Dietary Supplementation of Some Antioxidants as Attenuators of Heat Stress on Chicken Meat Characteristics

The study evaluated the effect of dietary chromium and vitamin C, Zinc, and sorrel wood powder supplements on chicken health and the nutritional, textural, and sensorial quality of chicken meat. A total of 120 Cobb 500 chickens (heat stress, 32 °C) were assigned into four treatments: control diet (C) and three test diets including 200 µg/kg diet chromium picolinate and supplemented with: 0.25 g vitamin C(VC)/kg diet (E1), 0.025 g Zn/kg diet (E2), and 10 g creeping wood sorrel powder (CWS)/kg diet (E3). Crude protein concentration increased in the breast meat from the E3 group; crude fat decreased in E1 and E3 compared to those fed the C diet. Dietary combinations of CrPic with VC, Zn, and CWS increased redness and decreased the luminosity parameter of breast meat compared with the C group. Dietary combinations of CrPic with VC and CWS lowered the hardness of breast meat. Significant positive correlation was found between hardness–gumminess (r = 0.891), gumminess–cohesiveness (r = 0.771), cohesiveness–resilience-EE (r = 0.861; r = 0.585), ash-L* (r = 0.426), and a negative one between ash–a* (r = 0.446). In conclusion, a dietary combination of CrPic with VC, Zn, and CWS as antioxidant sources could have a beneficial effect on quality without affecting sensory attributes.


Introduction
Chicken meat is the most accessible protein source for humans in most countries. However, the poultry sector may be limited by a series of problems, including high or low environmental temperature, high stocking density, etc. Heat stress is one of the main problems encountered within the poultry sector [1,2]. Heat stress affects nutritional quality, resulting in a lower protein content and higher fat deposition, etc. [3], and sensorial quality such as low water-holding capacity, higher brightness, and lower redness of meat, etc. [4]. In recent years, much attention has been paid to the inclusion of phytochemicals (vitamins, minerals, phytogenic feed additives) with antioxidant properties in the feed mixture, which would overcome the effects of heat stress on poultry health and on the quality of the meat they produce [5]. Some authors have stated that dietary Cr supplementation had a positive effect on meat quality by decreasing the fat content of the carcass, [6] and on carcass traits of broiler chicks in natural [7,8] or heat stress conditions [9]. Moreover, it has been suggested that combinations between Cr and other antioxidants (Zn, vitamin C) might have a synergistic action [2,10].
Vitamin C is a renowned water-soluble vitamin, with undeniable antioxidant activity. In heat-stressed broilers, dietary vitamin C supplementations have improved performance and humoral immunity [11].
Zinc is one of the most important components of the poultry diet, being a cofactor for enzymes with implication in the antioxidant defense system [12]. It was reported

Analysis
The proximate composition of the thigh and breast was determined according to the chemical methods specified by AOAC [16]. Dry matter (ISO 6496/2001) and Ash (ISO 2171/2010) were determined by gravimetric method, crude protein (ISO 5983-2/2009) was analyzed by Kjeldahl method, and ether extractives were performed by extraction in organic solvents (SR ISO 6492/2001).
Chicken thigh and breast color analyses were performed using a Konica Minolta CR-400 (Tokyo, Japan) colorimeter and the CIELAB trichromatic system, which determines lightness (L*), saturation index in green/red (a*), and saturation index in blue/yellow (b*) values. The analyses were performed according to the method described by Panaite [17] and Vlaicu et al. [18]. Each analysis was performed in triplicate to obtain an average colorimetric value.
Firmness was determined by a Warner-Bratzler shear test using a Perten TVT 6700 texturometer (Perten Instruments, Hägersten, Sweden). The principle of this test is the measurement of the force expressed in Newtons (N) necessary to shear a piece of meat. Sample cuts (three rectangular slices/group with 2.0 cm long × 1.0 cm wide × 1.0 cm high) were made parallel to the direction of the muscle fibers. Firmness was calculated from the maximum point of the curve obtained from the test.
The texture profile analyses (TPA) were performed by a double cycle compression using a Perten TVT 6700 texturometer (Perten Instruments, Hägersten, Sweden), equipped with a Compression Platen cell. Four portions of cylindrical form (15 mm high and 20 mm wide) were cut out from each meat sample. The double compression cycle test was applied using an aluminum cylinder probe of 20 mm diameter and was performed up to 50% Agriculture 2021, 11, 638 4 of 14 compression of the original portion height previously prepared. The variables analyzed were: hardness, which is the maximum force needed to compress the sample; springiness, which represents the ability of a sample to recover to its original form after removal of the compressing force; resilience, which is the ratio of the negative force input to positive force input during the first compression; cohesiveness, which is a ratio between the total energy required for the first and second compression; and gumminess, which is defined as the product of springiness, hardness, cohesiveness. Each analysis was carried out in triplicate.
The pH values of the thigh and breast samples were measured 24 h postmortem according to SR ISO 2917: 2007 using a Hach HQ30d pH-meter (Hach Company, Loveland, CO, USA). An aqueous homogenate (meat: distillated water, 1:1) was prepared and filtered according to the method described by Turcu et al. [19]. The measurements were performed in triplicate.
Principal component analysis (PCA) was performed with the proximate compositions and color and textural parameters of meat using Graph-Pad Prism v. 9.02 (San Diego, CA, USA) software package for Windows. Principal Component Analysis (PCA) was applied to assess the relationships between meat characteristics. The similarities and differences between measured variables can be seen in the loading plot. Close variables suggest direct correlations, whereas the opposed variables indicate indirect relationships between them.

Sensory Analysis of Meat
Consumer acceptance tests were performed at the Faculty of Food Engineering, "S , tefan cel Mare" University of Suceava. An acceptance test with hedonic scale was used for the sensorial evaluation, using a 5-point scale (5 = extremely like and 1 = extremely dislike). The raw breast and thigh meat (n = 6 samples/group) were rated for sensory attributes by a semi-trained panel of 13 members selected from the University community. The study was conducted at room temperature of 22 • C under normal daylight. The samples were evaluated for muscle fiber appearance, appearance and characteristics of fat, flavor, firmness, juiciness, and tenderness.

Statistical Analysis
The data were analyzed by one-way analysis of variance (ANOVA), and the means were compared applying Tukey's multiple range test using StatView program for Windows. The following statistical model was used: where Yi was the dependent variable, Ti is the treatment and ei is the error.
Significance was set at p < 0.05. The graphs highlighting the sensory attributes of chicken meat (breast and thigh) were plotted using the same statistical model with Graph-Pad Prism v. 9.02 (San Diego, CA, USA).

Biochemical Measurements
At the beginning of the trial, the mortality rate was 2.5% (starter phase), while at the end of the experiment no mortality was recorded (0%). Table 2 revealed the biochemical measurements performed on serum samples. Serum glucose was significantly lower (by 19.72%) in E2 group compared to E1 and insignificant (p > 0.05) compared to the other groups (C, E3). Diets did not affect (p > 0.05) the other parameters such as cholesterol, triglyceride, phosphorus, calcium, iron, AST, ALT, gamma GT.

Proximate Composition and Physicochemical Properties of Chicken Meat
Data on proximate composition of breast and thigh samples collected at the end of the experiment are presented in Table 3. In thigh meat, the concentrations of DM, CP and Ash did not differ (p > 0.05) between control group and experimental groups (E1, E2 and E3). However, the concentration of EE recorded significant differences (p < 0.05), being higher in E2 (CrPic + Zn) compared to C group.
In the analyzed chicken breast meat, there were significant increases (p < 0.05) in terms of CP and Ash and decreases in the EE. Thus, the dietary supplementation with CrPic + CWS determined the increase in crude protein concentration in breast meat compared to the C group; otherwise, the other groups had a statistically similar protein with group C. Additionally interesting is that CrPic + VC and CrPic + CWS caused a significant reduction in the concentration of crude fat in chicken breast compared to group C. The breast from E2 group had a significantly higher concentration of crude fat than E1 and E3, but similar to group C. Ash concentration was higher (p < 0.05) in groups E1 and E3 Agriculture 2021, 11, 638 6 of 14 than in C. The group E2 had a significantly lower Ash content than in E1 and E3, but at a comparable level to C. Table 4 shows the effects of diets on the color of thigh and breast muscle (pectoralis major). Supplementation of chicken diets with CrPic + VC (E1), CrPic + Zn (E2) and CrPic + CWS (E3) did not influence the value of the L* parameter of thigh meat. In contrast, the value of a* was significantly lower in the thigh collected from E1 than from E3 group. Yellowness parameter (b*) was also lower in the thigh samples from C group than in those from E1 group, while no difference was recorded in the E2 and E3 groups. Regarding the chicken breast, the L* parameter was significantly lower in the groups that included CrPic + VC, Cr + Zn, Cr + CWS in the diet than in the C group. Moreover, the values of a* parameter were higher in E1, E2 and E3 groups compared with the C group. The breast of chickens fed E1, E3 diets had a significant lower yellowness (b*) (p < 0.05) than those fed C diet. Notably is that the lowest value of b* parameter was recorded in the breast samples collected from E3 diet.

Meat Texture Parameters Determined by Shear with a Single Cycle and Double Cycle Compression
The chicken thigh meat recorded significant differences in the shear force parameter (p < 0.05) between experimental groups (E1, E2, and E3) and C group ( Table 5). The thigh meat collected from E1 was firmer compared to those collected from E2 and E3. The combined effect of chromium and vitamin C led to an increase in the firmness of the meat, the cutting force registering the lowest value (30.88 N). The thigh meat of chicken from group E3 was firmer compared to meat from group E2, but the differences were not statistically significant.
The analysis of the texture profile (TPA) of thigh samples resulting from the application of the double compression test shows that the parameters of hardness, elasticity, cohesiveness, and gumminess did not register significant (p > 0.05) differences between groups. Cohesiveness shows how well the sample retains its structure after compression and includes adhesive and cohesive forces as well as viscosity and elasticity. Gumminess, a secondary parameter in the analysis of the texture profile, is determined as a product between firmness and cohesiveness. The resilience of chicken thigh was significantly lower in the group that included CrPic + Zn (E2), CrPic + CWS (E3) in the diet compared to group C and E1. The lowest resilience value was obtained in group E2 (diet supplemented with CrPic + Zn). The pH values were lower (p < 0.05) in E2 and E3 compared to C group. The dietary inclusion of CrPic+ VC did not have a significant effect on the pH of the thigh meat. The shear force showed inconsistent patterns concerning the supplemented diets ( Table 6). It is observed that the breast meat obtained from groups E2 and E3 was characterized by a lower hardness (p < 0.05) compared to that from groups C and E1. The springiness of chicken breast collected from E2 and E3 groups did not differ significantly (p > 0.05) from that obtained for group C. Statistically significant differences were obtained between C and E1, the breast meat in group E1 showing a significantly lower elasticity (99.42%) compared to that of group C (99.74%). The resilience of chicken breast meat in groups E2 and E3 differs significantly (p < 0.05) from the values obtained in group E1. There were no differences between group C and the other groups. However, the lowest resilience value of breast meat (2.16 adm) was recorded in E2 group, which included CrPic + Zn in the diet. The cohesiveness and gumminess attributes of the chicken breast did not differ between groups. The inclusion of CrPic + VC in the chicken diet led to an increase in the pH of breast meat compared to C group. There were no significant differences in pH between C and E2 and E3. Diet supplementation with CrPic + Zn, and CrPic+ CWS respectively led to a significant decrease in pH value, compared to that of samples from E1.  Figure 1 shows the PCA plot for chemical characteristics, L*, a*, b* color parameters, textural parameters, and some sensorial characteristics.  Figure 1 shows the PCA plot for chemical characteristics, L*, a*, b* color parameters, textural parameters, and some sensorial characteristics. The first two PCs explain 50.81% of the total variance (PC1 = 32.30%, PC2 = 18.51%) for the evaluated characteristics. Some sensory characteristics such as muscle fiber appearance, appearance and fat characteristics, and smell were eliminated from the PCA because they have a low adequacy for PCs. The first principal component, PC1 was associated with chemical composition (CP, EE, and Ash) and L*, a*, b* color parameters. The second component, PC 2 was characterized by dry matter (DM), textural parameters (hardness and gumminess), and sensory characteristics (consistency, juiciness, and tenderness). Meat tenderness shows a weak negative correlation with EE (r = −0.342), which may be related to the intramuscular fat content that affects tenderness. Significant (p < 0.01) positive correlation was found between hardness and gumminess (r = 0.891), gumminess and cohesiveness (r = 0.771), between cohesiveness and resilience (r = 0.861), and EE (r = 0.585). A positive correlation was also found between Ash and L* (r = 0.426), and a negative one between Ash and a* (r = 0.446).

Relationship between Meat Characteristics
As Figures 2 and 3 show, the panelists indicated similar sensory attributes for chicken breast and thigh among groups. Thus, the dietary supplementation had not changed the consumer acceptance criteria. The first two PCs explain 50.81% of the total variance (PC1 = 32.30%, PC2 = 18.51%) for the evaluated characteristics. Some sensory characteristics such as muscle fiber appearance, appearance and fat characteristics, and smell were eliminated from the PCA because they have a low adequacy for PCs. The first principal component, PC1 was associated with chemical composition (CP, EE, and Ash) and L*, a*, b* color parameters. The second component, PC 2 was characterized by dry matter (DM), textural parameters (hardness and gumminess), and sensory characteristics (consistency, juiciness, and tenderness). Meat tenderness shows a weak negative correlation with EE (r = −0.342), which may be related to the intramuscular fat content that affects tenderness. Significant (p < 0.01) positive correlation was found between hardness and gumminess (r = 0.891), gumminess and cohesiveness (r = 0.771), between cohesiveness and resilience (r = 0.861), and EE (r = 0.585). A positive correlation was also found between Ash and L* (r = 0.426), and a negative one between Ash and a* (r = 0.446).
As Figures 2 and 3 show, the panelists indicated similar sensory attributes for chicken breast and thigh among groups. Thus, the dietary supplementation had not changed the consumer acceptance criteria.

Discussion
The implication of Cr in reducing glucose levels in heat-stressed broilers was well documented [20][21][22]. However, the literature is limited in studies regarding the effect of combinations of Cr with other antioxidant compounds. In this study, dietary supplementation with CrPic + Zn had a lowering effect in glucose level compared with CrPic + vitamin C. Nevertheless, there is evidence confirming that dietary Zn can decrease glucose level in heat-stressed broilers [23]. Contradictory results were reported by Abuajamieh et al. [24], showing that dietary organic zinc supplementation (50% and 100% of the Zn level from basal diet) in HS chicks increased blood glucose and additionally decreased blood calcium. Conversely, Saleh et al. [25] showed that Zn methionine supplementation (25, 50 and 100 mg/kg) in heat-stressed broiler diet significantly decreased plasma triacylglycerol, total cholesterol concentrations as compared to the control group. AST and glucose parameters did not record any difference. In this study, dietary supplementation with CrPic + VC or CrPic + CWS did not have any effect on serum biochemical parameters tested. In contrast, some researchers reported that combination of Cr with vitamin C has synergistic action and decreased glucose and cholesterol [10], while data using CrPic + CWS were not found. Perai et al. [26] showed that under stressful condition caused by transport, broiler chickens fed diet enriched in Cr + vit. C had a lower triglyceride level and a higher glucose level than before transport.
The dietary supplementation with CrPic + CWS determined an increase in crude protein concentration in breast meat compared to those fed C diet; otherwise, the other groups had a similar protein content with group C. Untea et al. [27] observed the positive influence of dietary chromium supplements (200, 400 µg/kg) on crude protein concentrations of breast meat in a study on broiler chickens raised under normal temperature conditions. Nevertheless, it is interesting to note that CrPic + VC and CrPic + CWS caused a significant reduction in the concentration of crude fat in chicken breast compared to group C. This reduction in crude fat is a good achievement because a high content of fat led to lipid oxidation, a key factor that negatively affects meat color and texture. Additionally, some researchers [28,29] have reported higher protein and lower fat content in breast meat, when chickens fed diets supplemented with 200 and 400 ppb Cr3+. The probable explanation for fat reducing effect could be the inhibitory potential on lipogenic activity in chick adipose tissue. Toghyani et al. [30] showed that under heat stress, broilers fed a diet enriched in Cr (500, 1000, 1500 ppb Cr nicotinate) recorded increases in crude protein of breast meat.
In this study, it was observed that the use of dietary VC, Zn, and creeping wood sorrel changed the meat color. The redness value (a*) of the thigh collected from E1 group was significantly lower than from E3 group. Therefore, the combination of CrPic + CWS had a favorable effect on thigh meat color compared to that of CrPic + VC. Nevertheless, the redness parameter was higher in E1, E2, and E3 groups compared to the C group. Definitely, those results showed that CrPic in combination with VC, Zn, and CWS improved the meat color. Moreover, the breast from E3 group had the highest concentration of crude protein and the highest redness. Myoglobin is the main heme protein in muscle tissue; perhaps an increase in myoglobin concentration has led to an increase in the redness of chicken breast color. According to Sałek et al. [31], Zn binds myoglobin and increase its oxygenation, which maintain the meat color. Consumers associate the increase in the redness parameter with a better quality of the meat [32]. Meat color is influenced by animal diet [33], its heme components concentration consisting of myoglobin, haemoglobin, cytochrome C [34,35], their oxidation-reduction state, chemical reactions, etc. [36]. The explanation of increasing the meat redness might be the antioxidant activity of supplements, being well-known that vit. C and Zn are involved in the redox reactions, delaying the meat oxidations processes. On the other hand, creeping wood sorrel contain phenols, but also large amounts of vitamin C, which could contribute to maintain the pigment stability of the meat. The luminosity parameter was significantly lower in the breast meat collected from groups that included CrPic + VC, CrPic + Zn, CrPic + CWS in the diet than in the C group. The lightness of the breast ranged from 46.93-51.38; redness from 0.61-1.49 and yellowness from 12. 13-14.40. The breast meat of chickens fed E1, E3 diets recorded a significant decrease in the yellowness parameter than those fed the C diet. Notably, the lowest value of b* parameter was recorded in the breast samples collected from E3 diet. Similar results were reported by Huang et al. [9] who studied the effect of three sources of Cr added in the diet of broiler chickens (Cr propionate, CrPro; Cr picolinate, CrPic; Cr chloride, CrCl3) and two concentrations of added Cr (0.4, or 2.0 mg of Cr/kg) on meat quality of broilers raised under heat stress. The authors reported that broilers supplemented Cr had decreased b* values of meat color in breast muscle. Peña et al. [37] showed no differences on breast color when included ascorbic acid (250, 500, and 1000 g/ton) + citric flavonoids in the diet of heat-stressed chickens.
Results from our study revealed that the dietary supplementation with combinations of CrPic with VC, Zn, and CWS had a beneficial effect on the mechanical strength of the muscle fiber. From a textural point of view, Tudoreanu [38] has found that meat firmness depends mainly on the structure of myofibrin and connective tissue, while Astruc [39] has shown that it depends on the amount of fat and collagen. The texture profile analysis (TPA) of thigh samples resulting from the application of the double compression test shows that the parameters of hardness, elasticity, cohesiveness, and gumminess did not register significant (p > 0.05) differences between groups. The resilience of chicken thigh was significantly lower in the group that included CrPic+ Zn (E2), CrPic + CWS (E3) in the diet compared to group C and E1. The lowest resilience value was obtained in group E2 diet was supplemented with CrPic + Zn. This observation could be related with the increase in EE in E2 than in C. The pH values of thigh meat were lower in E2 and E3 compared to C group. Normally, postmortem, the muscle undergoes various reactions, through which it is transformed into meat. For example, muscle glycogen is denatured by glycolysis, forming lactic acid. This reaction results in an increase in the acidity of meat. Heat stress can affect the pH of meat, increasing it, as a consequence of glycogen consumption in reserves and thus the production of lactic acid is reduced [40]. According to Listrat et al. [33], in poultry, the texture is strongly affected by the postmortem acidification kinetics of muscle.
The breast meat obtained from groups fed combination of CrPic + Zn and CrPic + CWS had a lower hardness (p < 0.05) compared to that from groups C and E1. This indicates that the caused an increase in the tenderness of the chicken breast compared to the control diet and the combination of CrPic + VC. Chromium, in combination with Zn or CWS may lead to a decrease in the mechanical strength of muscle fiber, having an effect of increasing its firmness. The breast meat in group E1 had a significantly lower elasticity (99.42%) compared to that of group C (99.74%). This result suggests that dietary supplementation with CrPic + VC leads to a significant decrease in the ability of the chicken breast meat to return to its original shape after compression. Nevertheless, the lowest resilience value of breast meat (2.16 adm) was recorded in E2 group, which included CrPic + Zn in the diet. This achievement could be correlated with the low hardness of the chicken breast meat in E2. The inclusion of CrPic + VC in the chicken diet led to an increase in the pH of breast meat compared to C group. pH is considered one of the crucial variables determining meat quality [41]. According to Berri et al. [42] the normal pH values of chicken meat at 15 min postmortem are around 6.2 to 6.5, whereas normal ultimate pH values are around 5.8 [43].
The results of PCA revealed that the assessed meat characteristics were clustered in function of similarity. The first component, PC1 underline a close association between EE, cohesiveness and a* parameters, variables which are negatively correlated with CP, Ash, L* and b*. The second component, PC2 distinguished between DM and a* parameters and between textural parameters, hardness and springiness, this fact being expectable. Additionally, an opposite relation can be observed between cohesiveness and springiness, meat springiness showing a direct relation with sensorial parameters, consistency, juiciness, and tenderness.
Sensory attributes of meat such as tenderness, juiciness, muscle appearance, are among the most critical attributes in consumer preferences [44]. In this regard, several studies conducted on consumers have shown that these attributes could be influenced by supplementation of broiler diets. For example, Velasco and Williams [45] showed that chicken meat quality could be improved by adding natural antioxidants. Some selected panelists have indicated an improvement in the flavor of thigh meat, reduced the metallic taste and the overall aftertaste as consequence of supplementing chicken diets with faba bean compared to a soy-based diet [46]. However, Suliman et al. [47] evaluated the sensory attributes of meat obtained from broilers fed diet supplemented with 1, 2, 3, 4, 5, and 6% clove seeds. The authors showed that the sensory attributes were not significantly different between the treatment groups except tenderness. However, in our study, according to panelists, the experimental diets had no influence on sensory attributes tested compared to the conventional diet.
Taken together, the achievement such as improvement of meat color could be probably attributed to the antioxidant potential of the tested supplements being involved in the redox reactions. However, the improvement of breast meat tenderness observed in this study as consequence of supplementing the broiler chicken diet with antioxidants is an important result as it strongly influences consumer satisfaction. Nevertheless, the influence of dietary antioxidants on the regulatory mechanisms that define metabolic and physiological changes in muscle tissue is complex, poorly understood, and further studies are needed to investigate it.

Conclusions
The dietary supplementation with CrPic + creeping wood sorrel determined an increase of crude protein concentration in breast meat, and CrPic + vitamin C and CrPic + creeping wood sorrel reduced the concentration of crude fat compared to those fed the C diet. Dietary supplementation with combinations of CrPic with vitamin C, Zn, and creeping wood sorrel improved the breast meat color, increasing the redness parameter and decreasing the luminosity parameter. Conversely, dietary supplementation with combinations of CrPic with vitamin C and creeping wood sorrel had a beneficial effect on textural parameters of chicken breast meat, increasing the tenderness. Furthermore, there was a clear relationship among proximate composition, color, and texture attributes of chicken meat. Taken together, this study revealed that a dietary combination of CrPic with vitamin C, Zn, creeping wood sorrel, respectively, as sources of antioxidants could attenuate the negative effects of heat stress on nutritional quality and texture without affecting the sensorial parameters of chicken meat.  Data Availability Statement: All data is contained within the article.

Conflicts of Interest:
The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.