*5.3. Gas Turbine*

The ammonia-fuelled gas turbine seems destined to become one of the key technologies in the sustainable energy economy of the future. In the 1960s, an early attempt was made to use ammonia as fuel in the gas turbine. However, due to nitrogen molecule in its structure, ammonia combustion is always associated with the formation of nitrogen oxides which exceed current standards [235,236]. Moreover, a longer residence time in the combustion chamber is required for ammonia to be completely combustible due to low laminar burning velocity, which makes it difficult to achieve stable combustion [16]. It also might cause an ammonia slip [237]. Therefore, ammonia-fuelled gas turbines were poorly studied in early development. However, the shift towards carbon-free alternative energy carriers has returned interest in ammonia, including the utilisation in power industries. Burning NH3 in turbines is the most promising direction of using ammonia as a carrier of surplus electricity generated from renewable energy to balance seasonal energy demand. Thus, many efforts have been devoted to overcoming these shortcomings.

Kobayashi et al. [238] have produced a review article covering the current ammonia combustion research and future directions. The study emphasised that the final product of ammonia combustion is not nitric oxides because the overall reaction is 4NH3 + 3O2 → 2N2 + 6H2O. This implies that the configuration of the parameters of the combustion system is crucial to perform the reaction according to this direction. Karabeyoglu et al. [239] developed a test rig and conducted a series of trials with a pre-burner system to partially crack ammonia into H2. The study revealed that ammonia combustion could be selfsustained when 10% cracking is applied. In 2014, Iki et al. [240] developed a 50 kWe micro gas-turbine system that enables a bi-fuel supply of kerosene and ammonia. The system was able to achieve over 25-kWe power generation by supplying about 10% heat from ammonia gas. In 2015, Iki et al. [241] were able to achieve 21 kWe by replacing the standard combustor with a prototype combustor. The gas turbine performance showed an efficiency of combustion up to 96% with NOx emission above 1000 ppm at 16% O2. Hayakawa et al. [242] investigated stretching limits for high-pressure flames and observed lower NO formation with higher mixture pressure. Okafor et al. [243], in their experiment and numerical calculations, concluded that predominant rate-limiting reactions in methane– ammonia flames are belonging to the ammonia oxidation path, which controls H and OH radicals. These radicals influence the burning velocity.

$$\text{NiO} + \text{NH}\_2 \rightarrow \text{NNH} + \text{OH} \tag{13}$$

$$\text{NH}\_2 + \text{O} \rightarrow \text{HNO} + \text{H} \tag{14}$$

$$\text{HNO} + \text{H} \rightarrow \text{NO} + \text{H}\_2 \tag{15}$$

The study also revealed that the NO concentration decreases when ammonia increases under rich conditions.

In 2016, Ito et al. [244] developed a gas-turbine combustion system with controlled emissions, which uses a mixture of NH3 and natural gas as the fuel. Combustion properties have been explored via the use of a swirl burner, generally employed in gas turbines, experimentally and numerically. Detailed compositions of the burner exhaust gas were measured under atmospheric pressure and lean fuel circumstances. The results show that with this system, the combustion efficiency above 97% can be achieved for an ammonia mixing ratio below 50%. The study also shows that with an increase of equivalency ratio, unburned species such as NH3, CO and THC decrease while NO and N2O emissions increase. The study concludes that low emissions and good combustion efficiency are difficult to obtain in a single-stage reactor.

In 2017, Onishi et al. [245] attempted to create novel ways for reducing NOx emissions while burning an ammonia-natural gas combination in a gas turbine combustor. The concept of low-emission combustion in two-stage combustion was examined numerically and experimentally. In the main zone, methane and ammonia are used as fuel. The secondary zone is then only supplied ammonia. The results indicated two methods for attaining low NOx combustion: rich-lean combustion and a combination of lean combustion and extra ammonia delivery. In the first technique, NOx is created only in the main zone when the fuel is abundant, and the burnt gas is diluted in the secondary zone by secondary air. As a result, primary zone NOx production dominates emission. A lean combustion state in the primary zone results in a low temperature and oxygen concentration in the secondary zone in the second approach. The NOx concentration at the combustor outlet is low as a result of these circumstances. These expected combustion qualities have been experimentally validated. The experimental findings showed that the NOx emission behaviour corresponded to the numerical results.

In 2018, Ito et al. [246] studied a mixture of ammonia and natural gas fuel in a 2 MWe gas turbine. Their results indicated that ammonia is suitable for use in large turbines. Before ammonia is fed to the combustor, the gas turbine power is raised up to 2 MWe using natural gas. The ammonia's heat input ratio to total fuel is used to calculate the ammonia feed to the engine. The result shows that the gas turbine engine's operation was shown to be steady across a wide variety of ammonia mixing ratios ranging from 0% to 20%. As the ratio of ammonia in the mixture increases, the concentration of NOx at the turbine outlet rises significantly up to 5% mixing ratio, then remains steady until it reaches 20%.


**3. Cavity detection in anisotropic regions: a field example**

Slates mining in Valongo (41o 11'N, 8o 30'W, 30 km North of Oporto, North Portugal) started in the nineteenth century. Since then many underground works were abandoned and no records are left about their whereabouts and extension. Slates of economic value occur in steeply dipping Ordovician schists where strong orientation effects of physical properties and anisotropy occur.

Increasing urban development pressure requires information about the position and extension of mining chambers but, documents about older works are scarce and field interventions demand the use of fast and non-invasive methods that is geophysics.

Electrical tomography techniques (ERTs) using the Wenner-Schlumberger array were used to investigate the dimensions of an old mining chamber. Today, the only evidence of the chamber at the surface is the shaft and the possible lateral limits of the underground works, depicted by the dashed line in **Figure 6**.

**Figure 6.** Mine chamber and ERT location in Valongo.

**Figure 6** also shows the mine entrance (central gray rectangle), the position of two ERTs, AB and CD, and the geological strike. Inspection of the shaft revealed that the mine is flooded and the groundwater level is very close to the surface. Thus, a large contrast between the cavity (conductive) and the surrounding rock (resistive) is expected. So a conductive anomaly is expected.

In the field, resistivity values can vary largely with the orientation θ of the line of electrodes,

The influence of anisotropy on resistivity measurements has been investigated [14, 15] and, herein, a case study on the location of old mining cavities in anisotropic media will be pre-

the nineteenth century. Since then many underground works were abandoned and no records are left about their whereabouts and extension. Slates of economic value occur in steeply dipping Ordovician schists where strong orientation effects of physical properties and anisot-

Increasing urban development pressure requires information about the position and extension of mining chambers but, documents about older works are scarce and field interventions

Electrical tomography techniques (ERTs) using the Wenner-Schlumberger array were used to investigate the dimensions of an old mining chamber. Today, the only evidence of the chamber at the surface is the shaft and the possible lateral limits of the underground works,

30'W, 30 km North of Oporto, North Portugal) started in

**Figure 5**, and the coefficient of anisotropy can reach values larger than 2 [9, 13].

**3. Cavity detection in anisotropic regions: a field example**

11'N, 8o

demand the use of fast and non-invasive methods that is geophysics.

sented [16].

122 Cave Investigation

ropy occur.

Slates mining in Valongo (41o

depicted by the dashed line in **Figure 6**.

**Figure 6.** Mine chamber and ERT location in Valongo.

Because of access difficulties, a first ERT, AB in **Figure 6**, was carried out parallel to the geological strike and, in these conditions, values corresponding to *ρ*l should have been measured. Data were inverted using the software RES2Dinv [12], and the model is shown in the left of **Figure 7**.

The model shows a resistive layer overlaying a conductive formation. The boundary, shown by the dashed line in the left of **Figure 7**, is at the same depth of the groundwater in the chamber entrance.

As the ERT orientation is parallel to the strike, the modelled resistivity should correspond to the longitudinal resistivity, *ρ*l . Therefore, longitudinal resistivity values must be higher above the groundwater level. However, they must decrease sharply below that level as a response to the water in the chamber and in the schists' foliation. Hence, this boundary is interpreted as the groundwater level.

Below this boundary, no more relevant information is obtained from the ERT model as only a conductive medium is shown.

Thus, tomography AB does not give evidence of the chamber and, if only this ERT was carried out, the location of the chamber would have been completely missed.

Therefore, it was decided to conduct a second ERT, CD in **Figure 6**. Because of access difficulties, CD orientation was at approximate 45° to the geological strike. The same field technique was used and model results are depicted in the right of **Figure 7**. In this case, a conductive body, bounded by the dashed lines, is clearly shown and stands out from the resistive surrounding rocks.

At shallow depths, a resistive layer is depicted with the same thickness in both ERTs. Bearing in mind the previous discussion, this layer corresponds to the resistive ground above the groundwater level.

**Figure 7.** ERT models in Valongo: left, AB (dashed line, groundwater level); right, CD (dashed line, interpreted cavity dimensions).

As the chamber is filled with water, the conductive anomaly should correspond to the mining chamber and, in fact, the proposed lateral boundaries match the expected limits of the dashed line in **Figure 6**. The higher resistivity values recorded away from the conductive anomaly refer to the schists' resistivity as measured with this ERT orientation.

Because of the orientation used, modelled resistivity values in CD do not correspond to the transverse resistivity of the schists. Therefore, it is not possible to calculate the anisotropy coefficient, but an indication of field anisotropy values can still be computed by calculating the square root of the ratio between the modelled resistivities for CD and AB.

Thus, a further section was constructed depicting the square root of the ratio between the resistivities modelled in the two ERTs.

**Figure 8** shows ratio values varying from 1 to more than 3.4. Thus, although this is not the anisotropy coefficient, field conditions are highly anisotropic.

The high resistive shallow layer, above the groundwater level, should correspond to ratio values close to 1. In this case, resistivity values modelled for AB and CD refer to a 'dry' or non-saturated area and, thus, should be similar. Hence, the dashed red line in **Figure 8** must correspond to the groundwater level as shown in **Figure 7** and, above this boundary, ratio values vary from 1 to 1.4. These values are considered to correspond to a low anisotropic medium [13, 17].

The central area of the section displays resistivity ratio values near 1 (or less than 1). This area corresponds to the position of the chamber depicted in the right of **Figure 7**. As the chamber is filled with water, resistivity values modelled for AB and CD ERTs should be similar as they correspond to data in the conductive chamber region.

It must be noted that ratio values less than 1 are depicted in **Figure 8**. This behaviour has been registered where structural anisotropy prevails [13, 17], such as in the vicinity of interfaces separating media with high resistivity contrast. In these circumstances, there is a rotation of the anisotropy axis and the so-called oblate anisotropy is observed [13]. This should be the

**Figure 8.** Ratio between modelled resistivities in Valongo; dashed line, proposed cavity limits.

case in the field example as the contact between the conductive chamber and the highly resistive surrounding rock corresponds to an interface separating two media with a very high resistivity contrast.

Away from the chamber position, ratio values increase as it should be expected. Therefore, one direction—AB—corresponds to the resistivity in the foliation planes (lower values), but the other direction—CD—is closer to the transverse resistivity (higher values).

There are many examples of the use of conventional ERT techniques in the location of mining cavities with evident success. However, the influence of formation anisotropy is seldom addressed and can mask ERT response. In this case, different field ERT orientations must be used to avoid misleading interpretations.

Often field space is restricted and only one ERT can be carried out. In this case, ERT orientation must be rather different from the strike of the geological formations and, in optimal conditions, ERT orientation should be perpendicular to the strike.

It is also possible to use electrode arrays that take into account orientation effects and anisotropy. Therefore, sets of linear arrays covering a wide range of directions can be used [14]. Alternatively, arrays of different geometry such as the square array have also been proposed [13].

However, the use of alternative arrays demands space and easy field access conditions and this requirement can be difficult to meet in urban areas.


**1. Introduction**

In the energy industry, every steel component is asked to have a long service duration (often longer than 30 years). The possibility to produce forged steel components characterized by high mechanical properties and competitive costs has gained more and more importance in the last years.

Medium-carbon steels have been widely used for primary energy-plant-production applications due to their excellent performances. This often needs to face with strong requirements in terms of severe service conditions (high temperature, high pressures, corrosion issues, nuclear irradiation, etc.) [1–4].

In the case of energy-sector applications (especially in oil and gas), corrosion resistance issues are often coupled with demanding mechanical requirements: in such cases, carbon steel is often cladded with stainless steels or even nickel superalloys [5–7].

Concerning mechanical properties, it is well-known that they strongly depend on grain size and steel alloying (through solution and precipitation hardening mechanisms), as discussed in the literature. Morito et al., reported the effect of microstructure refining [8]; Fan et al., discussed microstructural and precipitation effect on high-chromium steels microstructure [9]; Akbarzadeh et al., studied the hot workability of high-carbon and high-chromium steel [10]. The microstructure evolution in medium-carbon steel is well described in Reference [11], and the phase transformation evolution in high-strength low-alloyed steels is reported in Reference [12]. Some authors reported that limited amounts of retained austenite have a beneficial effect on mechanical properties [13]. It is reported that grain-size refinement should increase the yield strength up to three times [14–16]. Moreover, such a strengthening mechanism can allow the best balance of strength/toughness combination [17–19]. Solution-hardening mechanisms, if properly activated by the addition of elements, allows us to increase the wear, fatigue and high-temperature behavior [20–23].

As far as microstructure refinement is concerned, in recent years, there has been an intense focus on plastic-deformation processes, such us forging technologies [24–27]. In fact, it is reported that forging is a method for grain-size refinement via plastic-deformation accumulation. This enhances the material strength and toughness. The raw materials' internal cracks and defects can be reduced during forging, as well as mean grain size, thus allowing for a more uniform grain-size distribution [28,29]. Chaudhari et al. [30–33] reported that the grain size is clearly refined after steel multidirectional forging. This leads to the formation of fine austenite grains following the activation of continuous dynamic recrystallization and dynamic recovery. They showed that fine grains' formation is beneficial in terms of tensile strength and hardness enhancement. Gosh et al. [34] outlined that the material mechanical properties' best combination is found in the case of pure ferrite microstructure.

In high-Cr-bearing steels micro-alloying strengthening phenomena are reported to be effective to refine grains during thermomechanical processing. Kvackaj et al., analyzed austenite deformation behavior during thermomechanical processing of Nb–Ti micro-alloyed steel. They studied thermomechanical cycles aimed to favor the formation of (i) recrystallized austenite, (ii) un-recrystallized austenite, and (iii) ferrite-pearlite microstructures [35]. De Ardo reported micro-alloyed forged steels, emphasizing the possibility that pearlite-ferrite microstructures (formed during air cooling) can exhibit final strengths and fatigue resistances similar to those of the more expensive heat-treated steel [36]. Opiela reported the effect of thermomechanical processing on the microstructure and mechanical properties of Nb-Ti-V micro-alloyed steel [37]. Di Schino reported forging simulation and microstructure evolution of large-scale ingot in micro-alloyed tool steel [38]. Zitelli et al., recently discussed vanadium micro-alloyed steels for large-scale forged ingots, with the aim to balance production costs with the industrial demand of forged steels with high tensile properties. They demonstrated, at a laboratory scale, that ASTM A694 F70 forged-steel grade requirements can be fulfilled by a vanadium micro-alloyed steel with an addition of 0.15% V (plus proper heat treatment), consisting of a ferrite-pearlite microstructure [39]. Mancini et al., reported defect reduction and quality optimization by modeling plastic deformation and metallurgical evolution in ferritic stainless steels [40]. Moreover, substitutional strengthening mechanism in 7–12% Cr carbon alloyed steels has been widely studied and proved to have an excellent mechanical properties combination [41–44]. Among them, 7% Cr steels are nowadays proposed by many steelmakers for being quite promising in terms of properties/cost combination instead of the more common 9–12% steels.

Commonly, after casting, the steel ingots are deformed (forging) and heat treated. Such heat treatment, called preheating treatment or after-forging heat treatment, is aimed to develop the target microstructure for the desired mechanical properties. Finally, the heat-treatment sequence for high Cr steels (also called quality heat treatment) includes austenitization, followed by quenching and high-temperature tempering (Q & T). The microstructure of the final products typically consists of tempered martensite, undissolved carbonitrides, and/or re-precipitated carbonitrides [45]. The austenitizing temperature and soaking time determines the partitioning of carbon and alloying elements between the austenite and carbonitrides. In this processing step, a temperature increase leads to an increase of the carbonitrides dissolution degree with a positive effect on the mechanical properties [46–48]. After austenitization, cooling to below the martensite start temperature (Ms) leads to martensite formation. In its as-quenched martensitic condition, the steel is hard and brittle and may contain "pockets" of retained austenite (RA). The quenching step is followed by a high-temperature tempering step (in the range of 500–650 ◦C), to reduce brittleness and increase ductility and toughness by reducing residual stresses. All the parameters involved in a particular heat-treatment process strongly affect the steel final microstructure, e.g., austenitization temperature, holding time, quench medium, cooling rate, tempering temperature, and holding time. Well-defined heat-treatment guidelines are therefore required to assist the manufacturer in the selection of the correct heat treatment for achieving the target properties.

Ultimately, the microstructure evolution during the heat treatment of high-Cr steels is still not clearly understood. Thus, the understanding of microstructure evolution during heat treatment in wt.% Cr steel became a key factor for a safe long-term-service steel-microstructure design.

In this paper, the heat-treatment effect on a 7% Cr steel for forging is reported. Different heat treatments are considered. The resulting microstructure evolution is analyzed.

#### **2. Materials and Methods**

The steel nominal chemical composition is reported in Table 1.

**Table 1.** Main chemical composition of the considered materials (mass, %).

Specimens were withdrawn, starting from a sample-ingot for Continuous Cooling Temperature (CCT) diagrams determination. CCT diagrams were derived based on the dilatometry experiments carried out on 10 mm in length, 6 mm in diameter specimens, with controlled cooling rate. Microstructure analysis and hardness measurements of heat-treated specimens were performed by means of Light Microscopy (LM), after 4% Nital etching and Brinell indenter (HB). Prior austenite grain size was measured by image analysis software after samples etching in 10 g of CrO3, 50 g of NaOH, 1.5 g of picric acid, and 100 mL of distilled water solution, at room temperature, for 20 s. After etching, specimens were cleaned and dried.

The following heat treatments' cycles were the performed by means of dilatometer (Table 2 and Figure 1).

Heat treatments were designed with the aim to obtain different microstructures in terms of retained austenite content. Austenite transformation during the different steps of a laboratory manufacturing route that is reproducing an industrially relevant process was considered. In particular, the following four microstructural families were targeted:

In order to investigate the possible presence of retained austenite, X-Ray Diffraction (XRD) measurements were performed on heat-treated specimens. XRD analysis was carried out, using a Siemens D500 diffractometer (Siemens, Munich, Germany) equipped with Co-tube radiation and a graphite-monochromator on the detector side, with step size (2θ) of 0.05◦ and counting time of 5 s/step. The effect of possible texture was eliminated by a rotating fixture on the goniometer. Since low RA contents are expected in this class of steels, facing with XRD technique sensitivity, particular attention has been devoted to spectra postprocessing analysis. In particular, in order to avoid peaks overlapping, and thus to extract information also from "hidden peaks", spectra were processed by a tool specifically developed for peaks deconvolution (Figure 2). Three specimens were analyzed for each heat-treatment condition.

**Table 2.** Heat treatments' sequence.

Electron Backscattered Diffraction (EBSD) measurements were performed in selected cases, with the aim to detect the presence and position of retained austenite islands, by means of a JEOL field emission gun scanning electron microscope (FEG-SEM) (JEOL Ltd., Tokyo, Japan), using a 0.12 µm scanning step size. Retained austenite was revealed by building up phase maps, taking into account both face-centered cube (fcc) and body-centered cube (bcc) phases: automatic image analysis of such maps allowed us to determine retained austenite volume fraction. The precipitation state was analyzed by transmission electron microscope (TEM) on extraction replica specimens. The observations were performed with a JEOL 200CX transmission electron microscopy (JEOL Ltd., Tokyo, Japan). The hardness dependence upon heat treatments was assessed by means of Brinell hardness measurements.

**Figure 1.** (**a**) Heat-treatment cycle as performed on Q & P specimen; (**b**) heat-treatment cycle as performed on specimens from PT-γ-1 to PT-γ-4; (**c**) heat-treatment cycle as performed on specimens from PT-γ-5 to PT-γ-8.

**Figure 2.** Example of XRD peak deconvolution.

#### **3. Results and Discussion**

#### *3.1. Continuous Cooling Transformation*

CCT diagrams were determined after austenitization at two different temperatures: 980 and 1200 ◦C. The first temperature is typical of that commonly adopted during quality heat treatments, and the second one of the final forging-process passes (before cooling). A 30 min soaking time was considered. Dilatometric curves acquired in order to build up the steel CCT are reported in Figures 3 and 4, respectively. The green cooling curve reported in Figure 4 represents the first cooling undergone by all specimens reported in Table 2. This assures that, in the case of complete quenching, a fully martensitic microstructure was achieved (specimens Q and Q & T). On the other hand, this also assures that, in the case of partial quenching, a mixed martensite–austenite microstructure is obtained (specimens Q & P and PT-γ-1 to PT-γ-8).

**Figure 3.** Dilatometric curves of the considered steel: (**a**) austenitization at 980 ◦C; (**b**) austenitization at 1200 ◦C.

**Figure 4.** CCT diagrams of the considered steel (austenitization at 980 ◦C, blue curve; austenitization at 1200 ◦C, red curve)**.** Green cooling curve: first cooling undergone by all specimens reported in Table 2.

The results show that, even if prior austenite grain size were quite different, significative differences are not found in the cooling behavior (Figure 5). In particular 10 and 75 µm grain sizes were measured in specimens after austenitization at 980 and 1200 ◦C, respectively. These results were expected following the high intrinsic steel hardenability due to the high C and Cr content.

**Figure 5.** Austenitic grain size of two specimens after austenitization at 980 ◦C (**a**) and 1200 ◦C (**b**).

### *3.2. Retained Austenite Evolution*

Selected XRD spectra of specimens reported in Table 2 are shown in Figure 6. Even if retained austenite values are quite low, it is clear from the spectra reported in Figure 6 that it is possible to discriminate between specimens characterized by RA presence (e.g., Figure 6, specimen PT-γ-2) and specimens where XRD is not able to detect any RA (<2.0%) (e.g., Figure 6, specimen PT-γ-3). Retained austenite contents, as measured by means of XRD spectra analysis, are reported in Table 3. XRD measurements were confirmed, in selected cases, by phase maps analysis, as obtained by means of EBSD (Figure 7). In particular, 1.7% retained austenite content was measured in specimen PT-γ-2 by EBSD (to be compared to 2.5% as measured by XRD) and 1.2% in specimen PT-γ-3 (to be compared to <2.0% as measured by XRD). The low discrepancy obtained by EBSD with respect to XRD is justified by the two different adopted techniques [50]. The agreement between RA contents, as measured by the two different methods, makes the results reported in Table 3 consistent, even if their absolute values are quite low (in the proximity of XRD-technique sensitivity).

**Figure 6.** XRD spectra of selected heat-treated specimens: (**a**) specimen PT-γ-2 and (**b**) specimen PT-γ-3.

**Figure 7.** Phase maps by EBSD (**a**) specimen PT-γ-2 and (**b**) specimen PT-γ-**3**. Green areas correspond to retained austenite.

**Table 3.** Retained austenite content as a function of performed heat treatments (three measures for each specimen are reported).

Specimens *n*. PT-γ-3, PT-γ-7 and Q & T, double tempered at higher temperatures (>528 ◦C), show RA contents lower than 2.0%. All the other variants (reported in Table 3), with none or at a lower second tempering temperature, show higher RA contents. It is worth outlining that such results are independent from the microstructures obtained after cooling, according to Table 2. The other specimens show RA content ranging from 2.4% to 2.7%: such specimens were subjected to double tempering, but at a lower temperature (505 ◦C). The above considerations suggest that the double tempering heat treatment at higher temperatures (>528 ◦C) allows us to reactivate retained austenite that, during following cooling down to room temperature, decomposes in fine carbides

In fact, a very fine precipitation is detected in specimens PT-γ-3 and PT-γ-7 (average precipitation diameter *d*p = 14.6 nm and *d*p = 16.4 nm, respectively), as shown in Figure 8. Such precipitates were not detected in the other specimens. Finest precipitates (*d*p < 10 nm) were identified to be chromium carbides vanadium rich. Vanadium presence was not detected in coarser precipitates. The crystallographic lattice parameters' measurements obtained by diffraction patterns analysis allowed us to identify precipitates ranging *d*p < 5 nm as MC, precipitates ranging 5–10 nm as M2C. The largest precipitates were classified as M23C6 (ranging 70–200 nm) and M3C in the case of (*d*p > 200 nm). The Brinell hardness HB dependence on retained austenite content is reported in Figure 9. Results show that higher hardness values are found in the case of higher-retained-austenite-content specimens (hence lower tempering temperature). Results suggest therefore that the presence of retained austenite, also in a small amount, should exert a non-negligible effect. This means that manufacturers are called for a specific heat-treatment design, depending on the final properties required for different forged components.

**Figure 8.** TEM images describing the precipitation state of (**a**) specimen PT-γ-3 and (**b**) specimen PT-γ-7.

**Figure 9.** Hardness as a function of the retained austenite content.


*Review* **Fundamental Theory of Torsion Gravity**

**Luca Fabbri**

DIME, Metodi e Modelli Matematici, Università di Genova, Via all'Opera Pia 15, 16145 Genova, Italy<EMAIL_ADDRESS>
**Abstract:** In this work, we present the general differential geometry of a background in which the space–time has both torsion and curvature with internal symmetries being described by gauge fields, and that is equipped to couple spinorial matter fields having spin and energy as well as gauge currents: torsion will turn out to be equivalent to an axial-vector massive Proca field and, because the spinor can be decomposed in its two chiral projections, torsion can be thought as the mediator that keeps spinors in stable configurations; we will justify this claim by studying some limiting situations. We will then proceed with a second chapter, where the material presented in the first chapter will be applied to specific systems in order to solve problems that seems to affect theories without torsion: hence the problem of gravitational singularity formation and positivity of the energy are the most important, and they will also lead the way for a discussion about the Pauli exclusion principle and the concept of macroscopic approximation. In a third and final chapter, we are going to investigate, in the light of torsion dynamics, some of the open problems in the standard models of particles and cosmology which would not be easily solvable otherwise.

**Keywords:** torsion-gravity; electrodynamics; spinors

**Citation:** Fabbri, L. Fundamental Theory of Torsion Gravity. *Universe* **2021**, *7*, 305. https://doi.org/ 10.3390/universe7080305

Academic Editors: Antonino Del Popolo and Matteo Luca Ruggiero

Received: 8 July 2021 Accepted: 15 August 2021 Published: 18 August 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the author. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).


**3. Cybersecurity and real-time**

In the context of autonomous driving, ensuring cybersecurity and real-time capability is crucial. With the increasing networking and automation of vehicles, new challenges and questions arise that will be discussed in this section.

A central problem is ensuring end-to-end cybersecurity under real-time conditions. To do this, security measures must be implemented at all levels of the system, starting with the sensors and extending to communications and the cloud.

Examples include authentication and key exchange under real-time conditions. The typical use of asymmetric crypto methods is problematic for key renewal during

#### **Figure 2.**

*Challenges of parallelizability in key exchange with asymmetric cryptography.*

*Autonomous Driving and Cybersecurity by Design DOI: http://dx.doi.org/10.5772/intechopen.112042*

#### **Figure 3.**

*Example of parallelizing a MAC calculation.*

runtime due to their slow runtime. So, if you want to still have real-time capability, you must consider parallel key renewal during runtime (**Figure 2**). In addition, the use of parallelizable crypto algorithms can be an important building block; for example, authentication procedures, such as the Message Authentication Code (MAC) procedure, can be parallelized to guarantee real-time capability (**Figure 3**).

Another component is edge computing, where data processing and analysis take place in the vehicle instead of in the cloud, which can help optimize latency and data rates. This supports real-time guarantees by reducing the amount of data transmitted over the network and increasing the speed of response to events.

A major challenge arises from the fact that vehicles are in the field for a long period of time, so future systems should be prepared for changing crypto computing power and key length requirements by considering or balancing newer crypto techniques such as post-quantum cryptography etc. For example, the ongoing development of quantum computers poses a particular challenge by challenging the security of traditional asymmetric key exchange methods [10].

In summary, ensuring cybersecurity and real-time capability in autonomous driving is a challenging task that requires a combination of different technologies and concepts. The integration of edge computing, parallel key renewal and authentication, as well as the adaptation to future crypto requirements are key elements to ensure the security and performance of autonomous vehicles in the connected world.

### **4. Real-time management**

Our adaptive-real-time-manager (ARM) is an innovative concept that aims continuously assessing and optimizing the real-time capability of autonomous driving systems. This section discusses the basic design of the ARM and its advantages compared to existing solutions.

Factors such as vehicle environment, traffic conditions, visibility, and network connection quality influence the real-time capability of autonomous driving systems. The ARM constantly evaluates these factors and adjusts driving speed and strategy accordingly (**Figure 4**).

A crucial aspect of the ARM concept is the Round Trip Time (RTT) of the closed information chain from the vehicle's sensors and actuators to the cloud and back. The RTT varies depending on the preferred cybersecurity mechanisms, which can be selectively integrated at different security levels.

The ARM assesses the real-time capability of the respective closed information chain by considering the RTT and, if necessary, other system parameters. This enables optimal adjustment of driving speed and strategy to the respective conditions.

#### **Figure 4.**

*The ARM might suggest a speed of 50 km/h when the connection quality is good and the vehicle environment is favorable, but only 30 km/h when the connection quality is poor or the vehicle environment is more complex.*

The ARM can reduce the impact of traffic control systems on the real-time capability of autonomous driving systems. This is achieved by continuously adapting driving strategies and speeds to the current conditions and, if necessary, to the information provided by traffic control systems.

A real-world scenario illustrates this benefit of ARM: An autonomous vehicle stops before a green light at an intersection. One possible explanation for this behavior is that the intelligent traffic light has informed the autonomous driving system of the time remaining in the green phase. However, the ARM has suggested a driving speed that is not sufficient to cross the intersection without a collision, so in this case the vehicle waits for the next full green phase.

Compared to existing solutions, the ARM offers a more dynamic approach to real-time assessment and optimization of autonomous driving systems. The continuous analysis of influencing factors and the adaptation of driving speed and strategy increase the safety, efficiency and flexibility of these systems.

Another advantage of the ARM is the ability to selectively incorporate cybersecurity mechanisms at different security levels. This ensures data security and system integrity without unnecessarily compromising the real-time capability of the autonomous driving system.

In summary, the automITe Adaptive Real-time Manager offers a promising approach for addressing the challenges related to autonomous driving and cybersecurity. Continuous real-time assessment and optimization, selective incorporation of cybersecurity mechanisms, and enhanced interaction with infrastructure make the ARM a unique and forward-looking solution in this field. It remains to be seen how the ARM will prove itself in real application scenarios and what further developments and optimizations are possible in the future (**Figure 5**).

Together with the ARM there are two essential building blocks that can accelerate the integration into an ADS system:

• Cloud-Broker-Concept: This ensures independence from the cloud provider and a uniform interface on the ADS side to the cloud. An essential step here is the

*Autonomous Driving and Cybersecurity by Design DOI: http://dx.doi.org/10.5772/intechopen.112042*

#### **Figure 6.**

*Vehicle architecture with middleware and adaptive-real-time-manager in place.*

integration and the interface management of the cloud broker into the system of the ARM (see **Figure 6**).

• Simulation of the Adaptive Real-time Manager and the Cloud Broker: For this purpose, we are currently designing a driving simulator that can be used and extended to simulate the driving of a vehicle in a city, with all driving information obtained from the cloud. By using the simulator, the effort required for testing in the field can be reduced, as many shortcomings are already revealed by simulations.


*4.2. Respondents' Assessment of the Quality of Their Sectors' Efforts*

#### 4.2.1. Ratings of the Sector's Efforts to Prevent Work-Related Accidents

The second aim of the study was to examine respondents' perceptions of the quality of their sectors' efforts to prevent work-related accidents, and factors influencing this. As responsibilities for the prevention of work-related accidents are more clearly in the maritime sector, we expected respondents in the maritime sector to rate their efforts to prevent work related accidents as higher than respondents in the road sector (Hypothesis 2). This is compared in Figure 1, where we show results for the question: "How would you rate your own organizations' work with work-related accidents on a scale from 1 (= very deficient) to 7 (= very good).

**Figure 1.** How would you rate your own organizations' work with work-related accidents on a scale from 1 (= very deficient) to 7 (= very good) by sector. Shares from Road (N = 66) and the maritime sector (N = 46).

Figure 1 indicates that respondents from the road sector rate their own efforts to prevent work-related accidents as lower than respondents from the maritime sector. If we assume that scores ranging from 5–6 means "good", we see that 74% of the respondents in the maritime sector rate their own organization's work with work-related accidents as good, compared to 52% in the road sector. Correspondingly, 18% rate their organization's efforts as deficient in the road sector, compared to 4% in the maritime sector. The sector experts that we interviewed often referred to the rules governing safety in their sector and the enforcement of these rules when discussing their sectors' and organizations' efforts to prevent work-related accidents. Interviewees in the road sector referred, e.g., to the lacking enforcement of the Working Environment Act, while interviewees in the maritime sector referred to the SMS requirements of the ISM-code.


**5. Conclusions**

This study shows that in Al-Fe alloys the morphology of the intermetallic phase in the cast-state has a notable effect on the structural evolution during 20 revolutions of HPT. This feature can be even more important, than the concentration of Fe in the alloy. The initial size and dispersity of the particles, formed during crystallization, their volume fraction, and, respectively, the length of the interphase boundaries plays an important role in forming mechanical and electrical properties by HPT. The following specific conclusions are drawn from the results presented in this study:

**Author Contributions:** Conceptualization, M.M. and A.M.; methodology, X.S.; software, N.E.; writing—original draft preparation, A.M.; writing—review and editing, N.E., M.M. and X.S.; visualization, E.M. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by Russian Science Foundation, Grant number 20-79-10133.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Acknowledgments:** Authors are grateful to the personnel of the research and technology Joint Research Center, Nanotech, Ufa State Aviation Technical University, for their assistance with instrumental analysis.

**Conflicts of Interest:** The authors declare no conflict of interest.


[Nacalai Tesque (Kyoto, Japan)], TLC, precoated TLC plates with silica gel 60F254 [Merck (NJ, US), 0.25 mm] (ordinary phase), and silica gel RP-18 F254S (Merck, 0.25 mm) (reversed phase); reversed-phase HPTLC, precoated TLC plates with silica gel RP-18 WF254S. Detection was achieved by spraying with 1% Ce (SO4) 2–10% aqueous H2SO4 followed by heating.

#### Plant Material

Fresh leaves of A. sativum cultivated in Kochi prefecture, Japan, were obtained as commercial products purchased from Japan Agricultural Cooperatives (JA) farmers' market (Kochi, Japan) in April 2017. The plants were identified by the authors (H.M. and S.N.).

#### Extraction and Isolation

The fresh leaves of A. sativum (15.2 kg) were chopped and mixed with water, and then acetone was added to the mixture to be 80% acetone solution. The mixture was soaked for 4 days (96 h) at room temperature. Evaporation of the filtrate under reduced pressure provided acetone extract (1,500.37 g, 9.87%). The extract was partitioned between EtOAc and H2O (1:1, vol/vol) to obtain EtOAc fraction (41.98 g, 0.27%) and aqueous phase. The EtOAc-soluble fraction (41.98 g) was subjected to normal phase silica gel column chromatography [1,260 g, CHCl3-MeOH (1:0→ 100:1→ 50:1→ 30:1→ 10:1→ 0:1, vol/vol)] to give nine fractions {Fr.1 (1,471.6 mg), Fr.2 (715.5 mg), Fr.3 (7,193.2 mg), Fr.4 (8,339.2 mg), Fr.5 (4,085.8 mg), Fr.6 (1,334.9 mg), Fr.7 (4,367.0 mg), Fr.8 (617.7 mg), Fr.9 (5,841.2 mg)}. r. 5 (4,085.8 mg) was further separated by reversed-phase silica gel column chromatography [200 g, MeOH-H2O (2:8→ 4:6→ 6:4→ 8:2→ 1:0, vol/vol)] to give 13 fractions {Fr.5-1 (52.3 mg), Fr.5-2 (21.0 mg), Fr.5-3 (31.0 mg), Fr.5-4 (19.1 mg), Fr.5-5 (84.4 mg), Fr.5-6 (281.7 mg), Fr.5-7 (43.5 mg), Fr.5-8 (26.3 mg), Fr.5-9 (67.9 mg), Fr.5-10 (482.9 mg), Fr.5-11 (2,637.7 mg), Fr.5-12 (178.7 mg), Fr.5-13 (16.9 mg)}. Fr.5-5 (84.4 mg) was

purified by HPLC {mobile phase: MeOH-H2O (35:65, vol/vol) [YMC-triart PFP (250 × 10 mm i.d.)]} to give **1** (6.0 mg) and **2** (16.3 mg). Fr.5-6 (281.7 mg) was purified by HPLC {mobile phase: MeOH-H2O (50:50, vol/vol) [YMC-triart C18 (250 × 10 mm i.d.)]} to give **1** (14.6 mg), **2** (16.0 mg), **4** (24.2 mg), and **5** (12.3 mg). Fr.5-6-5 (37.1 mg) was purified by HPLC {mobile phase: MeOH-H2O (45:55, vol/vol) [YMCtriart C18 (250 × 10 mm i.d.)]} to give **3** (14.7 mg) and **4** (4.5 mg).

## Foliogarlic Disulfane A1 (1)

Yellow oil; [α] 25 D +160.9 (MeOH); HRESIMS: calcd for C12H16O6S2Na (M+Na)+: 343.0281, found: 343.0282; IR(ATR): 3,400, 2,975, 1,782 cm−1 ; 1H NMR (CD3OD), 13C NMR (CD3OD, 500 MHz): given in **Table 1**.

## Foliogarlic Disulfane A2 (2)

Yellow oil; [α] 25 D +139.0 (MeOH); HRESIMS: calcd for C12H16O6S2Na (M+Na)+: 343.0281, found: 343.0277; IR(ATR): 3,400, 2,970, 1,785 cm−1 ; 1H NMR (CD3OD, CDCl3), 13C NMR (CD3OD, CDCl3): given in **Table 1**.

## Foliogarlic Disulfane A3 (3)

Yellow oil; [α] 25 D −213.6 (MeOH); HRESIMS: calcd for C12H16O6S2Na (M+Na)+: 343.0281, found: 343.0282; IR(ATR): 3,400, 2,981, 1,785 cm−1 ; 1H NMR (CD3OD, CDCl3), 13C NMR (CD3OD, CDCl3): given in **Table 2**.

## Foliogarlic Trisulfane A1 (4)

Yellow oil; [α] 25 D +124.6 (MeOH); HRESIMS: calcd for C12H16O6S3Na (M+Na)+: 375.0001, found: 374.9998; IR(ATR): 3,400, 2,975, 1,780 cm−1 ; 1H NMR (CD3OD, CDCl3), 13C NMR (CD3OD, CDCl3): given in **Table 3**.

## Foliogarlic Trisulfane A2 (5)

Yellow oil; [α] 25 D −119.8 (MeOH); HRESIMS: calcd for C12H16O6S3Na (M+Na)+: 375.0001, found: 375.0003; IR(ATR): 3,400, 2,931, 1,789 cm−1 ; 1H NMR (CD3OD), 13C NMR (CD3OD): given in **Table 4**.

## DATA AVAILABILITY STATEMENT

All datasets generated for this study are included in the article/**Supplementary Material**.

## AUTHOR CONTRIBUTIONS

MF: isolation of constituents and Structure elucidation of new compounds. SeiN: Structure elucidation of new compounds and overall supervision in this study. HH and DN: isolation of constituents. SouN: Preparation of plant extracts and overall supervision in this study. TY: Structure elucidation of new compounds. HM: overall supervision in this study.

## REFERENCES

## FUNDING

This research was supported in part by a Ministry of Education, Culture, Sports, Science and Technology (MEXT)-Supported Program for the Strategic Research Foundation at Private Universities 2015–2019. This work was supported by JSPS KAKENHI Grant Number 17K08354 (SeiN).

## SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fchem. 2020.00282/full#supplementary-material

potent antithrombotic agent from garlic. J. Am. Chem. Soc. 106, 8295–8296. doi: 10.1021/ja00338a049

and scallions (Allium fistulosum L. var. caespitosum). J. Agric. Food Chem. 38, 1378–1381. doi: 10.1021/jf00096a017

**Conflict of Interest:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2020 Fukaya, Nakamura, Hayashida, Noguchi, Nakashima, Yoneda and Matsuda. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.


*2.7. Statistical Analysis*

The experimental values were analyzed by GLM procedure of SAS® 9.3 software (SAS Inst. Inc., Cary, NC, USA). The pen was used as the experimental unit for the analysis of growth performance, and an individual chicken was considered as the experimental unit for measuring the blood, meat quality, and carcass trait samplings. The difference of means was tested by Tukey test. The effect of dietary SK supplementation and stocking densities and their interactions were determined. A significant difference was expressed either *p* < 0.01 or *p* < 0.05.

#### **3. Results**

#### *3.1. Antioxidant Factors*

There was a significantly lower (*p* < 0.01) concentration of MDA in the serum with the supplementation of SK (Table 2). The serum concentrations of catalase (*p* < 0.05) and SOD (*p* < 0.05) were increased by supplementation of SK in the diet, while there was no difference between the SD treatments. The addition of dietary SK resulted in a greater (*p* < 0.01) TAC in the serum; however, the SD treatments did not change the TAC capacity. The low SD (*p* < 0.05) and supplementation of SK (*p* < 0.01) decreased the content of MDA in the leg muscle. The concentrations of catalase (*p* < 0.01) and SOD (*p* < 0.05) were increased by supplementation of SK in the diet, while there was a decrease in the concentration of SOD in the high SD treatment. There was an increased (*p* < 0.01) TAC in the leg muscle of

chickens with increased supplementation of SK; however, there were no significant effects of SD on TAC in the leg muscle.

**Table 2.** Effect of dietary SK and SD on antioxidant activity of serum and leg muscle in Korean native chicken.

SEM, standard error of means; SD, stocking density effect; SK, *Sophora koreensis* supplementation effect; SD × SK, stocking density × *Sophora koreensis* supplementation effect interaction; MDA, malondialdehyde; SOD, Superoxide dismutase; TAC, total antioxidant capacity.

#### *3.2. ABTS-Reducing Activity*

The antioxidant capacity result of serum indicated that ABTS-reducing activity was enhanced in the SK-supplemented treatments (Figure 1). No difference in ABTS-reducing activity was detected between the SD treatments. The SK-supplemented treatments showed an increased (*p* < 0.05) ABTS-reducing activity of leg muscles compared with the non-SKsupplemented treatments; however, there was a greater ABTS-reducing activity of leg muscle in the SK-supplemented treatments.

**Figure 1.** *Cont*.

**Figure 1.** *Cont*.

**Figure 1.** ABTS radical scavenging capacity (%) of different stocking density (SD) on serum (**a**) and leg muscle (**b**), and *Sophora koreensis* (SK) supplementation on serum (**c**) and leg muscle (**d**) of Korean native chicken. Non-SK, basal diet; SK, basal diet + 20 ppm SK; Asterisks (\*) indicate statistical significance (*p* < 0.05).

#### *3.3. Meat Color and Meat Quality*

The effect of diets and SD on breast meat color and quality of chickens was shown in Table 3. There were no breast meat redness, lightness, and yellowness responses to supplementation of SK in the diet and rearing in different stocking densities. The higher water holding capacity of breast muscle was shown for the SK-supplemented treatments (*p* < 0.01); however, there was no change among the SD treatments. A lower cooking loss and breast muscle pH was reported in the SK-supplemented treatments (*p* < 0.01), although there were no changes in cooking loss and pH of breast muscle between the SD treatments. The shear force was not affected by treatments.

#### *3.4. Growth Response, Carcass Traits, Immune Organ Ratio*

Table 4 shows the influences of diets and SD on growth performance, carcass traits, and relative organ weight. The effect of SK supplementation on improving final BW, BW gain, and feed intake of chickens was significant (*p* < 0.01). There was no difference in feed conversion ratio between the SK treatments. There was no difference in final BW and BW gain between the SD treatments; however, the feed intake and feed conversion ratio of chickens were adversely affected by the high SD. The carcass yield, breast meat, and drumsticks showed no dietary SK effects; however, a lower (*p* < 0.01) abdominal fat was shown in the SK-supplemented treatments. The carcass yield, breast meat, drumstick percentage, and abdominal fat were decreased in the high SD treatments. There was no change in the relative weight of the liver. There were significant interactions between the SD and SK in increasing the relative weight of the spleen and bursa Fabricius. The main effects shown were that the relative weight of the spleen and bursa Fabricius were

increased by supplementation of SK, but decreased by increasing the SD. There were no differences in the percentage of the spleen to bursa Fabricius and relative weight of thyroid between the treatments.

**Table 3.** Effect of dietary SK and SD on breast meat color and quality in Korean native chicken.

SEM, standard error of means; SD, stocking density effect; SK, *Sophora koreensis* supplementation effect; SD × SK, stocking density × *Sophora koreensis* supplementation effect interaction.

**Table 4.** Effect of dietary SK and SD on growth performance, carcass traits, and relative weights of organs in Korean native chicken.

SEM, standard error of means; SD, stocking density effect; SK, *Sophora koreensis* supplementation effect; SD × SK, stocking density × *Sophora koreensis* supplementation effect interaction; BW, bodyweight; FI, feed intake; FCR, feed conversion ratio.

#### *3.5. Blood Profile*

The effects of dietary SK on blood profile are shown in Table 5. Results indicated that total cholesterol and glucose levels were decreased in the high SD treatment but that the concentration of blood total cholesterol was increased in the SK-supplemented treatments. There was no change in concentration of total protein, triglyceride, GPT, GOT, albumin, phosphate, and calcium among the treatments. The blood corticosterone level was significantly higher (*p* < 0.05) in the high SD treatment; however, corticosterone level was decreased in the SK-supplemented treatments.

**Table 5.** Effect of dietary SK and SD on blood profile in Korean native chicken.

SEM, standard error of means; SD, stocking density effect; SK, *Sophora koreensis* supplementation effect; SD × SK, stocking density × *Sophora koreensis* supplementation effect interaction; GPT, glutamic pyruvic transaminase; GOT, glutamate oxaloacetate transaminase.

### **4. Discussion**

*Sophora koreensis* has been known as a traditional herb to treat rheumatoid issues in Korea because of its antioxidant capacity [13,24]. The antioxidant enzymes including SOD, GPx, and catalase are the first factors against antioxidant reactions [11,12]. Antioxidant effects of scopoletin were shown earlier [16,17]. An increase in the aforementioned enzyme's production capacity can improve the antioxidant system by controlling the production of ROS. Superoxide dismutase is an important enzyme in the protection of cells from adverse effects of ROS [25]. In the current study, decreased concentration of MDA and increased TAC in the serum of chickens treated with SK may be related to the increased concentrations of catalase and SOD, which reduce the formation of peroxides and hydroperoxides in fat tissues [7]. The increased activities of catalase and SOD in the serum of broiler chickens fed SK diets show its potential to scavenge free radicals during the stressful condition of high SD. It has been reported that the stressful condition decreases the SOD and catalase production, which in turn increases MDA production [26]. There is a positive correlation between high MDA concentrations and lipid peroxidation [7,8]. Meanwhile, the reduction of lipid peroxidation in the thigh muscle may be reflected in the decrease of MDA concentration in the SK treatments. Scopoletin also showed high inhibitory activities against anti-inflammatory cytokines by decreasing TNF-α, IL-1β, and IL-6 secretions [17,19,27]. It has been reported that the anti-inflammatory influences of scopoletin are associated with a decrease in free radicals production. The production of MDA, as an important indicator of lipid peroxidation, is due to the exposure of free radicals to the plasma membrane [7,8,28]. Lee et al. [29] reported that the MDA levels were significantly increased in mice under alcoholic food stress, but that the supplementation of scopoletin prevented the increase in MDA concentration compared with the control treatment. Several challenge experiments with high inflammatory condition confirmed that the decrease in inflammation mediates cell damage, lipid peroxidation, and increases the inactivation of antioxidant enzymes [9,10,28]. Several researchers have reported that high SD could increase the stress level and decrease the antioxidant status by increasing MDA and decreasing SOD concentrations in serum [1,2,30]. Although increased catalase and TAC seem to be consistent in the plasma and leg muscle in the SK-supplemented treatments, this trend is missing for chickens in the SD treatments. However, chickens in the SD treatments showed a higher MDA and lower SOD content in the leg muscle. Although the decrease in the SOD concentration was in line with the increased MDA in the leg muscle, the concentration of the aforementioned parameters in the serum was not in agreement with the leg muscle results.

Although scopoletin increases the antioxidant status [17], literature did not study the effects of supplemental scopoletin on meat quality related to SD stress in chickens focusing on the antioxidant capacity. Therefore, this study aimed to test the influences

of scopoletin on the antioxidant capacity of meat, plasma, and the possible interactions with the quality and color of meat. Although the scavenging capacity of scopoletin against ABTS in chicken meat has not been studied, an in vitro study on the antioxidant role of phenolic compounds reported that the α-diphenyl-β-picrylhydrazyl radical-scavenging activity of scopoletin was around 11,800 times higher than vitamin C, making it a potent antioxidant compound [31]. The increased scavenging capacity of ABTS of meat due to scopoletin was in line with an enhanced TAC in the leg muscle, indicating the reduction of oxidative damage in muscle tissues with the presence of scopoletin. Furthermore, an increased SOD, as well as decreased MDA, content in the serum and meat confirm the capability of scopoletin in decreasing oxidative stress. Therefore, the result of the current study showed that the supplementation of 20 ppm scopoletin adequately increased the ABTS-reducing activity in chickens.

In the present study, the abdominal fat percentage of chicken was shown to be lower in the SK-supplemented group than in the non-supplemented group, which was in line with the results reported by Rajaei et al. [32], who reported that the addition of 5 mL/L of noni juice, as a rich source of scopoletin, in drinking water had a significant effect in decreasing abdominal fat content in broiler chickens. Scopoletin is known as a stimulator of fatty acid oxidative genes including PPARa, Acsl1, CPT, Acox, and Acaa1a, and an inhibitor of lipogenic genes such as sterol regulatory element-binding protein-1c and fatty acid synthase in the white adipose tissue and liver in rat [29,33]. The reduced abdominal fat may be due to the supplementation of scopoletin as a bioactive component that reduces oxidative stress and improves carbohydrate and fat metabolism. Serum corticosterone can reflect the welfare status of chickens with the environment [34], and several production and behavioral parameters can be under the influence of corticosterone hormone. Hosseindoust et al. [5] reported that stress hormone is a decisive index for deposition of protein and carcass percentage in chickens. Supplementation with *Morinda citrifolia* L. as a rich source of scopoletin increased the absorption of amino acids, which subsequently increased the carcass rate of broiler chickens [32]. In the current study, the decrease of abdominal fat in the SK-supplemented treatments may not be due to the serum corticosterone because of the insignificant difference in breast meat and drumstick. However, chickens in the SD treatments showed a lower breast meat and drumstick percentage as well. The lower protein deposition may decrease the percentage of muscle to fat and be responsible for the higher relative abdominal fat. There is a positive correlation between corticosterone concentration and abdominal, thigh, or cervical adipose tissue's fat contents [35]; however, the degradation of skeletal muscle can be increased when the serum corticosterone concentration increases [36]. Therefore, the lower protein deposition in muscular organs including breast muscle and drumstick may be responsible for the higher relative abdominal fat.

The health status and BW of animals are under the adverse effect of SD stress, which may cause economic loss [1,2]. Corticosterone is one of the most common end-products of stress and will be secreted to the blood during stressful environments [5,34,37]. Long-time exposure to restraints disrupts the hypothalamic-pituitary-adrenal axis and increases the concentrations of plasma corticosterone [38]. Consistently, the current study showed that the supplementation of SK in the diet during SD stress led to the decrease of corticosterone in the serum. The excessive production of ROS compromises cell growth by degrading cytoskeletal proteins, as well as causing lipids peroxidation during stressful conditions [9]. Our result is in line with those from other researchers who have employed SD stress in poultry [34].

Our study showed that dietary SK supplementation improved the meat quality of chickens by increasing water holding capacity and decreasing cooking loss. The water holding capacity is known as a determinantal factor of meat quality [3]. The significantly lower cooking loss in SK-supplemented treatments may be due to the lower serum corticosterone level. It was reported that excessive levels of serum corticosterone not only reduced the BW gain through reducing anabolism and increasing catabolism processes [39], but also

induced lipid peroxidation [40,41], which may influence meat quality. The protective roles of antioxidant enzymes and controlling the effects of free radical damage seem essential to reduce the adverse influences of stocking density stress. Antioxidant properties of scopoletin may be reflected in the MDA concentration of meat. The lower concentration of serum corticosterone may indicate that the chickens fed SK diets had relatively lower stress regardless of SD because the SD did not decrease cooking loss of breast meat. There is a positive relationship between corticosterone production and the secretion of inflammatory cytokines including IL-1b, IL-6, IL-10, IL-12-a, and IL-18 [19,37], which can adversely affect meat quality. Corticosterone secretion has been shown to be a factor to decrease meat quality by degrading protein in muscle and decreasing the fatty acid transport protein expression [35]. Song et al. [42] stated that the concentration of uric acid, as a factor to show protein catabolism in tissues, increased in blood when the concentration of corticosterone increased in the blood. In addition, the production of ROS can be increased by corticosteroid hormones [43]. The high antioxidant status leads to lower exposure to hydroxyl and peroxyl radicals and possibly the protection of lipid tissues from oxidation through chelating free radicals [4,6]. The result of radical scavenging capacity in this study shows that scopoletin is a potent antioxidant factor to protect fatty acids oxidation and increase meat quality.

The BW gain of chickens in the high SD treatments was 11.9% lower than the low SD treatments. In addition, feed intake was 13.9% lower in the high SD treatments. The reduction of weight gain and feed intake may show that the SD stress adversely affected the performance of broiler chickens. There are important biomarkers such as corticosterone that decrease growth performance during a stressful period [34,38]. The greater BWG in SK-supplemented groups may be because of the antioxidant effects of scopoletin, which may stimulate protein synthesis.

#### **5. Conclusions**

In conclusion, to improve radical scavenging capacity and controlling lipids peroxidation, the antioxidant capacity of broiler chickens can be improved by SK supplementation during high SD stress. Dietary SK improved meat quality through increasing ABTS radical scavenging capacity in the serum and leg muscle. In addition, lower abdominal fat and higher immune organs weight were shown in chickens fed SK. Therefore, our study suggests that SK is a practically useful feed additive to improve the meat quality and weight gain of chickens regardless of SD stress.

**Author Contributions:** Conceptualization and writing—original draft preparation, A.H., S.H.H., H.K.K. and J.S.K.; methodology, A.H. and S.H.H.; software, H.K.K.; validation, S.H.H.; formal analysis, S.H.H.; investigation, S.H.H., H.K.K. and H.T.; resources, A.H.; data/table curation, H.K.K., H.T. and H.L.; writing—review and editing, J.Y.M., E.J.C. and J.M. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was carried out with the support of "Cooperative Research Program for Agriculture Science and Technology Development (Project No. PJ012821012021)" Rural Development Administration, Republic of Korea.

**Institutional Review Board Statement:** The experiment was approved by the Institutional Animal Care and Use Committee, Kangwon National University (KW-170519-1).

**Informed Consent Statement:** Informed consent was obtained from all subjects involved in the study.

**Conflicts of Interest:** The authors declare no conflict of interest.

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