Dietary Omega‐3 Fatty Acids Alter Hepatic Lipid Oxidation and Oxidative Stress Markers in Postpartum Dairy Cows

ABSTRACT

Omega‐3 (ω−3) polyunsaturated fatty acids are well recognized for their potential to influence metabolic adaptation in dairy cows during the transition period. This study evaluated the effects of calcium salts enriched with ω−3 fatty acids, including α‐linolenic acid and long‐chain ω−3 fatty acids such as eicosapentaenoic and docosahexaenoic acids, on hepatic lipid metabolism‐related proteins, oxidative stress biomarkers, and liver function in postpartum dairy cows. Prior to calving, cows received a total mixed ration supplemented with 0.400 kg dry matter (DM)/day of calcium salts derived from a flaxseed oil–fish oil mixture (60:40). After calving, cows were fed a partial mixed ration and received 0.650 kg DM/day of the same supplement mixed with a commercial concentrate during milking. Control cows received isoenergetic diets in which calcium salts were replaced with ground corn. Blood, urine, and liver samples were collected at −21 days relative to calving (used as a baseline covariate) and at +7, and +21 (±3) days postpartum, when treatment effects were evaluated. No differences were observed between treatments in conventional biochemical markers of liver function or hepatic triacylglycerol concentration. Plasma malondialdehyde concentrations were higher in ω−3‐supplemented cows, whereas other oxidative stress parameters were not affected. Supplementation with ω−3 fatty acids was associated with changes in the abundance of selected hepatic proteins involved in fatty acid metabolism, including increased acyl‐CoA oxidase 1 and 3‐hydroxy‐3‐methylglutaryl‐CoA synthase and reduced diacylglycerol O‐acyltransferase 1 abundance, whereas carnitine palmitoyltransferase 1 and peroxisome proliferator‐activated receptor alpha abundance were not affected. These findings indicate that ω−3‐enriched calcium salts induce molecular adjustments in hepatic lipid metabolism during early lactation without evidence of adverse effects on liver function.

1. Introduction

The transition period is one of the most critical phases in the productive cycle of dairy cows, spanning the weeks immediately before and after parturition. During this stage, the rapid increase in energy requirements associated with the onset of lactation is not fully matched by dry matter intake, resulting in a marked negative energy balance. This condition promotes adipose tissue lipolysis and elevated circulating concentrations of non‐esterified fatty acids (NEFA), which must be largely processed by the liver (Grummer 1995; Drackley 1999).

The liver plays a central role in the metabolic adaptation to early lactation by regulating NEFA uptake, oxidation, re‐esterification, and export. When NEFA influx exceeds hepatic oxidative and export capacities, excess fatty acids are redirected toward triacylglycerol (TAG) synthesis and storage, increasing the risk of hepatic lipid accumulation and impaired liver function (Drackley et al. 2001; Bobe et al. 2004). Within this metabolic context, nutritional strategies aimed at modulating hepatic fatty acid metabolism have gained increasing attention in dairy cow nutrition.

Hepatic lipid metabolism during the transition period is tightly regulated by transcription factors and enzymes. Peroxisome proliferator‐activated receptor alpha (PPARα) regulates mitochondrial and peroxisomal β‐oxidation through transcriptional control of genes such as carnitine palmitoyltransferase 1 (CPT1), which controls mitochondrial fatty acids entry, and acyl‐CoA oxidase 1 (ACOX1), a key enzyme of peroxisomal β‐oxidation (Loor et al. 2007; Bionaz et al. 2013). In parallel, mitochondrial ketogenesis is largely controlled by 3‐hydroxy‐3‐methylglutaryl‐CoA synthase (HMG‐CoA synthase), the rate‐limiting enzyme in ketone body synthesis (Nguyen et al. 2008). Conversely, enzymes involved in lipid esterification, including diacylglycerol O‐acyltransferase 1 (DGAT1) contribute to TAG synthesis when fatty acid supply exceeds oxidative and ketogenic capacity (Bobe et al. 2004; Angeli et al. 2019). In addition, the transition period is associated with increased generation of reactive oxygen and nitrogen species (RONS), contributing to oxidative stress and compromising metabolic homeostasis (Bernabucci et al. 2002; Sordillo and Aitken 2009). Because polyunsaturated fatty acids (PUFA) are particularly susceptible to oxidative modification, changes in dietary fatty acid composition may influence both hepatic lipid metabolism and oxidative status (Bernabucci et al. 2005).

Among dietary PUFA, omega‐3 (ω−3) fatty acids have received increasing interest due to their potential to modulate lipid metabolism and support metabolic adaptation during early lactation (Loor et al. 2005; Bionaz et al. 2013). Alpha‐linolenic acid (ALA, 18:3, ω−3) is the primary dietary ω−3 fatty acid, whereas eicosapentaenoic (EPA, 20:5, ω−3) and docosahexaenoic acids (DHA, 22:6, ω−3) are present in smaller amounts and may derive from both dietary sources or limited endogenous metabolism. Although dairy cows cannot synthesize ω−3 and ω−6 PUFA de novo, they retain a limited capacity to elongate and desaturate dietary precursors, and ruminal biohydrogenation further restricts intestinal availability (Jenkins et al. 2008; Angeli et al. 2021a). Grass‐based pasture diets can increase ALA intake and lower n‐6/n‐3 ratio compared with conventional total mixed ration (TMR) systems (Dewhurst et al. 2006; Elgersma et al. 2006); however, under intensive production conditions, additional supplementation may be required to ensure a consistent post‐ruminal supply of ω−3 PUFA.

In this context, we hypothesize that supplementation with calcium salts enriched with ω−3 fatty acids may represent a nutritional strategy to support hepatic metabolic adaptation during the transition period. Therefore, this study evaluated the effects of calcium salts enriched with ω−3 fatty acids, including ALA, EPA, and DHA, on lipid metabolism proteins, oxidative stress biomarkers, and hepatic function in transition dairy cows.

2. Materials and Methods

All procedures involving animal handling and care were reviewed and approved by the Ethics and Safety Committee of the College of Veterinary Sciences, National University of Litoral (Protocol No. 614/20). They were conducted in accordance with the Guide for the Care and Use of Agricultural Animals in Research and Teaching (Ag Guide 2020). Details of the experimental design have been reported previously (Roskopf et al. 2025). Briefly, forty Holstein cows in their second to fifth lactation and 21 ± 3 days before the expected calving date, were blocked according to expected parturition date, parity, body weight, body condition score, and milk yield in the previous lactation. Within each block, cows were randomly assigned to one of two treatments: Omega‐3 (ω−3) or Control (C). Two cows per group that developed illness during the experimental period were excluded from the analysis, resulting in 18 cows per treatment group. This final sample size was in agreement with the a priori sample size calculation, which was performed using an alpha level of 0.05, a statistical power of 0.80, and an effect size (f) of 0.40. The effect size was based on the ability to detect a minimum expected difference of 20% in protein expression (means: 1.0 vs. 1.2; SD: 0.25 in arbitrary units) using a repeated‐measures ANOVA with three measurements per cow (G*Power software). In addition, this sample size is consistent with previous studies evaluating biochemical and protein expression outcomes in dairy cows (Akbar et al. 2015; Mann et al. 2016; Pate and Cardoso 2018).

Cows were housed in dry‐lot pens equipped with shade structures and covered feeding areas, with unrestricted access to water, at the National Institute of Agricultural Technology (INTA) in Rafaela, Santa Fe, Argentina. Diets were formulated using NASEM (2021) software, according to the nutritional requirements of the cows (Table 1). Feed was offered ad libitum to each animal using individual feeders. During the 21 days before calving, the ω−3 group received a total mixed ration (TMR) that included 0.400 kg dry matter (DM)/day of calcium salts of ω−3 fatty acids. After calving, cows were fed a partial mixed ration (PMR) and, during milking (twice a day), received a commercial concentrate supplemented with 0.650 kg DM/day of the same calcium salts. The calcium salts were produced by TECNUAR SRL (Rosario, Santa Fe, Argentina) using a flaxseed oil‐fish oil mixture (60:40) and processed by saponification to protect fatty acids from ruminal biohydrogenation. The control group received the same diets as the ω−3 group, except that the ω−3 calcium salts were replaced with ground corn to ensure isoenergetic conditions (Table S1).

Table 1.

Antibodies and conditions used for Western blot assays.

Antibodies	Type	Suppliers	Dilution	Total protein/lane
Primary antibodies
CPT1‐L	Rabbit Polyclonal H‐95: sc‐20669	Santa Cruz Biotechnology, Inc., CA, USA	1/3000	40 µg
ACOX1	Rabbit Polyclonal H‐140: sc‐98499	Santa Cruz Biotechnology, Inc., CA, USA	1/3000	40 µg
PPARα	rabbit polyclonal H‐98: sc‐9000	Santa Cruz Biotechnology, Inc., CA, USA	1/1000	40 µg
DGAT1	Rabbit polyclonal ab54037	Abcam, Cambridge, UK	1/3000	40 µg
HMG‐CoA synthase	Mouse monoclonal B‐8: sc‐393256	Santa Cruz Biotechnology, Inc., CA, USA	1/20000	40 µg
Vinculin	Mouse monoclonal V284: MCA465GA	Bio‐Rad Laboratories, Inc., CA, USA	1/10000
Secondary antibodies
Mouse anti‐rabbit IgG	Monoclonal sc‐2357	Santa Cruz Biotechnology, Inc., CA, USA	1/10000
Mouse IgGκ binding protein	Binding protein: sc‐516102	Santa Cruz Biotechnology, Inc., CA, USA	1/10000

2.1. Milk Fatty Acid Profile

Individual milk samples (100 mL) were collected during the fifth week of lactation and stored at −24°C until analysis of the fatty acid profile. Fatty acid methyl esters (FAME) were prepared by transesterification using a methanolic potassium hydroxide solution as an intermediate step prior to saponification, following ISO 12966‐2 (2017). The milk fatty acid composition was determined by gas chromatography using a Shimadzu GC‐2014 system equipped with an automatic injector (AOC‐20i, Shimadzu, Japan) and a flame ionization detector (FID), as previously described by Masson et al. (2015).

2.2. Blood, Urine and Liver Sampling

Blood, urine and liver samples were collected at three time points: 21 days prior to the expected calving date (before the start of the experimental dietary treatments), and on days 7 and 21 postpartum (±3). Blood was obtained from the jugular vein into EDTA‐containing tubes and stored at 5°C until further processing. An additional sample was collected into tubes without anticoagulant to allow serum separation. Both samples were centrifuged for 10 min to separate plasma and serum, respectively, and then stored at −80°C until analysis.

Urine samples were obtained by gently stimulating the perineal area beneath the vulva and collected into sterile containers. The samples were immediately cooled at 4°C and subsequently stored at −80°C until analysis.

Liver biopsies were performed as described by Angeli et al. (2019). Briefly, under local anesthesia (15 mL of 2% lidocaine) and via a small incision in the right 11th intercostal space, approximately 500 mg of hepatic tissue was collected using a modified percutaneous biopsy needle. Samples were immediately snap‐frozen in liquid nitrogen and stored at –80°C until analysis.

2.3. Plasma Enzymes, Metabolites, Minerals and Liver TAG Content

Blood samples were analyzed for serum concentrations of albumin, total bilirubin (TBil), total cholesterol (TChol), calcium, magnesium and the enzymatic activities of aspartate aminotransferase (AST) and gamma‐glutamyl transferase (GGT) using commercial colorimetric kits (WIENER Lab., Rosario, Argentina) with a biochemical autoanalyzer (CM 250, WIENER Lab., Argentina).

Serum antioxidant enzyme activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH‐PX), as well as total antioxidant status (TAS), were determined using commercial colorimetric kits (Randox Laboratories Ltd., Ardmore, UK) and an ultrafast UV/Vis SPECTROstar Nano spectrometer (BMG LABTECH, Germany).

Hepatic TAG content was determined using a previously described methodology (Barcarolo et al. 2024). Briefly, a chloroform:methanol solution (2:1, v/v) was used to extract total lipids from liver homogenates as outlined by Folch et al. (1957). After removal of phospholipids by adsorption with silicic acid, TAG concentration in the liver was quantified enzymatically with a commercial kit (WIENER Lab, Rosario, Argentina).

2.4. Western Blot Analysis

Approximately 50 mg of frozen liver tissue was homogenized in a lysis buffer containing 0.1 mol/L phosphate‐buffered saline (PBS), 1% (v/v) octylphenyl‐polyethylene glycol (IGEPAL CA630), 50 mmol/L sodium fluoride, 1 mmol/L EDTA, 0.1% (w/v) sodium dodecyl sulfate (SDS), 0.5% (w/v) sodium deoxycholate (all reagents from Sigma‐Aldrich Corp., MO, USA), and a protease inhibitor cocktail (Complete Mini, Roche, Mannheim, Germany). Lysates were centrifuged at 12,000 x g for 20 min, and the resulting supernatants were collected and stored at −80°C. Protein concentrations were determined by the Lowry method using the Bio‐Rad Protein Assay Kit (Bio‐Rad Laboratories, CA, USA).

For Western blot analysis, proteins were denatured by boiling in Laemmli sample buffer for 5 min, separated on 10% (w/v) SDS‐polyacrylamide gels, and transferred to nitrocellulose membranes. Membranes were blocked for 1 h at room temperature with Tris‐buffered saline containing 0.05% (v/v) Tween‐20 (TBST; Sigma–Aldrich, USA) and 5% (w/v) non‐fat dry milk. They were then incubated overnight at 4°C with the appropriate primary antibodies (Table 2) (Angeli et al. 2019). After washing with TBST, membranes were incubated for 90 min at room temperature with peroxidase‐conjugated secondary antibodies (Table 2). Specific immunoreactive bands were visualized using a chemiluminescent reagent (ECL Prime Western Blotting System, GE Healthcare, Germany) and developed on Hyperfilm ECL (GE Healthcare, Germany). Vinculin was used as a loading control, processed in parallel with the same protocol and antibodies.

Table 2.

Plasma concentrations of albumin and total bilirubin (TBil), and activities of alkaline phosphatase (ALP), aspartate transaminase (AST) and gamma‐glutamyl transpeptidase (GGT) and hepatic triglyceride content (TG liver) of dairy cattle supplemented with omega‐3 (ω−3, n = 18) and not supplemented (C, n = 18) at days −21, 7 and 21 relative to partum.

		Prepartum	Postpartum days
		−21 (COV)	7	21	S	T	S × T
Albumin	ω−3	3.85 ± 0.04	3.54 ± 0.07	3.55 ± 0.09	0.62	0.49	0.68
(g/dL)	C	3.85 ± 0.09	3.47 ± 0.08	3.55 ± 0.09
TBil	ω−3	0.20 ± 0.02	0.24 ± 0.03	0.16 ± 0.01	0.45	0.02	0.27
(mg/dL)	C	0.17 ± 0.02	0.20 ± 0.03	0.17 ± 0.01
ALP	ω−3	133.42 ± 16.12	148.51 ± 13.03	105.38 ± 7.91	0.92	< 0.01	0.38
(IU/L)	C	157.14 ± 26.63	135.80 ± 14.41	113.04 ± 9.90
GGT	ω−3	16.42 ± 1.12	18.11 ± 1.78	25.99 ± 2.17	0.88	< 0.01	0.37
(IU/L)	C	16.35 ± 0.99	19.51 ± 2.32	23.41 ± 2.21
AST	ω−3	69.05 ± 2.59	90.26 ± 2.83	84.05 ± 3.55	0.07	0.01	0.44
(IU/L)	C	71.36 ± 2.73	86.39 ± 3.29	75.50 ± 3.55
Calcium	ω−3	9.60 ± 0.11	9.37 ± 0.15	9.72 ± 0.14	0.16	< 0.01	0.31
(mg/dL)	C	9.83 ± 0.17	8.97 ± 0.18	9.66 ± 0.17
Magnesium	ω−3	2.02 ± 0.03	1.91 ± 0.06	2.02 ± 0.06	0.20	0.04	0.71
(mg/dL)	C	1.99 ± 0.05	1.97 ± 0.07	2.13 ± 0.07
TG liver	ω−3	8.81 ± 1.51	13.54 ± 2.80	14.14 ± 2.60	0.43	0.57	0.90
(mg/g WT)	C	11.36 ± 1.22	15.29 ± 2.80	14.13 ± 2.59

Note: Values are expressed as mean ± SEM. The statistical effects of supplementation (S), time (T) and S × T are indicated. Bold values indicate statistical significance at p < 0.01.

Abbreviation: COV, covariate.

The Western blot assay was verified to be linear over a range of 20–80 µg of total protein. To account for inter‐membrane variability, a standard sample consisting of a pooled extract from six animals was included on each membrane and used to adjust band intensities across blots at increasing exposure times. Band intensities were quantified using Image‐Pro Plus software (version 3.0.1; Media Cybernetics, MD, USA). Protein abundance was expressed as the intensity of each target protein band relative to the corresponding vinculin band.

Antibody specificity was validated by the suppliers, as reported in the corresponding technical datasheets. Furthermore, the homology between the antigenic peptide recognized by each antibody and the bovine protein sequence was assessed using the Basic Local Alignment Search Tool (BLAST; http://www.ncbi.nlm.nih.gov/BLAST), enabling the identification of peptide alignment and confirmation of antigen recognition.

2.5. Oxidative Stress Biomarker Analysis

Liver and urine 3‐nitrotyrosine (3‐NT) and malondialdehyde (MDA) levels were determined by chromatographic methods coupled with mass spectrometry, as described by Barcarolo et al. (2022) and Angeli et al. (2023). Analyses were carried out using a Shimadzu ultra‐fast liquid chromatography system (UFLC‐XR; Shimadzu Co., Kyoto, Japan) with a Shim‐pack GIST C18 analytical column (3.0 μm, 100 × 4.0 mm) and a corresponding guard column (3.0 μm, 4.0 × 10 mm). The column compartment was maintained at 35°C, while the autosampler was kept at 4°C. Detection and quantification of analytes were performed with a QTrap 3200 triple quadrupole mass spectrometer (Sciex, USA), equipped with a turbo electrospray ionization (ESI) source.

2.6. Statistical Analysis

Data were analyzed using SPSS version 22.0 for Windows (SPSS Inc., IL, USA). A Generalized Linear Mixed Model (GLMM) was applied. Residual normality was assessed using the Shapiro–Wilk test. When residuals were non‐normal, a gamma distribution with a log link function was used; otherwise, a GLM with an identity link was applied. Fixed effects included supplementation (S), time (T), and the S × T interaction, while cow was included as a random effect and with the value of the day −21 as a covariate. When the S × T interaction was significant, pairwise comparisons between treatments at each time point were performed using marginal means with Bonferroni correction of multiple testing. The milk fatty acid profile was analyzed using a generalized linear model with an identity link function, including treatment included as a fixed effect. Statistical significance was set at p < 0.05. Results are expressed as mean ± standard error of the mean (SEM).

3. Results

3.1. Milk Fatty Acid Profile

Supplementation with calcium salts of ω−3 fatty acids in early‐lactation dairy cows modified the milk fatty acid profile (Table S2). Specifically, milk from cows in the ω−3 group showed a lower concentration of saturated fatty acids (66.11 vs. 70.04% for ω−3 and C, respectively; p < 0.01) and a higher concentration of unsaturated fatty acids (33.89 vs. 29.96% for ω−3 and C, respectively; p < 0.01).

The concentration of monounsaturated fatty acids was higher (p = 0.02) in milk fat from cows receiving the ω−3 treatment than in the control group (29.62 vs. 26.47 g/100 g of fatty acids). Similarly, the ω−3 treatment resulted in a greater concentration of PUFA (+18.6%; p < 0.01) compared with the control treatment.

Cows supplemented with calcium salts of ω−3 fatty acids exhibited a higher ω−3 fatty acid content (1.06 vs. 0.70 g/100 g of fatty acids; p < 0.01) and a lower ω−6: ω−3 fatty acid ratio (3:1 vs. 4:1 for ω−3 and C, respectively). The concentrations of linoleic acid (LA; C18:2 ω−6) and α‐linolenic acid (ALA; C18:3 ω−3) were also higher (+14.0 and + 50.8%, respectively; p < 0.01) in cows receiving the ω−3 treatment compared with the control group.

3.2. Plasma Enzymes, Metabolites and Liver TAG Content

Plasma albumin concentrations were not affected by treatment or sampling time, and no treatment × time interaction was detected (p > 0.05). In contrast, total bilirubin and alkaline phosphatase concentrations were significantly higher on day 7 postpartum compared with day 21 postpartum (p < 0.05), with no differences between treatments and no treatment × time interaction observed (p > 0.05).

AST activity was higher on day 7 postpartum compared with day 21 postpartum (p < 0.05). However, no overall time effect was detected, and no significant differences between treatments or treatment × time interactions were observed (p > 0.05). In contrast, GGT activity varied over time, with lower values detected on day 7 postpartum compared with day 21 postpartum (p < 0.05), and no effects of treatment or treatment × time interaction were detected (p > 0.05).

For the minerals analyzed, plasma magnesium and calcium concentrations were lower on day 7 postpartum compared with day 21 postpartum (p < 0.05), with no significant effects of treatment or treatment × time interaction detected for either mineral (p > 0.05) (Table 2).

Regarding antioxidant enzymes, no significant differences in SOD activity or TAS were detected over time or between treatment groups (p > 0.05). In contrast, GSH‐PX activity was higher on day 7 postpartum comparted with day 21 postpartum (p < 0.05), with no effects of supplementation or treatment × time interaction observed (p > 0.05) (Table 3).

Table 3.

Antioxidant enzymes activity and total antioxidant status (TAS) in dairy cattle supplemented with omega‐3 (ω−3, n = 18) and not supplemented (C, n = 18) at days −21, 7 and 21 relative to partum.

		Prepartum	Postpartum days
		−21 (COV)	7	21	S	T	S × T
GSH‐Px	ω−3	449.87 ± 30.55	414.54 ± 20.17	368.75 ± 24.94	0.37	< 0.01	0.40
(IU/g Hg)	C	458.24 ± 37.71	413.25 ± 23.78	329.12 ± 24.94
SOD	ω−3	453.81 ± 29.44	463.90 ± 31.24	450.09 ± 35.75	0.90	0.11	0.22
(IU/mL)	C	462.42 ± 37.82	505.51 ± 39.66	404.57 ± 35.20
TAS	ω−3	0.66 ± 0.03	0.54 ± 0.03	0.50 ± 0.02	0.84	0.37	0.64
(mmol/L)	C	0.60 ± 0.04	0.52 ± 0.03	0.51 ± 0.03

Note: Values are expressed as mean ± SEM. The statistical effects of supplementation (S), time (T) and S × T are indicated. Bold value indicate statistical significance at p < 0.01.

Abbreviations: GSH‐Px: glutathione peroxidase; SOD: superoxide dismutase; COV: covariate.

Finally, no differences in liver TAG content were observed between groups or by time (p > 0.05) (Table 2).

3.3. NT and MDA Concentrations

The oxidative stress biomarker 3‐NT did not differ in plasma or urine concentrations according to treatment or sampling time, an no treatment × time interaction was detected (p > 0.05). In contrast, hepatic 3‐NT concentrations were higher on day 7 postpartum compared with day 21 postpartum (p < 0.05), with no effects of supplementation or treatment × time interaction observed (p > 0.05).

MDA concentrations showed a significant treatment × time interaction in plasma, with higher MDA levels in the ω−3 group compared with the control group at day 21 postpartum (p < 0.05). In contrast, no effects of treatment, time, or treatment × time interaction were detected for urinary or hepatic MDA concentrations (p > 0.05) (Table 4).

Table 4.

Oxidative stress biomarker concentrations in plasma, urine and liver tissue of dairy cattle supplemented with omega‐3 (ω−3, n = 18) and not supplemented (C, n = 18) at days −21, 7 and 21 relative to partum.

		Prepartum	Postpartum
		−21 (COV)	7	21	S	T	S × T
3‐NT plasma	ω−3	34.22 ± 10.07	113.48 ± 10.24	111.77 ± 14.09	0.40	0.32	0.38
(mg/L)	C	24.03 ± 5.17	113.76 ± 11.96	91.36 ± 12.56
3‐NT urine	ω−3	0.010 ± 0.003	0.050 ± 0.005	0.042 ± 0.004	0.36	0.89	0.25
(mg/mg creat)	C	0.010 ± 0.003	0.039 ± 0.006	0.044 ± 0.006
3‐NT liver	ω−3	0.35 ± 0.11	0.71 ± 0.06	0.55 ± 0.07	0.85	0.01	0.56
(mg/kg WT)	C	0.28 ± 0.08	0.75 ± 0.09	0.50 ± 0.08
MDA plasma	ω−3	0.68 ± 0.03	0.45 ± 0.03	0.47 ± 0.02	0.13	0.01	< 0.01
(µmol/L)	C	0.60 ± 0.05	0.50 ± 0.03	0.35 ± 0.02
MDA urine	ω−3	0.015 ± 0.001	0.024 ± 0.004	0.027 ± 0.006	0.59	0.49	0.99
(µmol/mg creat)	C	0.020 ± 0.003	0.021 ± 0.004	0.024 ± 0.006
MDA liver	ω−3	19.99 ± 2.39	27.71 ± 2.47	24.06 ± 2.52	0.94	0.66	0.36
(µmol/kg WT)	C	28.09 ± 3.41	24.97 ± 2.71	26.26 ± 3.10

Note: Values are expressed as mean ± SEM. The statistical effects of supplementation (S), time (T) and S × T are indicated. Bold values indicate statistical significance at p < 0.01.

Abbreviations: 3‐NT: 3‐nitrotyrosine; MDA: malondialdehyde; WT: wet tissue; creat: creatinine; COV: covariate.

3.4. Liver Protein Abundance

Western blot analysis revealed differential effects of supplementation on the abundance of key proteins involved in liver lipid metabolism. Supplementation was associated with greater overall hepatic ACOX1 abundance in the ω−3 group [1.24 (95% CI: 1.07–1.41) ua] compared with the control group [0.96 (95% CI: 0.77–1.15) ua] (p < 0.05), with effects of time or treatment × time interaction (p > 0.05). In contrast, PPARα abundance did not differ according to treatment, sampling time, or treatment × time interaction (p > 0.05).

HMG‐CoA synthase exhibited a significant treatment × time interaction, with higher protein abundance on day 7 postpartum in the ω−3 group compared with the control group (p < 0.05), whereas no main effects of treatment or time were detected (p > 0.05).

In contrast, CPT1 abundance did not differ between treatments or across sampling times (p > 0.05). Finally, supplementation was associated with lower overall hepatic DGAT1 abundance in the ω−3 group [0.95 (95% CI: 0.79–1.11) ua] compared with the control group [1.20 (95% CI: 1.03–1.37) ua] (p < 0.05), with no effects of time (p > 0.05) (Figure 1).

Figure 1.

Protein abundance of hepatic PPARα, CPT1, ACOX1, HMG‐CoA synthase, and DGAT1 between day 14 prepartum and day 28 postpartum. Animals were grouped as ω−3 supplemented cows (ω−3; n = 18, black squares) and control cows (C; n = 18, black circles). Values are expressed as mean ± SEM. The statistical effects of supplementation (Suppl), time and Suppl × time (S × T) are indicated. Representative immunoblots of each determination; Molecular weight is shown on the right. Line 1, day 21 prepartum; lines 2 and 3, days 7 and 21 postpartum. PPARα: peroxisome proliferator‐activated receptor alpha; CPT1: carnitine palmitoyltransferase 1; ACOX1: acyl‐CoA oxidase 1; HMG‐CoA: hydroxymethylglutaryl‐CoA; DGAT1: diacylglycerol O‐acyltransferase 1; COV: covariate; au: arbitrary units.

4. Discussion

In dairy cattle, previous studies have reported positive effects of ω−3 fatty acids on reproductive performance and production parameters (Moallem 2018; Angeli et al. 2021a). Although several studies have evaluated the effects of n‐3 fatty acids on metabolism, inflammation, and performance in transition dairy cows (Ballou et al. 2009; Silvestre et al. 2011a, 2011b; Greco et al. 2015), the specific hepatic molecular mechanisms underlying these responses remain incompletely characterized, particularly with regard to lipid metabolism and oxidative pathways during the periparturient period. Given the profound metabolic adaptations that occur during the transition period, understanding how ω−3 fatty acids interact with oxidative balance, immune function, and hepatic metabolism is essential. This knowledge gap is particularly relevant, as nutritional strategies aimed at improving adaptation during this challenging stage may exert long‐term effects on both animal health and productivity.

In this context, research in human medicine has consistently demonstrated beneficial effects of ω−3 supplementation in mitigating oxidative stress (Heshmati et al. 2019). In the present study, the increase in plasma MDA concentration observed in the supplemented group is more likely related to greater PUFA intake rather than to a true increase in systemic oxidative stress, as no other oxidative or antioxidant parameters were affected. MDA is a final product of PUFA peroxidation, and increased lipid oxidation is expected with greater PUFA availability. As described by Voljč et al. (2011), PUFA supplementation increases lipid susceptibility to oxidation through the formation of peroxyl radicals, thereby leading to MDA production. Moreover, Guéraud et al. reported that MDA and other lipid peroxidation products may originate from dietary sources or be formed during digestion, particularly in PUFA‐rich diets. In addition, previous studies have reported positive associations between circulating NEFA, BHBA, and MDA concentrations (Li et al. 2016; Shi et al. 2015), suggesting that increased lipid mobilization and fatty acid availability may contribute to elevated MDA levels. Taken together, these findings support the interpretation that the higher MDA concentrations observed in the supplemented group reflect increased PUFA‐related lipid peroxidation rather than a generalized systemic oxidative stress response.

Consistent with this interpretation, ω−3 supplementation did not affect antioxidant components or oxidative stress markers in any of the biological matrices evaluated in postpartum dairy cows. To date, relatively few studies have specifically assessed oxidative stress responses to ω−3 supplementation in cattle. In this regard, Brozić et al. (2024) reported no changes in plasma MDA concentrations in Charolais cows supplemented during the last month of gestation, nor in their calves. Similarly, Sun et al. (2023) administered increasing levels of flaxseed oil to transition dairy cows and observed no significant effects on the evaluated antioxidant parameters.

Notably, despite the absence of detectable changes in oxidative stress markers, both Sun et al. (2023) and the present study reported improvements in milk production following ω−3 supplementation. This observation may indicate beneficial metabolic effects under conditions of increased energy demand. In this context, the greater milk yield and enhanced lipid mobilization observed in the present study could represent a higher metabolic challenge, which may be associated with an increased potential for reactive oxygen species (ROS) generation during early lactation.

Plasma metabolites have been widely investigated as indicators of liver function, particularly during the transition period, due to the central role of this organ at that stage (Bertoni, Trevisi 2013; Du et al. 2018). In the present study, concentrations of metabolites commonly used as indicators of hepatic health remained unchanged. Nevertheless, it is important to note that the absence of evident alterations in oxidative status or hepatic functionality biomarkers occurred in the context of higher milk production in cows supplemented with ω−3 (Roskopf et al. 2025). This could indicate an adequate adaptation to the increased energetic demands in this group, despite the potentially higher production of reactive oxygen species. Moreover, supplemented cows exhibited greater lipomobilization, as evidenced by increased plasma NEFA concentrations (Roskopf et al. 2025), without a corresponding increase in hepatic TAG content.

Previous studies have shown that during early lactation there is a substantial influx of NEFA to the liver and that, in ruminants, this influx may exceed hepatic oxidative capacity, favoring TAG esterification due to limitations in very‐low density lipoprotein secretion, despite activation of oxidative and ketogenic pathways (Emery et al. 1992; Bionaz et al. 2012). For this reason, the present study evaluated key enzymes involved in hepatic fatty acid metabolism. Although elevated NEFA concentrations have frequently been associated with increased hepatic TAG accumulation (Arshad, Santos 2022), this relationship was not observed in supplemented cows, suggesting differences in hepatic fatty acid metabolism. Previous studies from our group have shown greater abundance of the CPT1 enzyme in cows with lower hepatic TAG content (Angeli et al. 2019; Barcarolo et al. 2024); however, no differences in CPT1 abundance were observed in the present study.

Similarly, supplementation with ω−3 fatty acids did not modify hepatic abundance of PPARα. Although this nuclear receptor is activated by several PUFA, particularly EPA and DHA, which act as PPARα agonists (Zúñiga et al. 2011), its role in regulating lipid metabolism and inflammation has been more clearly established in non‐ruminants than in ruminants (Varga et al. 2011; Du et al. 2018). Most in vitro studies using bovine cell lines have reported PUFA‐induced changes primarily at the gene expression level (MacLaren et al. 2006; Abou‐Rjeileh et al. 2025). Moreover, in vivo studies using PPARα agonists have shown increased expression of PPARα target genes without concurrent changes in receptor abundance, suggesting that downstream gene activation may be a more appropriate indicator of PPARα activity (Busato et al. 2022; Litherland et al. 2010). In agreement with the present findings, previous studies from our group evaluating cows supplemented with antioxidant minerals and vitamins also reported no differences in hepatic PPARα abundance or associations with reproductive performance (Angeli et al. 2019; Barcarolo et al. 2024). Collectively, these results suggest that in dairy cows, hepatic lipid metabolism may be modulated through downstream pathways independently of detectable changes in PPARα protein abundance.

Consistent with this concept, supplemented cows exhibited greater overall hepatic ACOX1 abundance in supplemented cows. ACOX1 is a key enzyme regulating peroxisomal fatty acid β‐oxidation and is functionally linked to PPAR signaling (Xu et al. 2022). In ruminants, fatty acids oxidation occurs in both mitochondria and peroxisomes, with a more balanced contribution of both compartments compared with non‐ruminants (Grum et al. 1994). Therefore, increased ACOX1 abundance may represent an adaptive hepatic response that enhances peroxisomal oxidative capacity, facilitating the metabolism of elevated NEFA supply during the postpartum period. Given that PPARα protein abundance was unaffected, this upregulation may be mediated, at least in part, by alternative nuclear receptors involved in fatty acid oxidation, such as PPARδ. Recent evidence suggests that PPARδ activation can induce genes associated with peroxisomal β‐oxidation, including ACOX1, in response to increased fatty acid availability (Wang et al. 2018). However, PPARδ was not evaluated in the present study, and this regulatory mechanism warrants further investigation. Several studies have highlighted the complementary roles of mitochondrial and peroxisomal fatty acid oxidation in preventing excessive TAG accumulation within hepatocytes (Pinedo and Melendez 2022).

After β‐oxidation, acetyl‐CoA can either be fully oxidized through oxidative phosphorylation or diverted toward ketone body synthesis, such as BHBA. One of the key enzymes regulating this pathway is HMG‐CoA synthase, which catalyzes the rate‐limiting step of ketogenesis (Rodríguez et al. 2025). Ketogenic rate largely depends on the influx of oxidizable substrates to the liver (Rico and Barrientos‐Blanco 2024). Accordingly, the increased abundance of HMG‐CoA synthase observed in supplemented cows may be explained by the higher NEFA availability reaching the liver, which was accompanied by greater circulating BHBA concentration, consistent with enhances hepatic ketogenic flux. In transition dairy cows, ketogenesis is primarily substrate‐driven and represents an adaptive response to increased fatty acid supply, with circulating BHBA reflecting the balance between hepatic production and peripheral utilization (Drackley 1999, 2001). While in non‐ruminants HMG‐CoA synthase expression is hormonally regulated ‐being inhibited by insulin and stimulated by glucagon (Rico and Barrientos‐Blanco 2024) hormonal regulation of ketogenesis in ruminants appears to differ, with insulin playing a more prominent role than glucagon (She et al. 1999), particularly under the insulin‐resistant state characteristic of the periparturient dairy cow (Angeli et al. 2021b). Although insulin resistance was not directly evaluated in the present study, no differences in circulating insulin concentrations were observed between experimental groups (Roskopf et al. 2025).

Finally, overall hepatic DGAT1 protein abundance was lower in ω−3‐supplemented cows. DGAT1 catalyzes the final step of TAG synthesis, and its downregulation may limit hepatic TAG accumulation. In non‐ruminants, insulin stimulates DGAT1 expression through lipogenic factors such as the Sterol Regulatory Element‐Binding Protein 1c and Carbohydrate‐Responsive Element‐Binding Protein (Linden et al. 2018). Conversely, several studies in animal models and humans have demonstrated that EPA and DHA inhibit hepatic DGAT1 activity, thereby promoting fatty acid oxidation over TAG synthesis (Berge et al. 1999; Xu et al. 2015). This mechanism may be particularly relevant in dairy cows, which exhibit limited hepatic TAG export capacity and are therefore predisposed to lipid accumulation during early lactation (Andjelić et al. 2022; Barcarolo et al. 2022).

5. Conclusion

The biological effects of dietary PUFA have been widely documented across species and involve diverse regulatory mechanisms. In this study, supplementation with calcium salts enriched with ω−3 fatty acids during the transition period modulated the hepatic abundance of key proteins involved in fatty acid oxidation and esterification in postpartum dairy cows. These molecular responses were observed primarily during the postpartum period, when metabolic demands and lipid mobilization are greatest.

Despite the absence of major changes in conventional liver function indicators or hepatic TAG content, the modulation of enzymes related to peroxisomal β‐oxidation, ketogenesis, and lipid esterification indicates an adaptive adjustment in hepatic fatty acid metabolism. Changes in oxidative stress markers were consistent with the higher susceptibility of PUFA to lipid peroxidation, without evidence of compromised antioxidant defense. Overall, these findings support a role for ω−3‐enriched calcium salts in modulating hepatic metabolic responses during early lactation. Further studies are needed to elucidate the underlying mechanisms and to determine the physiological and productive relevance of these molecular adaptations under different feeding and management conditions.

Author Contributions

Investigation, methodology, data management, formal analysis, visualization, and drafting of the original manuscript: Gino Storani and Pablo Matías Roskopf; data management and formal analysis: Emmanuel Angeli and Lucas Emanuel Ribas; methodology, manuscript revision, and editing: Hugo Héctor Ortega, Eloy Eduardo Salado, and Gustavo Juan Hein; resources, supervision, project coordination, and manuscript revision: Eloy Eduardo Salado and Gustavo Juan Hein. All authors have reviewed and approved the final version of the manuscript for publication.

Ethics Statement

The authors confirm that the ethical policies of the journal, as noted on the journal's author guidelines page. All procedures involving animal handling and care were reviewed and approved by the Ethics and Safety Committee of the College of Veterinary Sciences, National University of Litoral (Protocol No. 614/20). They were conducted in accordance with the Guide for the Care and Use of Agricultural Animals in Research and Teaching (Ag Guide. 2020).

Conflicts of Interest

The authors declare no conflicts of interest.

Supporting information

Supporting File 1

Supporting File 2

Storani, G. , Roskopf P. M., Angeli E., et al. 2026. “Dietary omega‐3 fatty acids alter hepatic lipid oxidation and oxidative stress markers in postpartum dairy cows.” Journal of Animal Physiology and Animal Nutrition 110: 523–533. 10.1111/jpn.70062.

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.