Dietary essential oils modulate post‐mortem oxidative damage in trout fillets exposed to slaughter stress during frozen storage

TL;DR

This study shows that adding essential oils to trout diets helps preserve fish quality during frozen storage, even if it doesn't reduce initial stress from slaughter.

Contribution

The study reveals that dietary essential oils delay oxidative damage in trout fillets during frozen storage, despite not reducing immediate slaughter stress.

Findings

Air asphyxiation caused greater post-slaughter oxidative damage and texture loss compared to percussion.

Dietary essential oils delayed oxidative damage and preserved texture and color during 45 days of frozen storage.

Essential oils were more effective in preserving fillet quality under high-stress slaughter conditions like air asphyxiation.

Abstract

Slaughter is a critical phase in aquaculture that can severely compromise both animal welfare and product quality. Stress responses triggered during this stage may accelerate post‐mortem biochemical degradation and promote oxidative damage in fish fillets. Essential oils, known for their antioxidant and anti‐inflammatory properties, have been proposed as dietary supplements to help mitigate stress and preserve flesh quality. This study investigated the effects of dietary essential oil supplementation and different slaughter methods, air asphyxia and percussion, on stress biomarkers, oxidative processes, and fillet quality in rainbow trout (Oncorhynchus mykiss), both immediately after slaughter and during frozen storage.

Air asphyxiation significantly accelerated ATP degradation, increased lipid and protein oxidation products and caused texture loss in fillets assessed immediately post‐slaughter. These effects were markedly less pronounced in percussion‐slaughtered fish. Lipid mediators such as 12‐HpEPE+15‐HpEPE and PGD3 + PGE3 were elevated only in asphyxiated fish, providing potential biomarkers for slaughter‐induced stress. Proteomic analysis identified several glycolytic enzymes as highly responsive to air asphyxia. Dietary supplementation with essential oils (0.02%) did not mitigate the immediate physiological stress responses to slaughter and was associated with increased muscle protein oxidation at death. However, during 45 days of frozen storage, fillets from fish fed the supplemented diet exhibited delayed progression of oxidative damage, with improved texture and colour retention, especially under high‐stress conditions such as air asphyxia.

Although dietary essential oils did not reduce slaughter‐induced stress, they proved to be an effective strategy for delaying fish fillet degradation and preserving texture and colour during frozen storage. © 2026 The Author(s). Journal of the Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Figures

Lipid and protein oxidative damage after 30 (T30) and 45 (T45) days of frozen storage. (a) Total conjugate dienes (CD; mmol Hp kg−1 fat); (b) TBARS (mg MDA‐eq. kg−1 fillet). (c) Total protein carbonylation (a.u. [arbitrary units] μg−1 protein) in fillets from trout fed a diet with (MIX) or without (CTR) a supplementation with essential oils and slaughtered by asphyxia in air (ASP) or percussion (PER), at 30 and 45 days of frozen storage. (d–f) Fold changes in CD, TBARS and protein carbonylation, respectively, at 30 and 45 days of frozen storage relative to the initial time (T0). Bars represent means ± standard deviation (n = 5). Two‐way ANOVA assessed the effects of diet (D), slaughter method (S), and their interaction for each storage time. DT30/DT45 P < 0.05 indicates a significant effect of diet at 30 or 45 days. ST30/ST45 P < 0.05 indicates a significant effect of slaughter method at 30 or 45 days. Bars with different letters (a, b, c) denote significant differences (P < 0.05) between groups within the same storage time, as determined by Tukey–Kramer's post hoc test.

(a) Effect of diet × slaughter interaction found for carbonylation index of specific proteins of fillets from trout fed a diet with (MIX) or without (CTR) a supplementation with essential oils and slaughtered by asphyxia in air (ASP) or percussion (PER), after 45 days (T45) of frozen storage. (b) Fold change in total protein carbonylation for each protein and experimental group, calculated by dividing the level of protein carbonylation at 45 days of frozen storage by the level at 0 day (T45/T0). Bars represent means ± standard deviation (n = 5). Bars with different letters (a, b, c) denote significant differences (P < 0.05) between groups, as determined by Tukey–Kramer's post hoc test. PYGM, glycogen phosphorylase muscle form; PKM, pyruvate kinase isozymes M1/M2; ENO3, beta‐enolase; CKM, creatine kinase M‐type; ALDOA, fructose‐bisphosphate aldolase A; GAPDH, glyceraldehyde‐3‐phosphate dehydrogenase; LDHA, lactate dehydrogenase A‐chain; PGAM2, phosphoglycerate mutase 2; TPI1, triosephosphate isomerase; AK1, adenylate kinase isoenzyme 1.

Tables

Table 1. Nucleotides extracted from trout muscle and calculated K‐, H‐ and F‐values as affected by diet with (MIX) or without (CTR) a supplementation with essential oils and slaughter method (asphyxia in air, ASP; percussion, PER)

	CTR‐PER	MIX‐PER	CTR‐ASP	MIX‐ASP	RMSE	P‐value
						Diet, D	Slaughter, S	D × S
ADP	0.220	0.275	0.259	0.218	0.074	0.850	0.821	0.218
AMP	0.098	0.142	0.092	0.088	0.036	0.299	0.122	0.210
IMP	6.110	7.106	5.411	5.060	2.394	0.792	0.274	0.584
Ino	1.153	2.377	2.021	1.936	0.617	0.090	0.502	0.056
Hx	0.2ab	<LOD1b	0.328a	0.324a	0.156	0.288	0.020	0.310
K‐value	18.64b	24.63ab	29.18a	30.01a	5.967	0.264	0.024	0.373
F‐value	80.51a	74.22ab	69.40ab	68.66b	6.448	0.297	0.024	0.405
H‐value	3.13ab	—b	4.34a	4.40a	2.215	0.191	0.026	0.174

Table 2. Free fatty acids (DHA, EPA and ARA) and lipid mediators in plasma from trout fed a diet with (MIX) or without (CTR) a supplementation with essential oils and slaughtered by asphyxia in air (ASP) or percussion (PER), immediately after death

		CTR‐PER	MIX‐PER	CTR‐ASP	MIX‐ASP	RMSE	P‐value
							Diet, D	Slaughter, S	D × S
Free fatty acids	DHA	2.471	1.397	1.531	1.582	1.109	0.318	0.458	0.274
	EPA	0.959	0.537	0.631	0.578	0.274	0.070	0.258	0.152
	ARA	0.346	0.137	0.222	0.183	0.150	0.083	0.569	0.225
DHA‐derived mediators	17‐HDoHE	15.337	13.791	8.952	12.589	6.400	0.720	0.204	0.379
	11‐HDoHE	5.815	5.850	5.520	5.677	0.456	0.644	0.268	0.772
	4‐HDoHE	7.297	7.102	5.197	6.486	2.580	0.642	0.256	0.529
	Σ DHA‐derived hydroxides	28.449	26.743	19.669	24.752	8.657	0.669	0.183	0.394
EPA‐derived mediators	12‐HEPE	12.350	12.447	12.532	12.257	0.712	0.783	0.989	0.567
	15‐HEPE	1.648	1.381	1.273	1.465	0.268	0.762	0.244	0.074
	5‐HEPE	6.252a	5.538a	3.931b	4.853ab	1.369	0.868	0.026	0.200
	Σ EPA‐derived hydroxides *	20.205	19.366	17.736	18.575	1.829	0.978	0.060	0.308
	12‐HpEPE+15‐HpEPE	<LOD*	0.130	0.210	0.190	0.0919	0.697	0.429	0.104
	PGD3 + PGE3	<LOD † ,b	<LOD † ,b	0.137a	0.320a	0.163	0.226	0.007	0.226
17‐HDoHE/15‐HEPE ratio		9.35	9.71	7.02	8.81	3.987	0.556	0.379	0.694
11‐HDoHE/12‐HEPE ratio		0.47	0.47	0.44	0.46	0.045	0.544	0.321	0.634
4‐HDoHE/5‐HEPE ratio		1.17	1.25	1.32	1.33	0.318	0.764	0.446	0.826
ARA ARA‐derived mediators	LTB4	0.243	0.219	0.192	0.063	0.204	0.413	0.275	0.574
	11‐HETE	0.915a	0.984a	0.730b	0.653b	0.206	0.963	0.013	0.438
	PGE2	2.816a	2.768a	2.484b	2.330b	0.140	0.126	<0.0001	0.411

Table 3. Protein and lipid oxidation products and physical characteristics of fillets from trout fed a diet with (MIX) or without (CTR) a supplementation with essential oils and slaughtered by asphyxia in air (ASP) or percussion (PER), immediately after death

	CTR‐PER	MIX‐PER	CTR‐ASP	MIX‐ASP	RMSE	P‐value
						Diet, D	Slaughter, S	D × S
Total carbonyls	6.6c	14.3b	28.5a	50.3a	9.593	0.0283	0.0008	0.2375
Conjugated dienes	10.7	10.9	12.3	14.4	2.503	0.4743	0.1136	0.5171
TBARS	0.1	0.2	0.1	0.2	0.053	0.1226	0.8177	0.786
WHC, %	91.71ab	89.58ab	93.19a	88.01b	1.593	0.0041	0.9602	0.1354
Texture attributes
Hardness, N	5.09a	3.61b	3.22b	4.10a	0.859	0.5609	0.2011	0.0447
Cohesiveness	0.27	0.33	0.27	0.30	0.037	0.0887	0.3796	0.4813
Resilience	0.05	0.06	0.07	0.06	0.015	0.9189	0.7174	0.2983
Adhesiveness	0.40	0.36	0.20	0.42	0.119	0.2290	0.3206	0.0857
Colour parameters
L*	31.63ab	33.84ab	29.60b	34.39a	1.911	0.0132	0.5192	0.2762
a*	13.36a	10.60b	15.73a	10.68b	2.636	0.0334	0.4429	0.4738
b*	17.72ab	14.46b	21.54a	15.29b	3.460	0.0445	0.2778	0.4773

Table 4. Physical characteristics (WHC, colour parameters, texture attributes) of fillets from trout fed a diet with (MIX) or without (CTR) a supplementation with essential oils and slaughtered by asphyxia in air (ASP) or percussion (PER), at 0 time and after 30 and 45 days of frozen storage

Diet, D		CTR			MIX			RMSE	P‐value
Days of storage, T		0	30	45	0	30	45		D	T	D × T
ASP	WHC	93.19ab	97.50a	83.08b	88.01ab	83.27b	90.47b	4.524	0.085	0.296	0.005
	Colour parameters
	L*	29.60b	31.17b	38.24a	34.39ab	39.33a	39.44a	2.466	0.002	0.002	0.088
	a*`	15.73b	27.29a	16.76b	10.68b	18.36b	13.37b	3.028	0.002	0.001	0.302
	b*	21.54bc	38.63a	26.10bc	15.29c	27.94ab	21.98bc	4.370	0.005	0.001	0.440
	Texture attributes
	Hardness, N	3.22b	10.20a	4.29b	4.10b	5.91ab	4.70ab	1.668	0.227	0.002	0.037
	Cohesiveness	0.27c	0.33bc	0.36abc	0.30bc	0.47a	0.40ab	0.046	0.007	0.002	0.112
	Resilience	0.07b	0.07b	0.08b	0.06b	0.14a	0.10ab	0.022	0.021	0.009	0.032
	Adhesiveness	0.20	1.33	0.30	0.42	0.98	0.28	0.583	0.922	0.280	0.888
PER	WHC	91.71a	86.78a	80.73b	89.58a	85.20a	86.24a	4.260	0.769	0.038	0.262
	Colour parameters
	L*	31.63b	38.89a	33.18ab	33.84ab	39.16a	38.60ab	2.766	0.067	0.007	0.301
	a*	13.36bc	20.98ab	26.06a	10.60c	21.70a	19.55ab	3.143	0.079	0.0001	0.180
	b*	17.72bc	28.11abc	41.24a	14.4c	27.00abc	30.79ab	5.539	0.083	0.0002	0.344
	Texture attributes
	Hardness, N	5.09a	6.25a	5.19a	3.61b	5.61a	5.22a	0.511	0.014	0.001	0.074
	Cohesiveness	0.27b	0.40a	0.33ab	0.33ab	0.40a	0.35ab	0.033	0.112	0.001	0.363
	Resilience	0.05b	0.12a	0.07b	0.06b	0.12a	0.08b	0.016	0.331	<0.0001	0.764
	Adhesiveness	0.40a	0.13c	0.29abc	0.36ab	0.14ab	0.39a	0.084	0.548	0.001	0.356

Full text

INTRODUCTION

Essential oils are pure compounds derived from various plant parts such as leaves and roots. They are primarily composed of lipophilic secondary metabolites, including mono‐ and sesquiterpenes, allyl and isoallyl phenols.1 These compounds are widely recognized for their antiseptic, antibacterial, anti‐inflammatory and antioxidant properties, and have been used in traditional medicine for centuries.2 In recent years, their application in aquaculture has attracted growing interest, particularly as feed additives to enhance appetite, growth performance and immune responses in fish.3, 4 Several studies have assessed the effects of essential oil constituents, such as eucalyptol, carvacrol and thymol, either individually or in combination, when included in fish diets at low concentrations (typically 0.1–1%).5, 6 Reported benefits include improved feed efficiency, growth rate and serum protein levels.

In addition to these growth‐promoting effects, essential oils have shown potential to mitigate stress when used as anaesthetics or sedatives, or administered via the diet.5, 7 This is particularly important, as stress in fish can severely compromise both animal welfare and product quality. Acute or chronic stress may be induced by environmental and husbandry factors such as high stocking density, suboptimal water temperature or oxygen levels, handling and slaughter. These stressors trigger physiological and metabolic disruptions, including elevated cortisol and lactic acid levels, reduced muscle pH, protein denaturation, and impaired water holding capacity (WHC), collectively affecting immune function, growth and fillet quality.8

Moreover, stress can lead to oxidative stress, defined as an imbalance between the production of reactive oxygen species (ROS) and the organism's antioxidant defences, resulting in cellular damage to lipids, proteins and DNA. This negatively impacts fish health and contributes to undesirable changes in fillet texture, flavour and shelf life.9

Slaughter represents a particularly critical phase in aquaculture, with implications for both welfare and product quality.10 Among the methods still in use, air asphyxiation, where fish are removed from water and left to die from oxygen deprivation, is increasingly criticized as inhumane due to the prolonged time to loss of consciousness and the severe physiological distress it causes. Nevertheless, it remains common in some production systems, largely due to its operational simplicity and low cost.11 From a quality perspective, pre‐slaughter stress induced by air asphyxiation has been linked to accelerated post‐mortem changes such as ATP depletion and oxidative damage, promoting lipid and protein oxidation and, as previously noted, reducing fillet quality.12 These outcomes are largely due to hypoxia‐induced mitochondrial dysfunction, which impairs ROS elimination, electron transport and ATP synthesis.13

In this context, polyphenols present in essential oils exhibit antioxidant activity that may help prevent cellular damage caused by excessive ROS production during stressful events. For instance, dietary supplementation with essential oils (0.02–1 g kg^−1^ diet) has been shown to enhance antioxidant enzyme activity and reduce lipid peroxidation.7 However, data on the ability of essential oils to mitigate oxidative tissue damage in fish under stress remain limited.

Given these challenges, nutritional strategies are being explored to counteract the detrimental effects of pre‐slaughter stress. Essential oils represent a promising tool, not only to enhance antioxidant defences and reduce oxidative damage, but also to improve fish resilience during handling and slaughter‐stressful conditions that, while manageable, are often unavoidable. Therefore, the present study evaluates the effect of dietary supplementation with a commercial blend of essential oils on slaughter‐induced energetic stress and its capacity to prevent lipid and protein oxidation, with the goal of preserving fillet quality in rainbow trout (Oncorhynchus mykiss) during frozen storage at −10 °C.

MATERIALS AND METHODS

Experimental trial and sampling

The experiment was performed according to the ‘Regulations in Animal Experimentation’ of the Department of Agriculture, Food, Environment and Forestry (University of Florence, Italy); the slaughtering procedures were performed under standard farming conditions (Annex. 4.1, b of D. Lgs. 26/2014) and carried out by trained personnel.

A total of 864 juvenile rainbow trout (Oncorhynchus mykiss), 24 weeks old with an average weight of 9.50 ± 0.29 g, were reared at Fondazione Edmund Mach (San Michele all’Adige, Trento, Italy). A detailed description of the trial and the chemical composition of the administrated diets were previously reported.14 Briefly, fish were randomly assigned to two different dietary treatments. The control (CTR) diet consisted in a commercial feed (VITA and ECOFISH, according to the growth stage; Veronesi S.p.A., Verona, Italy), while the experimental feed was the CTR diet supplemented with a low level (0.02% w/w) of a commercial blend of essential oils (namely eucalyptol 3.0%, carvacrol 2.0% and thymol 0.5%) (MIX).

After 51 weeks of farming (when fish reached a live weight of 1016.5 and 1061.5 g in CTR and MIX groups, respectively), 16 fish per dietary group were subjected to two different slaughter procedures. A first group was represented by fish asphyxiated in air (ASP, n = 8) until visual loss of opercular ventilation, then percussively slaughtered by trained personnel. The second group of fish was directly stunned/slaughtered by percussion (PER, n = 8). Immediately after, blood was collected through the caudal vein and stored in heparinized syringes (1 mmol L^−1^ TRIS buffer with 30 units mL^−1^ sodium heparin). After this, fish were gutted and filleted. Overall, 16 fillets were obtained from each treatment (CTR‐PER, CTR‐ASP, MIX‐PER, MIX‐ASP) and stored at −10 °C for 45 days. The sampling times were set at day 0 (T0), day 30 (T30), and day 45 (T45); at each sampling day, five fillets per group were randomly selected, thawed overnight at 4 °C and sampled for the analyses described.

Chemical analysis

Nucleotides

Adenosine 5′‐triphosphate (ATP), adenosine 5′‐diphosphate (ADP), adenosine 5′‐monophosphate (AMP), inosine 5′‐monophosphate (IMP), inosine (Ino) and hypoxanthine (Hx) were extracted from 1 g of fish muscle at T0 using 10 mL of 6% perchloric acid, and analysed by high‐performance liquid chromatography according to Secci et al.15 K‐, F‐ and H‐values were calculated using the formulas described.16 K‐value (%) = 100 × ([Ino] + [Hx])/([ATP] + [ADP] + [AMP] + [IMP] + [Ino] + [Hx]); F‐value (%) =100 × [IMP]/([IMP] + [Ino] + [Hx]); H‐value (%) = 100 × [Hx]/([IMP] + [Ino] + [Hx]).

Fatty acid profiles

Total lipids were extracted from fish fillets at T0 following the Bligh and Dyer method,17 quantified gravimetrically, and converted to fatty acid methyl esters (FAMEs) using C19:0 as an internal standard. FAMEs were analysed via gas chromatography using a Supelco 37‐component FAME standard (Sigma‐Aldrich, St louis, MO, USA) for quantification.15

Lipid mediators

Plasma levels of free polyunsaturated fatty acids (PUFAs) including arachidonic acid (ARA), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), along with their derivatives, were quantified at T0.15 Briefly, 300 μL plasma was diluted in cold methanol, spiked with 11‐HETE‐d8, and processed through solid‐phase extraction. Lipid mediators were then analysed using liquid chromatography–tandem mass spectrometry (LC‐MS/MS) with electrospray ionization in negative mode, and quantified based on MS/MS transitions.

Lipid peroxidation products

From the lipid extract, primary lipid oxidation products (conjugated dienes) were measured spectrophotometrically at 232 nm and expressed as mmoles of hydroperoxides per kilogram of lipid.18 Secondary lipid oxidation products were determined through the 2‐thiobarbituric acid‐reactive substances (TBARS) assay,19 with results expressed as milligrams of malondialdehyde equivalents (MDA‐eq.) per kilogram of sample.

Protein carbonylation

Oxidative damage to proteins was measured at T0, T30 and T45, as previously described.20 Briefly, cranial muscle samples (400–500 mg, n = 5) were homogenized in a phosphate buffer (pH 6.0) with protease inhibitors. Protein carbonyl groups were labelled with fluorescein‐5‐thiosemicarbazide (FTSC), separated via sodium dodecyl sulfate–polyacrylamide gel electrophoresis and visualized under ultraviolet light. Total (lane gel intensity) and specific (band gel intensity) protein carbonylation levels were calculated using LabImage 1D software (Kapelan Bio‐Imaging, Leipzig, Germany), by comparing FTSC fluorescence to Coomassie staining. Carbonylated proteins were analysed by LC‐MS/MS following tryptic digestion, and identified using PEAKS DB software (Bioinformatics Solutions Inc., Waterloo, ON, Canada), searching the raw files against the UniProtKB/TrEMBL database with a FDR < 1%.

Physical analysis

Colour, texture and WHC

Physical characteristics of fish fillets were analysed at T0, T30 and T45. Colour measurements were taken at three points along the epaxial region (cranial, medial, caudal) using a Chroma Meter CR‐200 (Konica Minolta, Chiyoda, Japan) and expressed in CIELab coordinates: L* (lightness), a* (redness), and b* (yellowness).21 Texture was evaluated on two 3 × 3 cm fillet cranial sections using a Zwick Roell texturometer model (KAF‐TC 0901279, Zwick GmbH & Co. KG, Ulm, Germany) with a 1 kN load cell and 10 mm cylindrical probe. A compression test was performed at 30 mm min^−1^ until 50% deformation. Parameters such as hardness, cohesiveness, resilience and adhesiveness were calculated using the Test‐Xpert2 by Zwick Roell software, version 3.0. After that, the fish flesh was homogenised, and the percentage of water loss was calculated after centrifugation of 2 g of flesh, as detailed elsewhere.22

Statistics

All data were analysed using the General Linear Model procedure in SAS/STAT software, version 15.3 (SAS Institute, Cary, NC, USA).23 Two‐way analysis of variance (ANOVA) was used to test the effects of diet (CTR vs. MIX) and slaughter method (ASP vs. PER) on post‐mortem quality traits at T0. A second two‐way ANOVA tested the effects of diet and storage time (T0, T30, T45) within each slaughter method for evaluating protein oxidation, lipid peroxidation and physical characteristics over time. Tukey–Kramer's post hoc test was applied to compare means at P < 0.05. Each fish was treated as a biological replicate (n = 5).

RESULTS AND DISCUSSION

Immediate effects of dietary essential oils on slaughter‐induced energy stress and physicochemical parameters of trout fillets

Effects on nucleotides, plasma fatty acids and lipid mediators

All sample groups exhibited rapid ATP degradation, initiating the typical catabolic sequence associated with slaughter‐induced stress (Table 1). Consequently, ATP was not detected in significant amounts, with low concentrations of ADP and traces of AMP observed. The swift conversion of ATP into downstream catabolites was evidenced by high levels of IMP and Ino. IMP was the prominent nucleotide, accounting for 64% and 72% of the total pool in asphyxiated and percussively slaughtered trout, respectively. Conversely, MIX‐PER fish showed the highest content of Ino (D × S = 0.056). Based on the nucleotide concentrations, the K‐value was significantly higher (P < 0.05) in ASP (29.6%) than in PER (21.63%) groups. This result reflects the accelerated energy consumption in stressed fish, consistent with findings in Salmo carpio ^12^ and grass carp,24 where less stressful stunning methods resulted in lower K‐values. In line with this, both F‐ and H‐values, which relate IMP and Hx levels to their respective catabolites, were significantly higher and lower (P < 0.05), respectively, in PER (F: 77.37%; H: 1.57%) compared to ASP (F: 69.03%; H: 4.37%). These findings confirm that ASP strongly impacts energy metabolism. Notably, dietary essential oil supplementation partially mitigated slaughter‐induced stress, but only in the PER group.

Essential oils have often been tested as sedatives or anaesthetics in aquaculture (for a review, see Aydın and Barbas25) as alternatives to chemical agents. However, as previously reviewed,7 the efficacy of essential oils in mitigating stress varies depending on factors such as composition, concentration and administration mode. Few studies have explored the anti‐stress role of dietary essential oils in fish, and the available literature reveals inconsistent responses. For instance, dietary supplementation with 2 mL kg^−1^ of Aloysia triphylla extract (28.97% geranial, 20.78% β‐citral) in silver catfish for 21 days reduced plasma cortisol and lactate levels after handling stress for blood collection.26 However, it failed to attenuate the stress response in zebrafish subjected to handling for biometry.27 Similarly, Nile tilapia (Oreochromis niloticus) exposed to air for 3 min had similar plasma cortisol and glucose levels regardless of the different diets enriched in cinnamon (Cinnamomum sp.) (0.5–2.0 mL kg^−1^).28 In contrast, eucalyptol supplementation (0.5–1%) in Cyprinus carpio diets for 7 days effectively mitigated the increase in blood stress biomarkers induced by copper exposure,29 while oregano essential oil (60% carvacrol, 5% thymol) at 1–2 mL kg^−1^ partially improved welfare in high‐density reared (400 fish m^−3^) tilapia (O. niloticus).5

Under stress, catecholamine release activates triacylglycerol lipase, which may increase plasma triglycerides and free fatty acids.31 However, in this study, neither the plasma fatty acid profile (Supporting Information, Table S1) nor the levels of free ARA, EPA, and DHA (Table 2) were significantly affected by diet or slaughter method. Overall, DHA was the most abundant plasma fatty acid (~30%), followed by palmitic acid (C16:0, 18%), linoleic acid (C18:2n‐6, 11%) and oleic acid (C18:1n‐9, 10%). Consequently, PUFAs predominated over saturated and monounsaturated fatty acids. Nonetheless, essential oil supplementation tended to increase DHA levels relative to other PUFAs, such as linoleic acid and EPA, particularly in ASP groups, as indicated by significantly higher DHA/linoleic (P < 0.05) ratios and a trend toward higher DHA/EPA (P = 0.07) ratios. Free ARA and EPA also tended to be higher in CTR than in MIX groups (Table 2).

Lipid‐derived oxidized metabolites, formed through enzymatic and non‐enzymatic oxidation of PUFAs like ARA, EPA, and DHA, play important roles in physiological processes and are associated with immune functions and stress responses.32, 33 Among these metabolites, prostaglandins, synthesized from ARA via the cyclooxygenase (COX) pathway, modulate the hypothalamus–pituitary–interrenal (HPI) axis and regulate cortisol and corticosterone secretion in fish during the stress response.34 Consequently, the lipid composition of aquafeeds and supplementation with antioxidants have been investigated as strategies to mitigate the effects of various stressors.32 Regarding the inclusion of essential oils or herbal extracts in fish feed, the literature shows considerable variability in outcomes depending on the type of molecules used, their concentrations and the fish species involved.7

In this study, plasma lipid mediators were mainly derived from ARA, EPA and DHA, with higher concentrations of lipoxygenase (LOX)‐derived EPA and DHA metabolites (Table 2). Slaughter‐induced stress significantly influenced these profiles, while diet had no clear effect. Interestingly, the levels of EPA‐derived metabolites, 12(S)‐hydroperoxy‐5Z,8Z,10E,14Z,17Z‐eicosapentaenoic acid+15(S)‐hydroperoxy‐5Z,8Z,11Z,13E,17Z‐eicosapentaenoic acid (12‐HpEPE+15‐HpEPE) and especially the prostaglandins PGE3 + PGD3_,_ were useful for discriminating between the two slaughter methods. On one hand, EPA hydroperoxides were produced in high amounts in the ASP groups and were undetectable in the CTR‐PER group. Furthermore, the higher concentration of EPA hydroperoxides, together with the lower concentration of EPA hydroxides in ASP fish (P = 0.06; Table), suggests that stress during slaughtering may lead to reduced glutathione‐peroxidase detoxification activity. This decrease in endogenous antioxidant capacity in ASP trout could not be mitigated through dietary supplementation.

On the other hand, the exclusive detection of PGD3 and PGE3 in the ASP group (P < 0.05; Table 2) supports their use as reliable plasma stress markers in fish. In addition, the significantly lower levels of PGE2 and 11‐HETE in ASP trout compared to PER trout are consistent with the findings of Wu et al.,35 who reported that severe hypoxia significantly downregulates ARA metabolism in rainbow trout. Hence, these findings suggest that COX activity may shift their action from ARA toward EPA in stressed fish, favouring the production of anti‐inflammatory mediators. A similar compensatory mechanism has been identified in rats, where enzymatic activity was redirected toward EPA and DHA metabolites to restore tissue homeostasis in prediabetic rats.36

Regarding the diet, although statistical significance was not achieved, dietary supplementation with essential oils led to a 1.2‐fold increase in 5‐HEPE and total DHA hydroxides compared to the CTR diet in ASP trout. Additionally, COX‐derived metabolites from ARA, such as LTB4, were numerically lower in the MIX group (0.141 ng mL^−1^) compared to the CTR group (0.218 ng mL^−1^), suggesting a less pro‐inflammatory phenotype.

Therefore, although dietary supplementation with the commercial blend of essential oils did not fully counteract the negative effects of slaughter‐induced stress, as also reflected in the nucleotide data, the present results indicate a trend toward a less inflammatory profile in ASP trout fed the MIX diet.

Muscle lipid and protein oxidation, and physical characteristics

In accordance with previous reports highlighting that hypoxic slaughter methods increase stress and, consequently, oxidative damage in fish,12, 13 the data obtained in the present study confirm that asphyxiation significantly exacerbates oxidative damage to muscle proteins (Table 3). Notably, while protein oxidation, and particularly carbonylation, showed a significant increase (P < 0.0008), only a slight, non‐significant trend towards increased lipid oxidation was observed. This finding supports the conclusions of Hematyar et al.,38 who, after an extensive review of oxidative processes in fish, proposed that protein oxidation tends to predominate over lipid oxidation during post‐mortem changes in fish.

The data also indicate that muscle proteins are selectively affected by carbonylation. Among the various proteins present in fish muscle, a subset emerged as the primary oxidation targets: glycogen phosphorylase muscle form (PYGM), pyruvate kinase isozymes M1/M2 (PKM), beta‐enolase (ENO3), creatine kinase M‐type (CKM), fructose‐biphosphate aldolase muscle type (ALDOA), glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH), l‐lactate dehydrogenase A chain (LDHA), phosphoglycerate mutase 2 (PGAM2), triosephosphate isomerase (TPI1), and adenylate kinase isoenzyme 1 (AK1). Among these, lower‐molecular‐weight proteins, particularly metabolic enzymes such as CKM, GAPDH, LDHA, TPI1, AK1 and notably PGAM2, exhibited high susceptibility to ROS generated during hypoxia (Supporting Information, Fig. S1). The specific denaturation of some of these proteins has been directly linked to textural and colour changes in fish fillets. For instance, LDHA activity loss has been associated with early quality deterioration during frozen storage in various fish species,39 while CKM and PYGM have been positively correlated with muscle firmness in rainbow trout.40 Consistently, the CTR‐ASP group exhibited the lowest fillet hardness values among all groups (Table 3), significantly differing from CTR‐PER. This aligns with previous findings,35 which demonstrated that severe hypoxia (3.0 ± 0.05 mg L^−1^ dissolved O_2_ for 3 h) led to muscle fibre separation and cracking, reducing resistance to compression.

Although the effect on colour was moderate, asphyxia slaughter resulted in decreased L* values, likely due to both muscle structure disruption and protein oxidation. Given that proteins influence fillet pigmentation, their denaturation may partially explain the observed colour alterations.

In addition to slaughter conditions, dietary supplementation with essential oils also significantly increased total carbonyl levels immediately after death (P < 0.05; Table 3), although the increase caused by the asphyxia slaughter was markedly more pronounced. This observation is consistent with the nucleotide degradation patterns observed (Table 1). Specifically, protein carbonylation levels were 4.5‐fold higher in CTR‐ASP compared to CTR‐PER, whereas the effect attributed to essential oil supplementation was more moderate, showing a 2.2‐fold increase when comparing MIX‐PER to CTR‐PER. Importantly, the inclusion of essential oils in the diet of asphyxiated fish did not significantly enhance protein carbonyl formation, confirming that the primary driver of protein carbonylation is the hypoxia‐based slaughter method, rather than the essential oil blend. Regarding specific protein targets, essential oil supplementation appeared to induce a more uniform oxidative pattern across muscle proteins, without the selective sensitivity observed for the asphyxia slaughter (Supporting Information, Fig. S1).

As for the physical characteristics of the trout fillets (Table 3), essential oil supplementation had a subtle but significant influence. Specifically, the MIX group exhibited lower WHC, a* and b* values (P < 0.05), along with higher L* compared to the CTR fillets. WHC is a critical quality attribute, as it affects fluid retention during processing (yield) and storage (drip and thaw loss). The significantly lower WHC values in MIX trout, regardless of the slaughter method, may be attributable to the elevated carbonyl levels found in this group. Interestingly, the better textural properties observed in the MIX fillets could also be associated with the increased carbonylation. Indeed, a moderate and uniform degree of protein carbonylation, when not accompanied by other damaging oxidative effects, has been linked to enhanced muscle characteristics. For example, the firmness and cohesiveness of mackerel meat improved with the insolubilization of sarcoplasmic proteins during salt–vinegar curing, suggesting that the precipitation of these proteins contributed to better textural properties.41

Finally, the colour alterations detected with dietary supplementation of essential oils may also result, at least in part, from increased oxidation of sarcoplasmic proteins. While essential oils are not valuable sources of pigments, several studies have reported moderate changes in fillet colour following their inclusion in aquafeeds. For instance, it has been found that supplementing the basal diet of gilthead sea bream (Sparus aurata) with thymol (0.5 g kg^−1^ feed) and rosemary extract (0.6 g kg^−1^ feed) significantly reduced the b* value of the fillet, while carvacrol (0.5 g kg^−1^ feed) increased it.42 Similarly, it has been reported that supplementation with clove essential oil in Nile tilapia (O. niloticus) slightly affected flesh colour, reducing both a* and b* values.43

Effects of dietary essential oils on slaughter‐induced physicochemical parameters of trout fillets during frozen storage

Oxidative patterns and quality parameters observed during frozen storage

Both lipid and protein oxidation products (Fig. 1(a)–(c)) increased during frozen storage, as widely reported in the literature.38 However, both slaughter method and dietary supplementation significantly affected not only the quantity but also the formation rate of oxidation products (Fig. 1(d)–(f)). On one hand, fillets from fish subjected to asphyxia slaughter exhibited a significant increase in protein carbonylation, especially after 45 days of frozen storage (Fig. 1(c)). These fillets also presented the highest TBARS levels and the fastest TBARS formation rate (Fig. 1(e)).

Lipid and protein oxidative damage after 30 (T30) and 45 (T45) days of frozen storage. (a) Total conjugate dienes (CD; mmol Hp kg−1 fat); (b) TBARS (mg MDA‐eq. kg−1 fillet). (c) Total protein carbonylation (a.u. [arbitrary units] μg−1 protein) in fillets from trout fed a diet with (MIX) or without (CTR) a supplementation with essential oils and slaughtered by asphyxia in air (ASP) or percussion (PER), at 30 and 45 days of frozen storage. (d–f) Fold changes in CD, TBARS and protein carbonylation, respectively, at 30 and 45 days of frozen storage relative to the initial time (T0). Bars represent means ± standard deviation (n = 5). Two‐way ANOVA assessed the effects of diet (D), slaughter method (S), and their interaction for each storage time. DT30/DT45 P < 0.05 indicates a significant effect of diet at 30 or 45 days. ST30/ST45 P < 0.05 indicates a significant effect of slaughter method at 30 or 45 days. Bars with different letters (a, b, c) denote significant differences (P < 0.05) between groups within the same storage time, as determined by Tukey–Kramer's post hoc test.

On the other hand, dietary inclusion of essential oils significantly slowed (P < 0.05) the formation rates of both TBARS and protein carbonyls (Fig. 1(e),(f)), particularly in the asphyxiated fish. Consequently, a statistical trend (P < 0.10) toward reduced levels of lipid and protein oxidation products was observed after 45 days of frozen storage (Fig. 1(b),(c)). These results suggest a potential antioxidant effect of dietary essential oils, consistent with previous findings.43 Figure 2 illustrates the effects of slaughter method and dietary intervention on the carbonylation of specific muscle proteins during frozen storage. The data showed that slaughter‐induced stress selectively increased carbonylation (P < 0.05) of certain proteins, especially in ASP trout, as also reported.44 The most affected proteins were PYGM, PKM, ENO3, CKM, ALDOA, LDHA, and PGAM2. Dietary supplementation with essential oils exerted a protective effect, significantly slowing carbonylation progression over 45 days in both PER and ASP groups compared to their controls (P < 0.05). This protective effect was more pronounced in fish subjected to asphyxia. Notably, decreased carbonylation was observed in sarcoplasmic proteins such as PYGM, PKM, ENO3 and LDHA. Similarly, CKM, ALDOA, TPI1 and AK1 also tended toward lower carbonyl levels with essential oil supplementation. This selective protection may contribute directly to fillet quality, as previously discussed. In particular, the protection of PYGM and CKM, both positively associated with muscle firmness,40 may explain the more stable hardness observed in ASP fillets during frozen storage (Table 4).

(a) Effect of diet × slaughter interaction found for carbonylation index of specific proteins of fillets from trout fed a diet with (MIX) or without (CTR) a supplementation with essential oils and slaughtered by asphyxia in air (ASP) or percussion (PER), after 45 days (T45) of frozen storage. (b) Fold change in total protein carbonylation for each protein and experimental group, calculated by dividing the level of protein carbonylation at 45 days of frozen storage by the level at 0 day (T45/T0). Bars represent means ± standard deviation (n = 5). Bars with different letters (a, b, c) denote significant differences (P < 0.05) between groups, as determined by Tukey–Kramer's post hoc test. PYGM, glycogen phosphorylase muscle form; PKM, pyruvate kinase isozymes M1/M2; ENO3, beta‐enolase; CKM, creatine kinase M‐type; ALDOA, fructose‐bisphosphate aldolase A; GAPDH, glyceraldehyde‐3‐phosphate dehydrogenase; LDHA, lactate dehydrogenase A‐chain; PGAM2, phosphoglycerate mutase 2; TPI1, triosephosphate isomerase; AK1, adenylate kinase isoenzyme 1.

Regarding other quality parameters (Table 4), WHC declined while L*, b* and a* values increased over time, with significant changes detected in both CTR‐ASP and CTR‐PER groups. It is well established that frozen storage can compromise quality characteristics such as colour and WHC due to ice crystal formation, which damages cell membranes and promotes enzymatic activity related to lipid and protein oxidation.45 In this context, the observed reduction in WHC and the increases in L*, b* and a* values over time were expected outcomes. The slaughter method primarily influenced fillet colour throughout storage, consistent with observations made post‐mortem (Supporting Information, Table S2). However, although colour changes occurred in both ASP and PER fillets during storage, significant shifts in L*, a* and b* values manifested earlier in ASP samples.

In line with the oxidation data, essential oil supplementation helped mitigate these changes during frozen storage (Table 4), preventing WHC losses likely via antioxidant protection of muscle proteins. Furthermore, essential oils contributed to greater colour stability, particularly in the ASP fillets. Since colour changes are often driven by oxidative processes during storage, these results further support the antioxidant role of dietary essential oils, especially under the more oxidative conditions caused by asphyxia slaughter.

Overall, the significant reduction in carbonyl and lipid peroxidation product formation associated with essential oil supplementation appears to contribute to maintaining fillet quality during extended frozen storage and enhancing resistance to further degradation, as earlier shown.46 Previous studies also demonstrated the capacity of the same blend of essential oils to improve resistance to oxidative damage and reduced weight loss as liquid loss and WHC in fillets from percussion‐stunned rainbow trout during short‐term refrigerated storage.14

To conclude, the storage temperature of −10 °C selected in this study was chosen to represent suboptimal conditions, allowing oxidation differences between treatments to manifest within the experimental timeframe, in line with prior studies.47, 48 Additionally, these suboptimal conditions, often encountered in real‐life cold chains, where −18 °C is the standard set by the Codex Alimentarius and supported by the FAO,49 are not always consistently maintained. Numerous studies on rainbow trout and other fish species have demonstrated the strong temperature dependence of lipid and protein oxidation during frozen storage.50, 51, 52 Regarding lipid oxidation, lipolytic activity is generally considered markedly reduced only below approximately −30 °C; however, the reduction in activity between −10 and −20 °C is typically less pronounced than at lower temperatures.50 Concerning protein oxidation, at temperatures slightly below freezing, particularly in the −10 to −20 °C range, the freezing process temporarily accelerates oxidation. This acceleration is mainly due to the concentration of pro‐oxidant solutes and protein reactants, as well as the residual enzymatic activity at the sub‐zero temperatures.51 In line with the trends observed for lipid oxidation, the literature indicates that temperatures lower than −30 °C effectively inhibit protein oxidation.52 These findings confirm that storage at −10 °C accelerates both lipid and protein oxidation, but they also indicate that the utilized temperature reflects a suboptimal frozen‐storage condition rather than an extreme one that would distort comparative treatment effects. Assessing stability under these moderately challenging conditions provides an opportunity to observe whether essential oils can mitigate oxidation processes and thereby contribute to maintaining product quality during storage.

CONCLUSIONS

The present study confirms that slaughter is a critical stage affecting both animal welfare and fish product quality. Exposure to air asphyxia significantly impacted ATP degradation, the accumulation of lipid oxidation metabolites in plasma, protein carbonylation, and the textural properties of fillets assessed immediately post‐slaughter, underscoring the necessity of supporting ongoing efforts to replace this practice. Although the sample size was limited, the detection of 12‐HpEPE+15‐HpEPE and PGD3 + PGE3 in the plasma of fish subjected to air asphyxia effectively distinguished this method from alternative slaughtering practices. In addition, specific proteins, particularly CKM, GAPDH, LDHA, TPI, AK1, and especially PGAM2, appeared highly sensitive to air asphyxia. Dietary supplementation with essential oils did not fully mitigate the adverse effects of slaughter‐induced stress and was associated with increased oxidation of muscle proteins in fillets evaluated immediately after death. However, essential oil inclusion delayed the progression of protein oxidative damage, especially under high‐stress conditions such as air asphyxia, and contributed to preserving fillet quality during frozen storage. Nonetheless, further research is needed to clarify the role of essential oils, as dietary supplementation alone did not constitute an effective strategy to reduce slaughter stress, though it may help attenuate the detrimental effects of frozen storage.

FUNDING INFORMATION

This research was funded by the Xunta de Galicia–Axencia Galega de Innovación (GAIN) (IN607B 2023/05).

CONFLICT OF INTEREST

The authors declare that they have no conflicts of interest.

Supporting information

Table S1. Fatty acid profile of plasma (mg mL^−1^ plasma) of rainbow trout fed a diet with (MIX) or without (CTR) a supplementation with essential oils and slaughtered by asphyxia in air (ASP) or percussion (PER). Results are expressed as mean values (n = 5). Table S2. Water holding capacity (WHC, %), colour parameters (L*, a*, b*) values, textural attributes, total carbonyls (expressed as protein carbonylation index, arbitrary units), total lipids, and lipid oxidation items (conjugated dienes: mmol Hp kg^−1^ fat; TBARS: mg MDA‐eq. kg^−1^ fillet) of fillets from trout fed a diet with (MIX) or without (CTR) a supplementation with essential oils and slaughtered by asphyxia in air (ASP) or percussion (PER), at 30th and 45th days of frozen storage. Results are expressed as mean values (n = 5). Figure S1. Carbonylation of sarcoplasmic proteins of trout fillets immediately after death expressed as fold change means ± standard deviation in protein carbonylation between the effect of the diet [with (MIX) or without (CTR) a supplementation with essential oils] and the slaughter method [asphyxia in air (ASP) or percussion (PER)] immediately after death (T0). PYGM, Glycogen phosphorylase muscle form; PKM, pyruvate kinase isozymes M1/M2; ENO3, beta‐enolase; CKM, creatine kinase M‐type; ALDOA, fructose‐biphosphate aldolase muscle type; GAPDH, glyceraldehyde‐3‐phosphate dehydrogenase; LDHA, l‐lactate dehydrogenase A chain; PGAM2, phosphoglycerate mutase 2; TPI1, triosephosphate isomerase; AK1, adenylate kinase isoenzyme 1. *significant effect (P < 0.05; Student's t; n = 5) of the two main treatments on the specific proteins.