic acid:docosahexaenoic acid ratio versus the n-6 polyunsaturated fatty acid:n-3 polyunsaturated fatty acid ratio as nonalcoholic fatty liver disease biomarkers. Metab Syndr Relat Disord. 2022;20:69–78. https://doiorg.publicaciones.saludcastillayleon.es/10.1089/met.2021.0107. Epub 2021 Nov 2.

    Article  CAS  PubMed  Google Scholar 

  57. Jang H, Park K. Omega-3 and omega-6 polyunsaturated fatty acids and metabolic syndrome: A systematic review and meta-analysis. Clin Nutr. 2020;39:765–73. https://doiorg.publicaciones.saludcastillayleon.es/10.1016/j.clnu.2019.03.032.

    Article  CAS  PubMed  Google Scholar 

  58. Park S, Lee JJ, Lee J, Lee JK, Byun J, Kim I, Ha JH. Lowering n-6/ n-3 ratio as an important dietary intervention to prevent LPS-Inducible dyslipidemia and hepatic abnormalities in Ob/ob mice. Int J Mol Sci. 2022;23:6384. https://doiorg.publicaciones.saludcastillayleon.es/10.3390/ijms23126384.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  59. Pasciu V, Baralla E, Varoni MV, Demontis MP. Evaluation of curcuma and ginger mixture ability to prevent ROS production induced by bisphenol S: an in vitro study. Drug Chem Toxicol Jan. 2022;45:324–30. https://doiorg.publicaciones.saludcastillayleon.es/10.1080/01480545.2019.1690499.

    Article  CAS  Google Scholar 

  60. Pasciu V, Sotgiu FD, Porcu C, Berlinguer F. Effect of media with different glycerol concentrations on sheep red blood cells’ viability in vitro. Anim (Basel). 2021;11:1592. https://doiorg.publicaciones.saludcastillayleon.es/10.3390/ani11061592.

    Article  Google Scholar 

  61. Lewinska A, Wnuk M, Slota E, Bartosz G. Total anti-oxidant capacity of cell culture media. Clin Exp Pharmacol Physiol. 2007. https://doiorg.publicaciones.saludcastillayleon.es/10.1111/j.1440-1681.2007.04637.x.

    Article  PubMed  Google Scholar 

  62. Spanier A, Traylor R. A rapid, direct chemical assay for the quantitative determination of thiobarbituric acid reactive substances in Raw, cooked, and cooked/stored muscle foods. J Muscle Foods. 2007;2:165–76.

    Article  Google Scholar 

  63. Mischitelli M, Jemaà M, Almasry M, Faggio C, Lang F. Ca2 + Entry, oxidative stress, ceramide and suicidal erythrocyte death following Diosgenin treatment. Cell Physiol Biochem. 2016;39:1626–37. https://doiorg.publicaciones.saludcastillayleon.es/10.1159/000447864.

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

We would like to acknowledge Mara Donnaruma for support in the experiments.

Funding

This work has been developed within the framework of the project e.INS- Ecosystem of Innovation for Next Generation Sardinia (cod. ECS 00000038) funded by the Italian Ministry for Research and Education (MUR) under the National Recovery and Resilience Plan (NRRP) - MISSION 4 COMPONENT 2, “From research to business” INVESTMENT 1.5, “Creation and strengthening of Ecosystems of innovation” and construction of “Territorial R&D Leaders”.

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Authors and Affiliations

  1. Department of Veterinary Medicine, University of Sassari, Sassari, Italy

    Valeria Pasciu, Elena Baralla, Francesca Daniela Sotgiu & Fiammetta Berlinguer

  2. Department of Medicine, Surgery and Pharmacy, University of Sassari, Sassari, Italy

    Maria Nieddu

  3. Department of Agronomy, Biotechnology and Food, University of Navarre, Pamplona, Spain

    Ignacio Contreras-Solís

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Contributions

V.P. designed the study, performed the experiments, analysed data, prepared and revised the manuscript; M.N. designed the study, analysed data, prepared and revised the manuscript; E.B. analysed data, and revised the manuscript; F.D.S. collected RBCs samples and revised the manuscript; I.C.S. revised the manuscript; F.B. supervised the study, edited the manuscript, and was responsible for funding acquisition.

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Correspondence to Valeria Pasciu.

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Pasciu, V., Nieddu, M., Baralla, E. et al. Effect of PUFAs-ω3 and ω6 on oxidative stress of sheep erythrocytes. BMC Vet Res 21, 334 (2025). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s12917-025-04762-4

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