# Kinetic activity in slow-growing chickens: effect on antioxidant content, fatty acids profile, lipid oxidation and metabolism of blood and thigh muscles

S. Mattioli, E. Angelucci, L. Madeo, Claire Bonnefous, A. Cartoni Mancinelli, C. Ciarelli, Anne Collin, C. Signorini, A. Dal Bosco, Camille Oger, et al.

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## To cite this version:

S. Mattioli, E. Angelucci, L. Madeo, Claire Bonnefous, A. Cartoni Mancinelli, et al. Kinetic activity in slow-growing chickens: effect on antioxidant content, fatty acids profile, lipid oxidation and metabolism of blood and thigh muscles. Animal, 2025, 19 (7), pp.101569. 10.1016/j.animal.2025.101569. hal-05136054

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Submitted on 30 Jun 2025

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# Kinetic activity in slow-growing chickens: effect on antioxidant content, fatty acids profile, lipid oxidation and metabolism of blood and thigh muscles

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S. Mattioli<sup>a,*</sup>, E. Angelucci<sup>a</sup>, L. Madeo<sup>b</sup>, C. Bonnefous<sup>c,e</sup>, A. Cartoni Mancinelli<sup>a</sup>, C. Ciarelli<sup>d</sup>, A. Collin<sup>e</sup>, C. Signorini<sup>f</sup>, A. Dal Bosco<sup>a</sup>, C. Oger<sup>g</sup>, J.M. Galano<sup>g</sup>, T. Durand<sup>g</sup>, C. Castellini<sup>a</sup>

## ARTICLE INFO

**Article history:**

Received 10 October 2024

Revised 28 May 2025

Accepted 2 June 2025

Available online 9 June 2025

**Keywords:**

Bioactive compounds

Chicken

Foraging

Movement

Muscle

## ABSTRACT

The adaptation of chickens to extensive rearing systems depends on several factors, including genotype, and individual motivation for kinetic activity. The time spent outdoors is positively associated with the intake of grass and many bioactive compounds (e.g., tocols, carotenoids, polyphenols and polyunsaturated fatty acids). In this study, the relationships among kinetic activity, intake of bioactive compounds and body metabolism in outdoor-reared chickens were investigated. One hundred eighty five chicks of the Naked Neck genotype were reared indoors (60 birds) or in an outdoor range (120 birds, 20 chickens/pen, three replications/experimental group). The outdoor animals were monitored from 60 to 80 days of age through a ChickenGate placed 5 m from each shelter. The obtained results were used to discriminate high (OHA, crossing the Gate more than 250 times) and low (OLA, crossing the Gate less than 40 times) activity chickens. The grass intake in the different pens was estimated from 21 to 81 days of age. Blood and meat samples were collected at slaughtering in 12 chickens/group for lipid (TBARS, thio-barbituric acid reactive substances) and protein oxidation, antioxidants, reactive oxygen species (ROS), fatty acid profile and isoprostanoid evaluations. Furthermore, the thigh muscle was dissected to obtain the most glycolytic (PIL) and oxidative (PIFM) muscles. Blood showed lower levels of α-tocotrienol (0.40, 0.45 and 0.79 pmol/mL) and TBARS (15.80, 19.38 and 23.75 nmol MDA/mL) in OHA chickens, followed by OLA and indoor chickens. The trends of carbonyls, and ROS were not significantly affected by the kinetic activity of chickens. OHA resulted in lower levels of n-3 derivative isoprostanoids (on average 1.65 and 2.33-fold lower values of F<sub>3</sub>-isoprostanes and F<sub>4</sub>-neuroprostanes, respectively), than OLA and indoor chickens. The trend of antioxidants in meat was similar to that in blood; however, the two muscles of the thigh differed in several traits. Despite the almost double antioxidant content of PFIM with respect to PIL, the TBARS and carbonyl levels were also higher. OHA chickens presented higher total n-6 levels and n-6/n-3 ratios (16.96 vs 9.63 and 7.31 in OLA and indoor), whereas arachidonic acid, total n-3 and n-3 long-chain polyunsaturated fatty acids levels were lower. The present study highlights that more active animals ingested more grass than low-foraging animals, affecting the intake levels of the main antioxidants and fatty acids. However, this greater amount was not entirely transferred into the blood or muscles, probably due to the contemporary greater consumption of these compounds for antioxidant and energetic purposes.

© 2025 The Authors. Published by Elsevier B.V. on behalf of The animal Consortium. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

<footnote><sup>a</sup>Department of Agricultural, Environmental and Food Science, University of Perugia, Italy, Borgo XX Giugno, 74, 06125 Perugia, Italy</footnote>

<footnote><sup>b</sup>Experimental Zooprophylactic Institute of Umbria and Marche, Via San Costanzo, Perugia 06124, Italy</footnote>

<footnote><sup>c</sup>Department of Livestock Sciences, Research Institute of Organic Agriculture FiBL, Ackerstrasse 113, 5070 Frick, Switzerland</footnote>

<footnote><sup>d</sup>Department of Agronomy, Food, Natural Resources, Animal and Environment (DAFNAE), University of Padova, Viale dell'Università 16, 35020 Padova, Italy</footnote>

<footnote><sup>e</sup>INRAE, Université de Tours, BOA, 37380 Nouzilly, France</footnote>

<footnote><sup>f</sup>Department of Molecular and Developmental Medicine, University of Siena, Policlinico Santa Maria alle Scotte, Viale Bracci 16, 53100 Siena, Italy</footnote>

<footnote><sup>g</sup>Institut des Biomolécules Max Mousseron (IBMM), Pôle Chimie Balard Recherche, UMR 5247, CNRS, Université de Montpellier, ENSCM, 1919 route de Mende, 34293</footnote>

<footnote>Montpellier, France</footnote>

<footnote>* Corresponding author.</footnote>

<footnote>E-mail address: simona.mattioli@unipg.it (S. Mattioli).</footnote>
## Implications

EU regulation 848/2018 affirms that the organic system needs an outdoor run to implement the natural behaviours of chickens. However, different genotypes showed different attitudes towards exploring the outdoor run and then towards movement. This spontaneous activity affected the foraging behaviour of chickens and the intake of bioactive compounds. The not-adapted chickens exhibited a suffering profile, with negative effects on welfare, health and, ultimately, product quality. Accordingly, the EU Regulation recommends the use of slow-growing strains for organic systems and introduces the concept of adaptability to outdoor rearing.

## Introduction

The adaptation of chickens to extensive rearing systems (ERSs) depends on several factors, including genotype, reliable immune response, thermotolerance, outdoor run availability and individual motivation for kinetic activity (Zampiga et al., 2021; Mattioli et al., 2022; Cartoni Mancinelli et al., 2023). The physiological response of animals to the outdoor environment is essential for ensuring good health and welfare, but these environmentally induced responses can also have undesirable side effects (e.g., higher energy expenditure for non-productive actions and less efficiency in the conversion of dietary input; Bonnefous et al., 2023; Fiorilla et al., 2023). In particular, kinetic activity in chickens affects energy and muscle metabolism, storage ability and the antioxidant balance of the body (Mattioli et al., 2022). Furthermore, the time spent outdoors is positively associated with the intake of grass by many broiler strains (Collet et al., 2024). Because grass is a concentrate of bioactive compounds (e.g. tocopherols, carotenoids, polyphenols and fatty acids such as α-linolenic acid α-ALA, C18:3n-3; Dal Bosco et al., 2012, 2016; Mattioli et al., 2022), its intake by chickens contributes to the consumption of these important substances for both animal well-being (homeostasis of the body; Rauw, 2008; Cartoni Mancinelli et al., 2022) and meat quality (lipid stability and shelf life; Funaro et al., 2014; Baéza et al., 2022). Obviously, locomotor activity is negatively correlated with the amount of time spent indoor (Collet et al., 2024); indeed, when very active chickens are confined to a closed space (e.g., an indoor system) they exhibit a suffer profile, enhancing the oxidative processes and the production of free radicals and reactive oxygen species (ROS) by different pathways (Fiorilla et al., 2023; Collet et al., 2024). An excess of energy expenditure requires a further fuel received by the β-oxidation of long-chain (<20 C) polyunsaturated fatty acids (LCP) (Bargut et al., 2014; Coleman, 2019). To reduce the loss of LCP both as metabolic energy substrates, but, in particular as oxidative substrate, an additional antioxidant protection is needed, because an antioxidant acts as a scavenger removing oxidation products without blocking energy metabolism. Indeed, lipid oxidation is one of the most crucial quality parameters in meat, leading to the deterioration of the nutrient profile, flavour, texture and appearance of food and a shortened shelf-life. Malondialdehyde (MDA) is the most common and unspecific marker of lipid oxidation (Fellenberg and Speisky, 2006), whereas isoprostanoids are considered very specific and sensitive. Isoprostanoids, are prostaglandin-like substances, derived from FA esterified of cell membrane phospholipids induced by ROS (Milne et al., 2015), and they are released in a free form in biological fluids produced. The main markers of isoprostanoids are F₂-isoprostanes (F₂-IsoPs), derived from the non-enzymatic oxidation of arachidonic acid (AA, C20:4n-6), and F₃-isoprostanes (F₃-IsoPs) and F₄-neuroprostanes (F₄-NeuroPs), derived from the non-enzymatic oxidation of eicosapentaenoic (EPA, C20:5n-3) and docosahexaenoic (DHA, C22:6n-3) acids, respectively (Milne et al., 2015;

Pisoschi and Pop, 2015; Ahmed et al., 2020). Paying attention to a rearing system (i.e., ERS), which allows chickens to run without considering the oxidative thrust generated by excessive and/or unsuitable kinetic activity of chickens (i.e. use of fast-growing genotypes, or not trained/walking birds; Failla et al., 2021), has led to the development of low-quality products.

The hypothesis of this research is that the kinetic behaviours of chicken genotype may affect their adaptability to a rearing system. Thus, the objective of this research was to study the relationships among kinetic activity, intake of bioactive compounds and body metabolism (antioxidant and fatty acid profile, lipid oxidation, and thigh muscle metabolism) in outdoor-reared chickens, and we compared the obtained results to those acquired by indoor-reared chickens of the same genotypes.

## Material and methods

### *Study design*

One hundred eighty chicks (male/female 1:1) of the Naked Neck slow-growing genotype were reared indoors (60 birds) or with an outdoor range (120 birds) in a commercial facility. Chickens from both groups were raised at the same time from March 2022 to June 2022. All methods are reported in accordance with ARRIVE guidelines for the reporting of animal experiments. This study was carried out in accordance with the Guiding Principles in the Use of Animals and approved by the Italian Ministry (authorisation n°850/2021-PR) and the Animal Ethics Monitoring Committee of the Experimental Zooprophylactic Institute of Umbria and Marche (Protocol ID: 7F31D.20). Three replications of each rearing system were used. Twenty chickens/pen (three replications/experimental group) were allocated and four animals died in the first 3 weeks of age. The indoor (a shelter for the night) and outdoor densities of the animals were 0.10 m²/bird and 4 m²/bird respectively, according to organic regulations (EC Regulation no. 834/2007 and 889/2008). From 1 to 21 d of age, the birds were housed in an environmentally controlled poultry facility with a temperature range of 30–32 °C and a relative humidity of 65–70% (monitored for 24 h). From 22 d of age, the chickens were kept inside a barn, and after this period, the outdoor groups had access to an outdoor run with spontaneous grass. The temperature and humidity of the outdoor

<table><caption>**Table 1**
Chemical composition and main bioactive compounds of grower feed of chicken and grass.</caption><thead><tr><th>Item</th><th>Units</th><th>Grower feed</th><th>Grass</th></tr></thead><tbody><tr><td><strong>Chemical composition</strong></td><td></td><td></td><td></td></tr><tr><td>Moisture</td><td>%</td><td>12.00</td><td>78.61</td></tr><tr><td>CP</td><td>% of DM</td><td>18.41</td><td>8.34</td></tr><tr><td>Ether extract</td><td>"</td><td>4.55</td><td>2.11</td></tr><tr><td>Ash</td><td>"</td><td>5.78</td><td>7.85</td></tr><tr><td>Crude fibre</td><td>"</td><td>3.60</td><td>23.2</td></tr><tr><td>NDF</td><td>—</td><td>10.1</td><td>60.90</td></tr><tr><td>ADF</td><td>—</td><td>5.06</td><td>39.81</td></tr><tr><td>ADL</td><td>—</td><td>1.11</td><td>5.81</td></tr><tr><td>Cellulose</td><td>"</td><td>3.56</td><td>34.0</td></tr><tr><td>Hemicellulose</td><td>"</td><td>5.05</td><td>21.09</td></tr><tr><td>Metabolisable energy</td><td>kcal/kg</td><td>3295.94</td><td>1876.00</td></tr><tr><td><strong>Bioactive compounds</strong></td><td></td><td></td><td></td></tr><tr><td>Retinol</td><td>mg/kg of DM</td><td>14.50</td><td>—</td></tr><tr><td>α-Tocopherol</td><td>"</td><td>63.00</td><td>350.50</td></tr><tr><td>B-carotene</td><td>"</td><td>1.00</td><td>210.10</td></tr><tr><td>Total carotenes</td><td>"</td><td>2.21</td><td>401.60</td></tr><tr><td>LA</td><td>g/100 g of DM</td><td>0.35</td><td>0.82</td></tr><tr><td>ALA</td><td>"</td><td>0.034</td><td>0.86</td></tr></tbody></table>

N = 3 replicates.

Abbreviations: LA: linoleic acid; ALA: α-linolenic acid.
area were 18.3 ± 6.5 °C and 59.5 ± 14.3%, respectively. The pasture was not treated with pesticides. The animals were fed ad libitum the same diet (starter at 1–20 d and grower at 21–81 d). The main chemical characteristics (% d.m.) of the grower diet are reported in Table 1.

All chickens were individually identified with a ring provided with an alphanumeric code chip. For the outdoor chickens, a monitoring system called ChickenGate (Lunageber Engineer, Perugia, Italy; Fig. 1) was placed 5 m from each shelter. This system was characterised by 20 antennas (one every 20 cm) that covered the entire width of the pen. This system was able to read the chips of each chicken and, consequently, record the chickens that moved away at a distance greater than 5 m from the shelter. All the data were stored in a memory sim placed in the control unit of the system.

The outdoor animals were monitored from 60 to 80 d of age. The obtained results were used to discriminate high-activity (OHA) and low-activity (OLA) chickens. Specifically, the OHA chickens were those that, after 10 days of observation, crossed ChickenGate more than 250 times, whereas the OLA chickens were those that crossed ChickenGate fewer than 40 times. Approximately 33% of the animals were grouped in the OHA, 30% were in the OLA, and the remaining (approximately 36%) had a mean value of 102 crossings.

The grass intake (GI) was estimated according to the modified method of Lantinga et al. (2004) by measuring the mass of the grass present at the start of each cycle, the mass of the grass grown in the exclusion pens (0.50 × 0.50 m, one per replications) and the grass at the end of the cycle according to the following equation:

<figure><img src="image_4.png" /><figcaption>**Fig. 1.** Plan of ChickenGate monitoring system. The outdoor monitoring system (Lunageber Engineer) was placed at 5 m of distance from each shelter. This system was characterised by 20 antennas (one every 20 cm), which covered the entire width of the pen. This system was able to read the chips (green ring on the leg) of each chicken and, consequently, to record the chickens that moved away at a distance greater than 5 m from the shelter. All the data were stored in a memory sim placed in the control unit of the system. The OHA were the animals that, in 20 days of observation, crossed the ChickenGate more than 250 times whereas, the OLA were those animals that crossed the ChickenGate less than 40 times.</figcaption></figure>

$$GI = (GMs - GMe) + \left[ \frac{[1 - (GMe/GMs)] - \text{In}[GMe/GMs]}{\text{GMu} - \text{GMs}} \right] \times \text{GMu}$$

where GMs = grass present before birds entered each pen; GMe = grass that remained at the end of the trial; and GMu = undisbursted forage mass from the exclusion pens.

The grass intake estimated from the different pens (GI/d/number of chicks), was referred to 60 d of outdoor access (from 21 to 81 d). Our previous studies revealed that walking activity was positively correlated with the foraging activity of chickens (Bosco et al., 2014; Mattioli et al., 2021a; Bonnefous et al., 2022); thus, the grass intake of the two outdoor groups (OHA and OLA) was estimated starting from the mean grass intake of the pens adjusted for the specific kinetic activity as detailed in Mattioli et al. (2022). Moreover, the nutrient intake in outdoor chickens was normalised for the chickens reared indoors. The kinetic activity of the indoor group was not monitored because numerous previous trials revealed a very low distance covered by chickens (Buijs et al., 2010; Dal Bosco et al., 2010; Rasmussen et al., 2024), approximately 100-fold lower than that covered in outdoor conditions. All chickens/group were weighed weekly (Fig. S1), and feed consumption was also recorded by weight difference (total feed provided by week minus feed remain in the feeder). In particular, every replicate was recorded by weighing the quantity of feed provided minus the feed that remained at the end of the week to measure the daily feed intake (g/d/chicken).

## Slaughter, blood and meat sampling

Twelve chickens/group were selected based on average weight (indoor chickens weighing 2 430 ± 78.23 g) and ChickenGate results (OLA chickens crossed the ChickenGate on average 22 ± 7 times; OHA chickens crossed the ChickenGate on average 250 ± 18 times) and slaughtered at 81 days of age, in a commercial slaughtering house 12 h after feed withdrawal. Birds were electrically stunned (110 V; 350 Hz), killed, and after bleeding, the carcasses were placed in water at 56.5 °C for 1 min) and then plucked and eviscerated (non-edible viscera, including the intestines, proventriculus, gall bladder, spleen, oesophagus, and full crop were removed); successively, carcasses were transported to the laboratory of the Department of Agricultural, Environmental and Food Science, where according to Failla et al. (2021), the more glycolytic (M. iliotibialis lateralis postacetabularis, PIL) and oxidative muscles (M. puboischiofemoralis pars medialis, PIFM) were excised from the left thigh.

Blood samples were taken at slaughtering in four chicken replications, selected as previously reported and collected in heparinised vacutainers to obtain the plasma for measuring the in vivo oxidative parameters, or in empty tubes to recover the serum for fatty acid determination and immunity traits. After collection, the blood samples were immediately transported to the laboratory of the Department of Agricultural, Food and Environmental Science, where the plasma tubes were centrifuged at 1 500 × g for 10 min at +4 °C, and the serum tubes were left to separate for 2 h at room temperature (Mattioli et al., 2021a). Finally, the plasma and serum samples were stored at -80 °C until analysis (at least 2 weeks later).

## Chemical analysis of feed and grass

In Supplementary Table S1 was reported the definition and reference in ATOL, Chebi or Vertebrate trait ontology of the measured traits. Feed and grass analyses were performed on three samples from the three replicates/group.
The moisture content was determined by oven drying 5 g of feed and grass at 105 °C overnight (AOAC, 1995). The CP content was measured by Kjeldahl nitrogen analysis (AOAC, 1995). Ether extracts were quantified using a Soxhlet (SER 148, VELP Scientifica, Monza-Brianza, Italy), with diethyl ether as the solvent. The ash content was determined by combustion for 3 h at 550 °C of samples resulting from dryness. The crude fibre content was determined as described by AOAC (1995). NDF, ADF, and ADL contents were determined according to Van Soest et al. (1991). Cellulose and hemicellulose were calculated as differences starting from NDF, ADF and ADL (i.e., cellulose = ADF-ADL; hemicellulose=NDF-ADF). Metabolisable energy was estimated following the methods reported by Carré et al. (1990).

The levels of α-tocopherol, carotenoids and retinol were quantified using an HPLC system (Hitachi Primade, Milan, Italy) according to Hewavitharana et al. (2004) extraction method, and the quantification was performed using a HPLC system as detailed in Mattioli et al. (2022) (Hitachi Primade: cooling autosampler 1210, pump 1110, fluorimetric detector 1440, diode array detector 1430 and a Synergi Hydro-RP column, Phenomenex, Bologna, Italy). The antioxidant contents in the feed and grass are expressed as mg/kg. The average amount of each bioactive compound was used to calculate the daily intake (µg/d). The summed amounts of the intake of tocols (tocotrienols + tocopherols) and carotenes (lutein + zeaxanthin) in the grass and feed were also calculated.

Fatty acid intake was evaluated from the lipid fraction extracted from feed and grass following the method reported by Folch et al. (1957). To measure the fatty acid methyl esters, the lipid extract was dried with a rotary evaporator, and 1 mL of n-hexane was added. Finally, the transmethylation procedure was performed with 0.5 mL of 2 M KOH methanol solution at 60 °C for 15 min. One mL was added to the gas chromatography system (CP 3800 VARIAN, Milan, Italy) equipped with an FID detector and a capillary column of 100 m length × 0.25 mm × 0.2 µm film (Supelco, 189 Bellefonte, PA). To calculate the amount of each fatty acid, heneicosanoic acid was used as the internal standard (C21:0, Merck analytical standard; Milan, Italy).

## *In vivo oxidative status evaluations*

The following traits were analysed in the plasma and serum samples. The extent of plasma lipid peroxidation (0.5 mL) was evaluated by measuring the absorbance of thiobarbituric acid reactive substances, using a spectrophotometer set at 532 nm (Shimadzu Corporation UV-2550, Kyoto, Japan). The quantification used a tetraethoxypropane calibration curve in sodium acetate buffer (pH = 3.5). The results are expressed as nmol of MDA/mL of plasma. The detection of protein carbonyl groups followed the method of Dalle-Donne et al. (2003), with 2,4-dinitrofenilhidrazina (DNPH) used as the reactive group. The carbonyl content was determined starting from 0.25 mL of serum from the absorbance at 366 nm using the same spectrophotometer previously reported, and a molar absorption coefficient of 22 000 M × 1/cm was used for the quantification as detailed in Mattioli et al. (2021b). The results are expressed as nmol/mg of protein as previously reported.

The tocols (α-tocopherol and its isoforms γ and δ, and α and γ-tocotrienol) and retinol were measured according to Schüep and Rettenmaier (1994). Briefly, 0.2 mL of plasma was mixed with 1 mL of water and 4 mL of an ethanol solution of 0.06% butylated hydroxytoluene (BHT). The mixture was saponified with water/potassium hydroxide (KOH, 60%) at 70 °C for 30 min and extracted with hexane/ethyl acetate (9/1, v/v). Following centrifugation, 2 mL of the supernatant was dried under N₂ and resuspended in 200 µL of acetonitrile. The pellet was re-extracted twice. A 50 µL

volume of filtrate was then injected into the HPLC/FD system previously described on a Sinergy Hydro-RP column (4 µm, 4.6 × 100 mm; Phenomenex, Bologna, Italy). The flow rate was set at 2 mL/min. All tocopherols and tocotrienols were identified using the FD detector reported for fed and grass and quantified using external calibration curves of pure standard solutions in ethanol (Sigma-Aldrich, Bornem, Belgium). The total score was used for statistical analysis. Retinol was analysed with the same HPLC system using a UV-VIS spectrophotometer detector (Jasco UV2075 Plus) set at 325 nm. Retinol was identified and quantified by comparing the sample with a pure commercial standard in chloroform (Sigma-Aldrich, Steinheim, Germany; Genay, France).

The level of ROS in the plasma was evaluated with a commercial kit following the manufacturer's protocol (Diacron, Grosseto, Italy) and expressed as mmol H₂O₂.

Lipid extraction from the blood serum was performed according to the method of Folch et al. (1957), and esterification was carried out following the procedure of Christie (1982). The transmethylation procedure was conducted using heneicosanoic acid methyl esters (Sigma-Aldrich) as an internal standard. The fatty acid (FA) composition was determined using a Varian gas-chromatograph (CP-3800) equipped with a flame ionisation detector and a capillary column of film (Supelco, Belle-Fonte, PA, USA). Helium was used as the carrier gas at a flow rate of 0.6 mL/min. The split ratio was 1:20. Individual FAMEs were identified by comparing the relative retention times of the peaks in each sample with those of a standard mixture (Mixture GLC Nu-check 674, Elysian, MN 56028 USA). The FAs are expressed as a percentage of the total FA content. The average amount of each FA was used to calculate the sum of the total SFAs, monounsaturated fatty acids (MUFAs), and n-3 and n-6 PUFAs. The LC-PUFAs included EPA, n-3 DPA, and DHA acids for n-3 and AA and n-6 DPA acid for n-6.

The levels of F₂⁻, F₃-IsoPs and F₄-NeuroPs were determined in the plasma of the blood samples (0.5 mL) which were present in free form (i.e. no longer esterified to their respective precursor fatty acids). The plasma samples were diluted (1:3, v:v) with acidified water (pH 3) and then mixed with 500 pg of the internal standard (tetradodeuterated prostaglandin F₂α, PGF₂α-d₄). Two solid-phase extraction steps were then performed. Each sample was purified by extraction with an octadecylsilane (C₁₈) cartridge, and the resulting eluate was further purified with an aminopropyl (NH₂) cartridge. In each of the two extraction phases, the conditioning, washing and extraction phases were performed as previously described by Guy et al. (2014). The final eluates from the second extraction on the NH₂ cartridge were derivatised to convert the carboxyl group of isoprostanoids to pentafluorobenzyl ester and the hydroxyl group to trimethylsilyl ether, as previously reported (Signorini et al., 2003), and the quantification was performed by gas chromatography in combination with tandem mass spectrometry using a negative chemical ionisation method (GC/NICI-MS/MS). The F₂⁻, F₃-IsoPs and F₄-NeuroPs were identified as ions with *m/z* 299, 297, and 323, respectively, (Galano et al., 2017) and quantified using calibration curves.

## *Oxidative status of the meat and fatty acid profiles*

All the oxidative parameters and fatty acid profiles were analysed in duplicate on thawing samples starting from a minimum quantity of 2 g. However, when the sample availability was lower than this quantity (i.e. PFIM muscle), an adaptation of the methods reported was used, modifying the quantity of solution proportionally and maintaining a recovery rate as specified for the analysis.

Antioxidants. The α and γ, δ-tocopherol, α and γ-tocotrienol, and retinol contents of the meat were quantified using the HPLC system described above, according to Hewavitharana et al.
(2004) as specified for feed and grass. The recovery rate ranged from 97 to 98% for the different tocol isoforms, whereas for retinol was 96.6%.

Lipid oxidation was evaluated using the same spectrophotometer reported for the *in vivo* evaluations, and a 1,1,3,3-tetraethoxypropane calibration curve (Ke et al., 1977). The oxidation products were quantified as malondialdehyde equivalents (µg MDA/g). The limit of detection (LOD) of malondialdehyde was 0.0017 µg MDA/mL. Carbonyl derivatives of proteins were detected according to Mattioli et al., (2024). The carbonyl content of the resulting supernatants was evaluated spectrophotometrically (Shimadzu Corporation UV-2550, Kyoto, Japan) as reported for blood; values are expressed as nmol of carbonyl/mg of protein in the guanidine chloride solution. The LOD of carbonyls was 0.0162 nmol/mL.

The same procedure reported for feed and grass was used (Folch et al., 1957). The average amount of each fatty acid was used to calculate the total saturated (SFA), total monounsaturated (MUFA), and polyunsaturated (PUFA) acids from the n-3 and n-6 series. The n-6/n-3 fatty acid ratio was also calculated. The recovery rate of the internal standard (heneicosanoic acid) was 97.2%.

From the FA profile, the conversion efficiency into n-3 long -chain fatty acids (LCP) and the activity index (Failla et al., 2021) were calculated. Higher values of the index indicate more intense chicken locomotor activity.

$$ \text{Activity index (AI)} = \frac{[\text{PIL (EPA + DPA + DHA)}/\text{ALA}]}{[\text{PIFM (EPA + DPA + DHA)}/\text{ALA}]} $$

where PIL: Iliotibialis lateralis postacetabularis muscle, PIFM Puboischiofemoralis pars medialis muscle, EPA: ecosapentaenoic acid, DPA: docosapentaenoic acid; DHA: docosahexaenoic acid; ALA: α-linolenic acid.

## Statistical analysis of data

The normality of the data was evaluated using the sk-test procedure (Stata, version 14.2, 2018). All the data were analysed with linear models using ANOVA from the same package. The intake levels of grass and the main nutrients (ALA, LA, retinol, carotenoids and α-tocopherol) and chicken blood traits were evaluated with a one-way model (treatment: Indoor, OLA, OHA; Model a).

The effect of thigh muscle (PIL, PIFM) was evaluated with a two-way model comprising the effect of treatment and muscle type (Model b) and their interaction.

The following models were used:

$$ \text{a)} \text{Yi} = \mu + \alpha_i + \text{ei} $$

$$ \text{b)} \text{Yij} = \mu + \alpha_i + \beta_j + \alpha\beta_{ij} + \text{eij} $$

where μ is the population mean, αi is the fixed effect of treatment (i = Indoor, OLA, OHA), βj is the fixed effect of thigh muscles (j = PIL and PIFM), αβij is the interaction between treatment and muscle type, and ei or eij is the error term. The significance of differences was corrected with the Bonferroni correction (P < 0.05).

## Results

The dietary ingredients are reported in the companion paper (Mattioli et al., 2024). The chemical composition and main bioactive compounds of the grower feed and grass are shown in Table 1. In addition to retinol, the grass (expressed as d.m.) was richer in bioactive compounds than the feed. The estimated intakes of feed, grass and main PUFAs and antioxidants are shown in Table 2 and Fig. 2. The outdoor chickens ate different amounts of grass with contemporary increases in bioactive compounds such as fatty acids

(15 and 29% ALA; 11 and 21% LA, respectively, in OLA and OHA), total carotenoids (6.6 and 21.5 times) and α-T (+35 and 69%, respectively).

The amount of antioxidants in the blood revealed lower levels of tocotrienols (δ, γ, α-T₃) in highly kinetic animals, whereas the other tocols maintained similar values (P > 0.05; Table 3). The thiobarbituric acid reactive substances values were significantly lower in OHA chickens, followed by OLA chickens and indoor chickens. The trends of carbonyls and ROS were not significantly affected by kinetic activity. The main isoprostanoids (Fig. 3) in the blood were F₂-IsoPs, followed by F₃-IsoPs and F₄-NeuroPs; OHA chickens presented lower levels of n-3 derivatives (F₃-IsoPs and F₄-NeuroPs), followed by OLA and indoor chickens. The trend of antioxidants in meat was similar to that in blood (Table 4). The different kinetic activities had significant effects only on thiobarbituric acid reactive substances, carbonyls and retinol. In particular, outdoor animals (OLA and OHA) presented lower thiobarbituric acid reactive substances and retinol values and higher carbonyls.

The more glycolytic and oxidative muscles of the leg differed in several traits (retinol, α-T, Σ Tocols, thiobarbituric acid reactive substances, carbonyls). Despite the higher antioxidant content of PIFM than of PIL, the thiobarbituric acid reactive substances and carbonyl levels were also higher. The kinetic activity and grass intake of chickens affected the PUFA profile (Table 5) of the blood. OHA chickens presented relatively high levels of LA and total n-6 and n-6/n-3 ratios, whereas AA, total n-3 and LCPn-3 were relatively low; the conversion rate of ALA into LCP (P = 0.13) was also relatively low. The kinetic activity affected the PUFA profiles of the thigh muscles analysed (Table 6). ALA was lower in both the muscles of outdoor chickens and oxidative thigh muscles, as was LCP n-3 (Table 6). The conversion rates of ALA into LCPn-3 were similar in the indoor and outdoor groups, whereas the activity indices were greater in OHA and OLA chickens.

## Discussion

Different levels of kinetic activity (OHA vs OLA) under outdoor conditions affected almost all the traits recorded in this trial. Previous experiment has shown that the scavenging behaviour of chickens under outdoor conditions is time-consistent and is associated with the personality traits of individual animals (Ferreira et al., 2022, 2024). Moreover, it is widely known that the presence of outdoor runs, with respect to the indoor conditions, greatly improves the kinetic activity of chickens (Dal Bosco et al., 2010; Rasmussen et al., 2024). As previously stated, the activity of chickens outdoors affects both the intake of nutrients and muscle metabolism. Our previous studies (Mattioli et al., 2017; Bonnefous et al., 2022; Guarino Amato and Castellini, 2022) revealed that the walking activity of chickens was accompanied by foraging behaviour and that high-foraging animals ate more grass than low-foraging animals.

It should be noted that the estimation of grass intake in chickens is a very challenging goal. Behaviour studies (e.g. time spent in eating grass), analysis of the crop and gizzard content which requires the killing of animals and is not replicable and exclusion pens (our choice) are the most common procedures used. In our opinion, the exclusion pen, that is frequently used in scientific papers (Ponte et al., 2008a, b), although does not permit to distinguish the different feeds ingested outdoor (grass, insects, stones, etc.) and it is affected by some chicken activity (e.g. scratching) is an useful method for estimation of the grass intake. The present results showed that the levels of some antioxidants in the blood and muscles did not always follow the bioactive compound intake of the experimental groups (retinol -3.8 and 8.3%; α-T +35 and 70%, carotenoids +7- and 14-fold, respectively, in OLA and OHA).
<caption>**Table 2**</caption>

Daily feed consumption and estimated intake of grass and bioactive compounds until chicken slaughtering.

<table><thead><tr><th>Item</th><th>Units</th><th>Indoor</th><th>OLA</th><th>OHA</th><th>RMSE</th><th>P-value</th></tr></thead><tbody><tr><td>Feed intake</td><td>g DM/d</td><td>117.00</td><td>110.05</td><td>110.05</td><td>10.54</td><td>0.590</td></tr><tr><td>Grass intake<sup>a</sup></td><td>g DM/d</td><td>—</td><td>8.91<sup>a</sup></td><td>29.00<sup>b</sup></td><td>1.73</td><td>&lt;0.001</td></tr><tr><td>LA</td><td>total intake g</td><td>3.26<sup>a</sup></td><td>3.51<sup>a</sup></td><td>4.33<sup>b</sup></td><td>0.37</td><td>0.026</td></tr><tr><td>ALA</td><td>total intake mg</td><td>2.70<sup>a</sup></td><td>2.99<sup>b</sup></td><td>3.90<sup>c</sup></td><td>0.32</td><td>&lt;0.001</td></tr><tr><td>Retinol</td><td>total intake mg</td><td>1834.23</td><td>1764.12</td><td>1680.04</td><td>176.00</td><td>0.594</td></tr><tr><td>Carotenoids</td><td>total intake mg</td><td>32.62<sup>a</sup></td><td>216.96<sup>b</sup></td><td>702.71<sup>c</sup></td><td>42.50</td><td>&lt;0.001</td></tr><tr><td>α-Tocopherol</td><td>total intake mg</td><td>540.93<sup>a</sup></td><td>682.74<sup>b</sup></td><td>1084.22<sup>c</sup></td><td>80.29</td><td>&lt;0.001</td></tr></tbody></table>

N = 3 replicates/groups.

Abbreviations: OHA: outdoor high active chickens; OLA: outdoor low active chickens. ALA: α-linolenic acid; LA: linoleic acid.

Values within a row with different superscripts differ significantly at *P* < 0.05.

<figure><img src="image_5.png" /><figcaption>**Fig. 2.** Trend (%) of nutrient intake in outdoor (OLA and OHA) chickens normalised for the chickens reared indoor. *: *P* < 0.05; N = 3 replicates/group; OHA: outdoor high active chickens; OLA: outdoor low active chickens. ALA: α-linolenic acid; LA: linoleic acid; α-T: α-Tocopherol.</figcaption></figure>

<caption>**Table 3**</caption>

Main antioxidants and oxidative status in chicken blood.

<table><thead><tr><th>Item</th><th>Units</th><th>INDOOR</th><th>OLA</th><th>OHA</th><th>RMSE</th><th>P-value</th></tr></thead><tbody><tr><td>Retinol</td><td>pmol/mL</td><td>2.21</td><td>2.16</td><td>1.75</td><td>0.19</td><td>0.363</td></tr><tr><td>δ-T<sub>3</sub></td><td>"</td><td>0.12<sup>b</sup></td><td>0.003<sup>a</sup></td><td>0.003<sup>a</sup></td><td>0.004</td><td>&lt;0.001</td></tr><tr><td>γ-T<sub>3</sub></td><td>"</td><td>0.0008<sup>c</sup></td><td>0.0002<sup>a</sup></td><td>0.0005<sup>b</sup></td><td>0.0003</td><td>&lt;0.001</td></tr><tr><td>α-T<sub>3</sub></td><td>"</td><td>0.79<sup>b</sup></td><td>0.45<sup>a</sup></td><td>0.40<sup>a</sup></td><td>0.20</td><td>&lt;0.001</td></tr><tr><td>δ-T</td><td>"</td><td>0.89</td><td>0.46</td><td>1.03</td><td>0.73</td><td>0.423</td></tr><tr><td>γ-T</td><td>"</td><td>0.03</td><td>0.03</td><td>0.03</td><td>0.01</td><td>0.234</td></tr><tr><td>α-T</td><td>"</td><td>1.58</td><td>1.73</td><td>1.50</td><td>0.57</td><td>0.554</td></tr><tr><td>Σ Tocols</td><td>"</td><td>3.31</td><td>2.78</td><td>3.00</td><td>1.10</td><td>0.649</td></tr><tr><td>TBARS</td><td>nmol MDA/mL</td><td>23.75<sup>b</sup></td><td>19.38<sup>a</sup></td><td>15.80<sup>a</sup></td><td>3.04</td><td>&lt;0.001</td></tr><tr><td>Carbonyls</td><td>nmol/mg protein</td><td>0.38</td><td>0.41</td><td>0.45</td><td>0.01</td><td>0.506</td></tr><tr><td>ROMS</td><td>mmol H<sub>2</sub>O<sub>2</sub></td><td>0.51</td><td>0.24</td><td>0.48</td><td>0.51</td><td>0.184</td></tr></tbody></table>

N = 12 chickens/group.

Abbreviations: OHA: outdoor high active chickens; OLA: outdoor low active chickens. T<sub>3</sub>: tocotrienol; T: tocopherol; TBARS: Thiobarbituric reactive substances; ROMS: reactive oxygen species; MDA: malondialdehyde.

Values within a row with different superscripts differ significantly at *P* < 0.05.

In particular, the first notable outcome is the lack of relationships between the intake of carotenoids and the retinol in the tissues analysed (blood and muscles; Çalışlar, 2019). In poultry, the main carotenoids (β-carotene, lutein and zeaxanthin) with vitamin A activities are α- and β-carotenes, and cryptoxanthin, where the lat-

ter has a conversion efficiency of vitamin A of approximately half that of β-carotene. Although the dietary intake of carotenoids and β-carotene in the outdoor groups was greater than those in the indoor groups, the retinol levels in the blood and muscles were similar (*P* > 0.05). This discrepancy can be explained by two

<footnote><sup>a</sup> The intake is referred to as 60 days of outdoor access.</footnote>
<figure><img src="image_6.png" /><figcaption>**Fig. 3.** Isoprostanoid (pg/mL) concentration in chicken blood. *: P < 0.05; N = 12 chickens/group; OHA: outdoor high active chickens; OLA: outdoor low active chickens. F2-isoPs: F2-Isoprostanes; F3-isoPs: F3-isoprostanes; F4-NeuroPs: F4-Neuoprostane.</figcaption></figure>

<table><caption>**Table 4**

Oxidative status in two thigh muscles (PFIM- oxidative and PIL – Glycolytic) of chickens.</caption><thead><tr><th rowspan="2">Item</th><th rowspan="2">Units</th><th colspan="2">Indoor</th><th colspan="2">OLA</th><th colspan="2">OHA</th><th rowspan="2">RMSE</th><th colspan="3">P-value</th></tr><tr><th>PFIM</th><th>PIL</th><th>PFIM</th><th>PIL</th><th>PFIM</th><th>PIL</th><th>Treatment</th><th>Muscle</th><th>TxM</th></tr></thead><tbody><tr><td>Retinol</td><td>µg/g</td><td>0.92</td><td>0.61</td><td>0.63</td><td>0.42</td><td>0.81</td><td>0.54</td><td>0.29</td><td>0.031</td><td>0.027</td><td>0.247</td></tr><tr><td>γ-T<sub>3</sub></td><td>"</td><td>0.02</td><td>0.01</td><td>0.01</td><td>0.01</td><td>0.01</td><td>0.01</td><td>0.01</td><td>0.311</td><td>0.286</td><td>0.354</td></tr><tr><td>α-T<sub>3</sub></td><td>"</td><td>1.00</td><td>0.85</td><td>0.30</td><td>0.27</td><td>0.90</td><td>0.71</td><td>0.45</td><td>0.405</td><td>0.357</td><td>0.284</td></tr><tr><td>δ-T</td><td>"</td><td>0.55</td><td>0.36</td><td>0.44</td><td>0.29</td><td>0.43</td><td>0.28</td><td>0.08</td><td>0.663</td><td>0.572</td><td>0.505</td></tr><tr><td>γ-T</td><td>"</td><td>2.22</td><td>1.48</td><td>1.84</td><td>1.23</td><td>2.34</td><td>1.56</td><td>0.70</td><td>0.557</td><td>0.214</td><td>0.306</td></tr><tr><td>α-T</td><td>"</td><td>7.32</td><td>2.44</td><td>7.35</td><td>2.45</td><td>7.41</td><td>2.47</td><td>1.90</td><td>0.424</td><td>0.012</td><td>0.586</td></tr><tr><td>Σ Tocols</td><td>"</td><td>11.22</td><td>5.04</td><td>10.00</td><td>4.21</td><td>11.16</td><td>4.97</td><td>2.80</td><td>0.332</td><td>0.010</td><td>0.412</td></tr><tr><td>TBARS</td><td>µg MDA/g</td><td>0.19</td><td>0.12</td><td>0.12</td><td>0.08</td><td>0.13</td><td>0.09</td><td>0.05</td><td>0.044</td><td>0.010</td><td>0.334</td></tr><tr><td>Carbonyls</td><td>nmol/mg protein</td><td>5.06</td><td>3.37</td><td>7.06</td><td>4.71</td><td>7.21</td><td>4.80</td><td>2.10</td><td>0.016</td><td>0.010</td><td>0.153</td></tr></tbody></table>

N = 12 chickens/group;

Abbreviations: OHA: outdoor high active chickens; OLA: outdoor low active chickens. PFIM: Oxidative muscle; PIL: glycolytic muscle; T<sub>3</sub>: Tocotrienol; T: Tocopherol; TBARS: Thiobarbituric reactive substances; MDA: malondialdehyde.

<table><caption>**Table 5**

Polyunsaturated fatty acid profile (% of total FA) in chicken blood.</caption><thead><tr><th>Item</th><th>INDOOR</th><th>OLA</th><th>OHA</th><th>RMSE</th><th>P-value</th></tr></thead><tbody><tr><td>C18:2 n-6, LA</td><td>16.64<sup>a</sup></td><td>15.99<sup>a</sup></td><td>22.22<sup>b</sup></td><td>3.88</td><td>0.005</td></tr><tr><td>C18:3 n-3, α-ALA</td><td>0.64</td><td>0.58</td><td>0.47</td><td>0.02</td><td>0.071</td></tr><tr><td>C20:4 n-6, AA</td><td>6.56<sup>b</sup></td><td>5.89<sup>a</sup></td><td>4.93<sup>a</sup></td><td>0.43</td><td>0.010</td></tr><tr><td>C20:5 n-3, EPA</td><td>0.66<sup>b</sup></td><td>0.40<sup>a</sup></td><td>0.46<sup>a</sup></td><td>0.04</td><td>0.002</td></tr><tr><td>C22:5 n-3, DPA</td><td>0.68<sup>b</sup></td><td>0.32<sup>a</sup></td><td>0.42<sup>a</sup></td><td>0.041</td><td>0.004</td></tr><tr><td>C22:6 n-3, DHA</td><td>1.23<sup>b</sup></td><td>0.86<sup>a</sup></td><td>0.75<sup>a</sup></td><td>0.10</td><td>0.001</td></tr><tr><td>SFA</td><td>38.02</td><td>40.70</td><td>39.35</td><td>1.015</td><td>0.580</td></tr><tr><td>MUFA</td><td>23.77</td><td>21.75</td><td>20.58</td><td>0.55</td><td>0.059</td></tr><tr><td>PUFA</td><td>27.14</td><td>25.48</td><td>32.90</td><td>1.38</td><td>0.086</td></tr><tr><td>n-6</td><td>23.48<sup>a</sup></td><td>21.96<sup>a</sup></td><td>30.36<sup>b</sup></td><td>4.58</td><td>0.001</td></tr><tr><td>n-3</td><td>3.22<sup>b</sup></td><td>2.17<sup>a</sup></td><td>2.10<sup>a</sup></td><td>0.45</td><td>0.001</td></tr><tr><td>LCpn-3</td><td>2.58<sup>b</sup></td><td>1.59<sup>a</sup></td><td>1.64<sup>a</sup></td><td>0.54</td><td>0.003</td></tr><tr><td>n-6/n-3</td><td>7.31<sup>a</sup></td><td>9.63<sup>a</sup></td><td>16.96<sup>b</sup></td><td>5.73</td><td>0.006</td></tr><tr><td>Conversion rate (LCpn-3/ALA)</td><td>4.51</td><td>2.96</td><td>3.48</td><td>1.50</td><td>0.130</td></tr></tbody></table>

N = 12 chickens/group.

Abbreviations: OHA: outdoor high active chickens; OLA: outdoor low active chickens. C18:2 n-6, LA: linoleic acid; α-ALA: α-linolenic acid; AA: arachidonic acid; EPA: eicosapentaenoic acid; DPA: docosapentaenoic acid; DHA: docosahexaenoic acid; SFA: saturated fatty acids; MUFA: monounsaturated fatty acids; PUFA: polyunsaturated fatty acids; LCPn-3: long chain n-3.

Values within a row with different superscripts differ significantly at *P* < 0.05.
<table><caption>**Table 6**
PUFA profile (% of total FA) and estimated indices in the two thigh muscles (PFIM – oxidative and PIL – Glycolytic) of chickens.</caption><thead><tr><th rowspan="2">Item</th><th colspan="2">Indoor</th><th colspan="2">OLA</th><th colspan="2">OHA</th><th rowspan="2">RMSE</th><th colspan="3">P-value</th></tr><tr><th>PFIM</th><th>PIL</th><th>PFIM</th><th>PIL</th><th>PFIM</th><th>PIL</th><th>Treatment</th><th>Muscle</th><th>TxM</th></tr></thead><tbody><tr><td>C18:2 n-6, LA</td><td>22.90</td><td>21.60</td><td>21.10</td><td>19.70</td><td>22.80</td><td>21.50</td><td>1.12</td><td>0.350</td><td>0.001</td><td>0.151</td></tr><tr><td>C18:3 n-3, α-ALA</td><td>0.89</td><td>0.74</td><td>0.77</td><td>0.62</td><td>0.79</td><td>0.64</td><td>0.08</td><td>0.040</td><td>0.001</td><td>0.061</td></tr><tr><td>C20:4 n-6, AA</td><td>4.03</td><td>6.05</td><td>4.15</td><td>6.18</td><td>4.72</td><td>6.74</td><td>1.48</td><td>0.611</td><td>0.003</td><td>0.281</td></tr><tr><td>C20:5 n-3, EPA</td><td>0.87<sup>a</sup></td><td>1.37<sup>b</sup></td><td>0.84<sup>ab</sup></td><td>1.34<sup>b</sup></td><td>1.00<sup>b</sup></td><td>1.52<sup>c</sup></td><td>0.27</td><td>0.501</td><td>0.002</td><td>0.044</td></tr><tr><td>C22:5 n-3, DPA</td><td>0.33<sup>a</sup></td><td>0.61<sup>b</sup></td><td>0.29<sup>a</sup></td><td>0.56<sup>b</sup></td><td>0.45<sup>ab</sup></td><td>0.74<sup>c</sup></td><td>0.16</td><td>0.050</td><td>0.001</td><td>0.046</td></tr><tr><td>C22:6 n-3, DHA</td><td>1.45</td><td>2.00</td><td>0.89</td><td>1.57</td><td>0.99</td><td>1.77</td><td>0.56</td><td>0.112</td><td>0.002</td><td>0.227</td></tr><tr><td>SFA</td><td>34.46</td><td>34.28</td><td>35.69</td><td>35.51</td><td>34.12</td><td>33.93</td><td>1.20</td><td>0.354</td><td>0.270</td><td>0.185</td></tr><tr><td>MUFA</td><td>30.68</td><td>27.87</td><td>32.59</td><td>29.78</td><td>30.89</td><td>28.08</td><td>3.14</td><td>0.334</td><td>0.025</td><td>0.223</td></tr><tr><td>PUFA</td><td>30.65</td><td>32.42</td><td>28.17</td><td>30.13</td><td>30.95</td><td>32.97</td><td>2.38</td><td>0.276</td><td>0.015</td><td>0.175</td></tr><tr><td>n-6</td><td>27.11</td><td>27.70</td><td>25.38</td><td>26.04</td><td>27.71</td><td>28.30</td><td>2.45</td><td>0.492</td><td>0.195</td><td>0.285</td></tr><tr><td>n-3</td><td>3.54</td><td>4.72</td><td>2.79</td><td>4.19</td><td>3.20</td><td>4.67</td><td>0.63</td><td>0.022</td><td>0.001</td><td>0.110</td></tr><tr><td>n-6/n-3</td><td>7.66</td><td>5.87</td><td>9.10</td><td>6.21</td><td>8.66</td><td>6.06</td><td>0.55</td><td>0.030</td><td>0.042</td><td>0.102</td></tr><tr><td>Long Chain PUFA n-3</td><td>2.65<sup>a</sup></td><td>3.89<sup>b</sup></td><td>2.02<sup>a</sup></td><td>3.47<sup>b</sup></td><td>2.44<sup>a</sup></td><td>4.03<sup>b</sup></td><td>0.64</td><td>0.105</td><td>0.001</td><td>0.025</td></tr><tr><td>Conversion rate (LCpn-3/ALA)</td><td>2.98</td><td>5.37</td><td>2.62</td><td>5.60</td><td>3.07</td><td>6.30</td><td>1.10</td><td>0.194</td><td>0.001</td><td>0.141</td></tr><tr><td>Activity index</td><td>2.40</td><td></td><td>2.95</td><td></td><td>3.37</td><td></td><td>0.96</td><td>0.017</td><td>—</td><td>—</td></tr></tbody></table>

N = 12 chickens/group.

Abbreviations: OHA: outdoor high active chickens; OLA: outdoor low active chickens. C18:2 n-6. LA: linoleic acid; α-ALA: α-linolenic acid; AA: arachidonic acid; EPA: eicosapentaenoic acid; DPA: docosapentaenoic acid; DHA: docosahexaenoic acid; SFA: saturated fatty acids; MUFA: monounsaturated fatty acids; PUFA: polyunsaturated fatty acids; LCPn-3: long chain PUFAn-3. T = treatment (indoor, OLA or OHA) and M = thigh muscle (PFIM or PIL).

Values within a row with different superscripts differ significantly at *P* < 0.05 for TxM.

different hypotheses: a low conversion rate of carotenoids in retinol or/and high conversion of carotenoids in outdoor animals for antioxidant activity.

According to Olson (1990), animals require 100–200 IU of vitamin A per kilogram of BW per day; the retinol level in the commercial feed greatly exceeds the requirements thus, probably only low or no conversion of provitamin A into vitamin A is necessary. Moreover, it is likely that carotenoids, mainly in outdoor chickens, are used for antioxidant purposes as carotenoids are considered the first scavengers of free radicals (Khan et al., 2023). This hypothesis seems appropriate because, despite the expected effects of kinetic activity on ROS production and lipid oxidation, both outdoor groups presented lower thiobarbituric acid reactive substances values in the blood (*P* < 0.01) and muscles, which is surprising considering the greater oxidative pressure induced by muscle activity. Naturally, the different compartments (blood and muscle) specifically react to the kinetic activity of chickens. In particular, the blood of OHA showed a lower thiobarbituric acid reactive substances than that of OLA because in the blood, there is a continuous counterbalancing of products derived from lipid oxidation by the way of blood oxidants. In the muscle, the picture observed is an *ex post* situation, which derives from *in vivo* traits but also from other mechanisms that occurred postmortem which could not be balanced.

With respect to tocols, it is difficult to interpret the trends of single isoforms; however, when focusing on α-T, the most representative isoform in vegetables (Navyashree, 2024), it could be emphasised that although there was a relatively high dietary intake of α-T in outdoor chickens, the levels in the blood and muscles were almost the same. Accordingly, as theorised for carotenoids, the current results could be explained by the use of α-T for antioxidant protection (Bartolini et al., 2021). Among the four isomers, α-tocopherol has the strongest antioxidant properties. Exclusively, photosynthetic organisms can produce tocopherols, which is why tocopherols are found in the green tissues of plants; thus, for animals and humans, it is essential to obtain tocopherols through feed/food. Through its ability to stop the lipo-oxidation cycle stage of chain propagation, tocopherols also function as potent free radical traps (scavengers of oxygen and lipid radicals) (Galli et al., 2017).

However, the most relevant effect of the different kinetic activities was on the FA content of thigh muscle (oxidative vs glycolytic

and blood. The intake and blood level of LA were greater in OHA chickens than in OLA and indoor chickens. ALA intake was also greater in OHA chickens than in OLA and indoor chickens. However, these differences were not observed in blood or muscles, suggesting a strong turnover of this FA in chicken metabolism (Poureslami et al., 2010; Cartoni Mancinelli et al., 2022). Furthermore, all the LCP derivatives (LCP n-3 and AA) were lower in OLA and OHA. Assuming the same conversion rate (elongation/desaturation) of ALA into LCPn-3 in all the groups, we hypothesise that in oxidative muscles (i.e., PFIM), high kinetic activity activates the consumption of LCPn-3 for energetic purposes. This assumption, confirmed by the higher activity index in OLA and mainly in OHA, clearly indicates that OHA animals partially used LCPn-3 via β-oxidation. Accordingly, it should be noted that oxidative muscles use the lipid as preferentially energetic substrates, explaining also the higher FA turnover (Lilburn et al., 2019).

The use of LCP in OHA animals also affected the trend of isoprostanoids. The main isoprostanoids found in chicken blood were F₂-IsoPs, followed by F₃-IsoPs and F₄-NeuroPs. High-kinetic animals presented lower levels of n-3 isoprostanoid derivatives (F₃-IsoPs and F₄-NeuroPs), followed by OLA and Indoor chickens. Iso-prostanoids are considered biomarkers of oxidative stress and have recently been associated with exercise-induced stress (Nikolaidis et al., 2011). In particular, the authors reported that acute exercise-induced stress in humans results in short-term growth of F₂-IsoP in plasma. Continuous exercise also increased F₂-IsoP in skeletal muscles, whereas further long-term exercise reduced F₂-IsoP in urine, probably because of the limited presence of AA, the F₂ precursor. Similarly, Garcia-Flores et al. (2016), reported that the response of elite triathletes training at moderate altitude increased F₄-NeuroP-s- and F₂-IsoPs-related oxidative damage to the central nervous system in the urine with respect to the training.

Conversely, in the present research, we found no difference (*P* > 0.05) in the F₂-IsoPs of the kinetic chickens compared with those of the Indoor chickens, although the AA concentrations were lower. This trend suggests that exercise, which is a spontaneous activity in slow-growing chicken strains adapted to movement, did not generate significant oxidative stress, resulting in isoprostanoid production (Montuschi et al., 2004). In addition, the F₃-IsoPs and F₄-NeuroPs presented lower values in OHA than in OLA because of, on the one hand, the lower presence of their pre-
cursors (EPA and DHA) and, on the other hand, the use of EPA and DHA as energetic "fuels" for movement. The lack of weight difference at slaughter confirms this statement (2 469 vs 2 456 vs 2 430 g, respectively, in indoor, OLA and OHA chickens; Fig. S1) even if it is possible that the estimated feed intake in the different groups (OLA vs OHA) could be affected by the experimental design. Previous results indicate that genetic strains strongly affect the training and adaptation of the body to movement and that slow-growing chicken strains respond progressively to kinetic activity (Castellini et al., 2006, 2016; Lindqvist, 2008; Mancinelli et al., 2020; Cartoni Mancinelli et al., 2021).

## Conclusion

The present study highlights that the kinetic activity of chickens largely affects the fatty acid profile, lipid oxidation and metabolism of blood and thigh muscles. In particular, more active animals ingested more grass than low-foraging animals, affecting the intake of the main antioxidants and fatty acids. However, this greater amount has not been entirely transferred into tissues (blood and muscles), probably due to the contemporary greater consumption of these compounds for antioxidant and energetic purposes. These findings are supported by the fact that, despite the probable greater oxidative pressure induced by muscle activity on ROS production and lipid oxidation, both outdoor groups presented lower thiobarbituric acid reactive substances values, indicating the important role of grass intake in the redox balance of the body.

Another interesting result is the assessment of the different kinetic effects on the FA of thigh muscles. In particular, more active chickens (OHA) may partially use LCPn-3 via β-oxidation for energetic purposes. Accordingly, a complex framework should be considered to evaluate the suitability of a genetic chicken strain to the outdoor environment. Indeed, with respect to indoor system, a different pattern of traits must be assessed to ensure the good health and welfare of animals and to able to produce high-quality meat (e.g., LCP protected from oxidation). Further studies are in progress to estimate β-oxidation in the muscle of different chicken strains.

## Supplementary material

Supplementary Material for this article (https://doi.org/10.1016/j.animal.2025.101569) can be found at the foot of the online page, in the Appendix section.

## Ethics approval

This study was carried out in accordance with the Guiding Principles in the Use of Animals and approved by the Italian Ministry (authorisation n°850/2021-PR) and the Animal Ethics Monitoring Committee of the Experimental Zooprophylactic Institute of Umbria and Marche (Protocol ID: 7F31D.20).

## Data and model availability statement

Data were not deposited in an official repository. The data that support the study findings are available from the authors upon request.

## Declaration of Generative AI and AI-assisted technologies in the writing process

During the preparation of this work the author(s) did not use any AI and AI-assisted technologies.

## Author ORCIDs

Simona Mattioli: https://orcid.org/0000-0001-5063-6785.

Elisa Angelucci: https://orcid.org/0000-0002-0040-5257.

Laura Medeo: https://orcid.org/0000-0002-2406-1348.

Claire Bonnefous: https://orcid.org/0000-0002-3522-5686.

Alice Cartoni Mancinelli: https://orcid.org/0000-0001-9888-9079.

Claudia Ciarelli: https://orcid.org/0009-0001-9633-8641.

Anne Collin: https://orcid.org/0000-0002-3410-6108.

Cinzia Signorini: https://orcid.org/0000-0003-0006-9414.

Alessandro Dal Bosco: https://orcid.org/0000-0002-4021-2281.

Camille Oger: https://orcid.org/0000-0002-5177-5792.

Jean-Marie Galano: https://orcid.org/0000-0001-6412-4967.

Thierry Durand: https://orcid.org/0000-0001-6086-7296.

Cesare Castellini: https://orcid.org/0000-0002-6134-0901.

## CRediT authorship contribution statement

S. Mattioli: Writing – original draft, Visualisation, Validation, Supervision, Software, Methodology, Investigation, Data curation, Conceptualisation. E. Angelucci: Writing – review & editing, Visualisation, Validation, Supervision, Software, Methodology, Investigation, Formal analysis, Data curation. L. Madeo: Writing – review & editing, Methodology, Formal analysis, Data curation. C. Bonnefous: Writing – review & editing, Methodology, Formal analysis, Data curation. A. Cartoni Mancinelli: Writing – review & editing, Methodology, Investigation, Data curation, Conceptualisation. C. Ciarelli: Writing – review & editing, Methodology, Investigation, Formal analysis, Data curation. A. Collin: Writing – review & editing, Resources, Methodology, Investigation, Funding acquisition, Conceptualisation. C. Signorini: Writing – review & editing, Methodology, Formal analysis, Data curation. A. Dal Bosco: Writing – review & editing, Supervision, Resources, Investigation, Conceptualisation. C. Oger: Writing – review & editing, Methodology. J. M. Galano: Writing – review & editing, Methodology. T. Durand: Writing – review & editing, Methodology. C. Castellini: Writing – original draft, Visualisation, Validation, Supervision, Software, Resources, Project administration, Methodology, Investigation, Funding acquisition, Data curation, Conceptualisation.

## Declaration of interest

The authors have declared that no competing interests exist.

## Acknowledgements

Authors wish to thank Mr. Giovanni Migni and Ms. Cinzia Boldrini for their contributions to animal handling and ungraduated students of Perugia University for their interest in following the present experimentation. Authors also thank breeders' companies (Aviagen, Hubbard and Lohmann) for providing animals for experimentation and PPILOW project partners for their positive opinion.

## Financial support statement

The project has received funding from the European Union's Horizon 2020 Research and Innovation Programme under grant agreement N816172.
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