**Bioactive Compounds of Barbatimão (***Stryphnodendron* **sp.) as Dietary Additive in Lamb Diets**

**Cristiane R. Barbosa 1, Jessica C. Pantoja 1, Tatiane Fernandes 1, Renata A. Chagas 1, Carla G. Souza 1, Aylpy R. D. Santos 1, Marcio R. Souza <sup>2</sup> and Fernando M. Vargas Jr. 1,\***


**Abstract:** This study aimed to evaluate barbatimão bark extracts as a feed additive and substitute for lasalocid sodium (LAS) for feedlot lambs. Lambs were distributed into three treatments: LAS (0.018 g of lasalocid sodium), DBB (1.500 g of dried and milled barbatimão bark), and BHE (0.300 g of barbatimão hydroalcoholic extract). There was no effect (*p* = 0.32) of the inclusion of DBB and BHE extracts on the average daily gain. Inclusion of BHE in lamb diets reduced (*p* < 0.05) the fatness score compared to LAS, which was similar to DBB. The BHE decreased the yellowness intensity and hue angle (*p* < 0.05) of meat compared to the LAS. Animals that consumed DBB and BHE had a reduced (*p* = 0.04) total cholesterol level. Thus, the use of barbatimão bark extracts can replace lasalocid sodium in the diet of feedlot lambs, with no detrimental effects on performance or metabolic parameters.

**Keywords:** ruminants; tannins; bioactive compounds; saponins; feed additives

#### **1. Introduction**

Lasalocid sodium (LAS) is a synthetic product used as an additive in animal feed, related to its potential to mitigate methane (CH4) emissions, improve animal performance [1], and increase production profitability. However, the market is increasingly demanding, resulting in the rejection of chemicals in animal protein production, due to the evolution of antibiotic-resistant pathogens [2]. In the search for substitutes for synthetic products, the animal feed additive industry has intensified its investments in biocompounds [3]. These biocompounds modify ruminal subtract availability and microbial ecosystem, thus reducing CH4 emissions [4]. The natural additive effects are related to the type of diet (high or low concentrate), concentration and amount of ingested additive, mode of action in the gastrointestinal tract, and physiological state of the animal [3,5]. In addition, the composition of biocompounds in plants can be affected in several ways, from the plant development to the final extraction [6].

Barbatimão (*Stryphnodendron* sp.) is a plant native to the Brazilian savanna and produces several chemical metabolites in its secondary metabolism, such as tannins and saponins [7]. These biocompounds have antimicrobial, healing, anti-inflammatory, and antioxidant activities; thus, it is a plant used by communities in traditional medicine [8,9]. Tannins are phenolic compounds with properties that precipitate proteins [10]. High doses of tannins (>5% in dry matter; DM) in a ruminant diet may lead to a reduction in DM intake, digestibility, and performance [11]. However, at moderate doses, tannins potentiate nutrient use efficiency, due to the greater availability of these nutrients in the small intestine [3]. Saponins are active photochemical components of plants, which are part of the plant defense system, and that have an antimicrobial and antioxidant potential [8,12] that can affect ruminal microorganisms. In the literature, several studies have shown

**Citation:** Barbosa, C.R.; Pantoja, J.C.; Fernandes, T.; Chagas, R.A.; Souza, C.G.; Santos, A.R.D.; Souza, M.R.; Vargas, F.M., Jr. Bioactive Compounds of Barbatimão (*Stryphnodendron* sp.) as Dietary Additive in Lamb Diets. *Agriculture* **2023**, *13*, 664. https://doi.org/ 10.3390/agriculture13030664

Academic Editors: Arabela Elena Untea, Mihaela Saracila and Petru Alexandru Vlaicu

Received: 23 February 2023 Revised: 7 March 2023 Accepted: 8 March 2023 Published: 13 March 2023

**Copyright:** © 2023 by the authors. 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/).

that the inclusion of sources of tannins and saponins in ruminant diets can reduce enteric methane production, improving feed efficiency and animal performance [11,13,14].

In this context, barbatimão extract has advantages because of its various active components and modes of action, being a natural and safe option as an additive [15]. Thus, the objective of this study was to evaluate the effects of the use of barbatimão bark extracts, as a food additive in place of LAS, on the metabolic parameters, performance, and carcass characteristics of feedlot lambs.

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

#### *2.1. Location and Experimental Facilities*

The experiment was carried out at the Federal University of Grande Dourados. The Animal Use Ethical Committee of the Federal University of Grande Dourados, Brazil, approved the experimental animal procedures (Protocol 032.2020).

#### *2.2. Collection, Production, and Phytochemical Analysis of Extracts*

Barbatimão barks (*Stryphnodendron rotundifolium*) were manually collected from several trees during the summer season in the morning and dried in a forced ventilation oven at 55 ◦C for 72 h. Then, the material was milled in a Willey mill with a 2 mm sieve, to obtain the dry barbatimão bark (DBB). Barbatimão hydroalcoholic extract (BHE) was obtained by submersion of 250 g of DBB in a water–ethanol solution 50:50 [16] and incubated in a water bath at 60 ◦C for 60 min. Hot filtration was performed in a funnel with four layers of cheesecloth and dried (40 ◦C) to a constant weight. Then, the material was macerated until a powder granulometry was obtained.

The extracts were submitted to phytochemical evaluation [17], to confirm the secondary metabolite classes [18]. The presence of triterpenes and steroids was confirmed through hydrolysis of the dry methanolic extract. This procedure was performed with potassium hydroxide (0.5 mol/L) and with reflux for 1 h. Then, the compounds were extracted with ethyl ether and then submitted to a Liebermann–Burchard reaction. To determine the presence of secondary metabolite classes, the reactions of characterization intensities were classified as follows: negative reaction (− = 0%), partial intensity (±/+ = 10%), low intensity (+ = 50%), medium intensity (++ = 75%), and high intensity (+++ = 100%) [18]. The extracts were solubilized at a concentration of 1 mg/mL in methanol, to analyze phenolic compounds and flavonoids [19]. The content of phenolic compounds was determined based on the Folin–Ciocalteau colorimetric method [19]. The tannin content was determined using the Folin–Denis spectrophotometric method, with tannic acid as a reference [20].

#### *2.3. Animals, Diets, and Experimental Design*

Twenty-four lambs, non-castrated males, with 150 ± 4.59 days of age and 21.2 ± 3.63 kg of body weight (BW) were used. At the beginning of the experiment, the lambs were weighed, identified, and dewormed (Baycox, 1 mL/3.5 kg BW). The lambs were allocated to individual covered stalls with an area of 2.2 m2 (1.5 m × 1.5 m), with a cement floor lined with rice husk. They had free access to water and had access to ground oat hay (*A. sativa*) and concentrate ad libitum for 14 days for adaptation.

After the adaptation phase, the experimental period began, consisting of three periods of 14 days, counting 42 days of performance evaluation plus six days up to the slaughter, totaling 48 days for the experiment. The experimental diet (Table 1) was formulated based on soybean meal, ground corn, oat hay, and specific commercial mineral supplement for sheep, according to the National Research Council (NRC) [21], to meet the requirement of lamb weight gain of 300 g/animal/day, with the dry matter intake estimated at 3.5% of BW. The forage:concentrate ratio was 20:80. The total diet was offered at 07:00 and 12:00, adjusting the amount provided every three days, considering 5% of leftovers.


**Table 1.** Ingredients and chemical composition of the experimental diet.

The experimental designed used randomized blocks, with three treatments and eight repetitions. The lambs were blocked based on BW, and the treatments were randomly distributed within each block. The treatments tested were addition of DBB, 1.50 g/animal/day; addition of BHE, 0.300 g/animal/day; and addition of LAS, 0.018 g/animal/day. DBB and BHE were mixed with 30 g of concentrate and supplied as a top-dress at morning meals, to ensure total intake. For LAS, 0.150 g/kg of the ionophore (Taurotec-Zoetis, Campinas, São Paulo, Brazil; 15% of lasalocid sodium) was mixed into the diet.

#### *2.4. Animal Performance*

The dry matter intake was determined daily from the difference between the offered feed and the leftovers. The leftovers were weighed before the morning meal. A composite sample of diet ingredients and leftovers was collected per period per animal and stored in a freezer at −20 ◦C. At the end of the collection period, the samples were pre-dried in a forced ventilation oven at 55 ◦C for 72 h. Lastly, the samples were milled (1 mm) in a Willey mill for further analysis.

The lambs were weighed on the first day (initial body weight) and at the end of each experimental period, with fasting of solids and liquid in the previous 12 h. The average daily gain was measured from the difference in BW between the beginning and the end of the experimental period, divided by the number of days. The relation between dry matter intake and average daily gain was used to determine the feed conversion ratio. The body condition at slaughter was evaluated by two specialists, based on a scale from 1 to 5, with a 0.5 variation.

#### *2.5. Chemical Composition*

Diet and extract samples were analyzed for dry matter content (ID 934.01), ash (ID 930.05), crude protein (CP, ID 981.10), and ether extract (ID 920.39) [22]. Neutral detergent fiber and acid detergent fiber contents were evaluated according to Van Soest [23].

#### *2.6. Slaughter and Carcass Evaluation*

All slaughter procedures were performed according to the Regulation of Industrial and Sanitary Inspection of Products of Animal Origin and the rules of Technical Regulation of Methods of Insensitization for the Humanitarian Slaughter of Butcher Animals [24]. The lambs were slaughtered at the end of the experiment, after 62 days in the feedlot (14 of adaptation + 48 of the experiment). The lambs were submitted to solid fasting for 16 h and weighed to determine the body weight at slaughter. The lambs were desensitized by electronarcosis, suspended by the hind legs, and the carotid arteries and jugular vein were sectioned for bleeding, then they were skinned and eviscerated. The full and empty gastrointestinal tract, bladder, and gallbladder were weighed, and the weight of the abiotic components was obtained from the difference between the full and empty weights. The empty body weight was determined from the subtraction of the abiotic components from body weight at slaughter. Carcass conformation and fatness indexes were determined by two specialists, based on a scale from 1 to 5, with a 0.5 variation, where: 1—Very poor; 1.5—Poor; 2—Acceptable; 2.5—Average; 3—Good; 3.5—Very Good; 4—Superior; 4.5—Very Superior, and 5—Excellent [25].

The carcasses were weighed to obtain the hot carcass weight. The carcasses were suspended by the leg tendons and stored in a cold room at 4 ◦C for 24 h, and were posteriorly weighed to obtain the cold carcass weight. Then, the yields of the hot carcass and cold carcass and the loss by cooling were determined [26].

The meat color in the Longissimus thoracis et lumborum muscle was determined [27] using a digital colorimeter (Minolta CR-400, Minolta Co., Osaka, Japan), calibrated in the CIELAB system. The luminosity (L\*), red intensity color (a\*), and yellow intensity color (b\*) [28] were measured. The saturation index (chroma; C\*) was determined according to the equation:

$$\mathbf{C}\* = \sqrt{(\mathbf{a}\*^2) + \left(\mathbf{b}\*^2\right)}\tag{1}$$

The definition of metric hue angle (HUE) was determined according to the equation:

$$\text{HUE} = \arctan(\text{b}^\star/\text{a}^\star) \tag{2}$$

#### *2.7. Metabolic Parameters*

Blood samples were taken by jugular vein puncture, four hours after morning feeding, on the 11th day of each experimental period with a vacutainer with heparin. The samples were centrifuged immediately after collection at 3000 rpm for 15 min, and the plasma was frozen for further analysis. The plasma glucose concentration was determined by the enzymatic-colorimetric method of glucose-oxidase, using a commercial kit (Sigma C.C.). Total cholesterol was evaluated using a Cholesterol Labtest Diagnóstica commercial kit. The concentration of urea, aspartate aminotransferase, and alanine aminotransferase were evaluated using Diagnóstica commercial kits.

Urine was sampled after slaughter. Creatinine and urea were evaluated using Gold Diagnóstica commercial kits. The colorimetric method was used for allantoin determination [29]. Blood and urine tests were performed at the Laboratory of the Veterinary Hospital of the University Center of Grande Dourados. Creatinin was used to estimate the total urine excretion.

#### *2.8. Statistical Analysis*

The data were analyzed using MIXED PROC from the Statistical Package of SAS (SAS University Edition), except for the data from carcass conformation and fatness, which were analyzed by proc npar1way (SAS University Edition). The means were compared using the Tukey test. Significance was declared when *p* < 0.05. The statistical model included treatment as a fixed effect and the block as a random effect.

#### **3. Results**

The high tannin contents of barbatimão bark extracts (Table 2) did not interfere in acceptability because it did not reduce the lamb dry matter intake (*p* = 0.56; Table 3). Barbatimão bark extract's effects were similar to (LAS), and there was no change in the performance variables average daily gain, feed conversion ratio, initial body weight, body weight at slaughter, empty body weight, and body condition at slaughter (*p* > 0.05) with the addition of DBB and BHE (Table 3). The inclusion of DBB and BHE did not influence the variables related to body weight at slaughter and yield (*p* > 0.05), such as hot carcass weight, cold carcass weight, loss by cooling, hot carcass yield, cold carcass yield, and carcass conformation of lambs. The BHE showed a lower carcass fatness index (*p* = 0.04).


**Table 2.** Chemical composition and secondary metabolic compounds of dry barbatimão bark (DBB) and barbatimão hydroalcoholic extract (BHE).

\* The presence of secondary metabolic compounds was classified as follows: negative reaction (− = 0%), partial intensity (±/+ = 10%), low intensity (+ = 50%), medium intensity (++ = 75%), and high intensity (+++ = 100%).

**Table 3.** Productive performance characteristics and carcass evaluation of lambs fed with diets containing lasalocid sodium (LAS), dried barbatimão bark (DBB), or dry barbatimão hydroalcoholic extract (BHE).


<sup>1</sup> Standard error mean; <sup>2</sup> Conformation estimated by a scale of 1 (no fat) to 5 (excess fat); <sup>3</sup> Estimated fatness by a scale of 1 (no fat) to 5 (excess fat); a, b Averages followed by different letters on the same line differ (*p* < 0.05) from each other.

The parameters of luminosity, red intensity, and chroma were not influenced (*p* > 0.05) by the DBB and BHE (Table 4). However, the BHE lambs had a lower intensity of yellow and hue angle compared to the other treatments. The barbatimão bark extracts in the diets did not influence (*p* > 0.05) the levels of glucose and urea in the blood and urea and allantoin in urine of the lambs (Table 5). The barbatimão bark extracts reduced (*p* = 0.01) the total blood cholesterol levels compared to the LAS treatment. The barbatimão bark extracts did not influence (*p* > 0.05) the aspartate aminotransferase and alanine aminotransferase levels.


**Table 4.** Meat color of lambs fed with diets containing lasalocid sodium (LAS), dried barbatimão bark (DBB), or dry barbatimão hydroalcoholic extract (BHE).

<sup>1</sup> Standard error mean; a, b Averages followed by different letters on the same line differ (*p* < 0.05) from each other.

**Table 5.** Blood and urinary parameters of lambs fed with diets containing lasalocid sodium (LAS), dried barbatimão bark (DBB), or dry barbatimão hydroalcoholic extract (BHE).


<sup>1</sup> Standard error mean; a, b Averages followed by different letters on the same line differ (*p* < 0.05) from each other.

#### **4. Discussion**

Normally, a high tannin content is associated with harmful effects, such as reduced feed intake [30]. However, if the supply is moderate, there are no harmful impacts of intake [5], as occurred in this study. Barbatimão bark extract's effects were similar to LAS, by reducing ruminal proteolysis and promoting the flow of dietary protein into the duodenum. This was inferred to be because the flow of dietary protein with microbial protein increased the availability of nitrogen compounds for protein synthesis in the animal [31]. In addition, the presence of tannins and saponins in the barbatimão bark extracts was associated with reduced energy expenditure, justified by the ability of these phenolic compounds to reduce the process of methanogenesis [32]. Consequently, there is reduction in energy losses by decreasing ruminal methane production [4]. Studies with sheep fed a diet containing less than 50 g of tannins/kg of dry matter showed no effect on daily dry matter intake, presenting a higher feed efficiency and daily weight gain than a treatment without tannins [30,33]. Furthermore, carcass characteristics and meat quality were not affected by tannins [34].

Regardless of the treatment, body weight at slaughter and carcass yields were similar, and this may indicate a positive relation of body weight at slaughter with carcass yield. All treatments had an average within the appropriate variation range, from 40 to 50% for sheep [35]. Loss by cooling was not influenced by treatments, with established indices for sheep ranging from 1 to 7% [36]. Loss by cooling is related to the carcass fatness classification, because it is related to age, nutritional management, live weight, and carcass conformation [37].

The lower carcass fatness index on BHE may be related to the level of extract used in the diet, resulting in a lower influence of barbatimão biocompounds on lambs. The BHE carcasses had an acceptable fatness index (2 to 2.4), with an average of 2.4, followed by DBB, with a medium fatness index (2.5 to 3.0) and LAS with a fatness index considered good (3.0 to 3.4) [25]. Carcass fatness index is directly connected to adiposity, which predicts the tissue composition of the carcass [38]. In addition, it reduces fluid loss and shortening of muscle fibers and increases meat darkening during the cooling process [39].

The BHE may have influenced the meat color through the activity of barbatimão biocompounds, probably due to the level of extract used, since this additive has an antioxidant

action, which can interfere in the meat color [40]. Meat values of b\* (9.44), C\* (25.29), and HUE (21.92) of feedlot Pantaneiros lambs reported in the literature [41] differ from the values of this study, which were lower.

To verify the metabolic alteration, the blood and urinary parameters were evaluated. Barbatimão bark extracts reduced the total blood cholesterol levels compared to the LAS treatment. Cholesterol levels are indicative of energy balance [42]. The reduction in blood cholesterol concentration can be explained by a possible decrease in rumen acetate production, since acetate is a precursor to cholesterol synthesis in ruminants [43]. The barbatimão tannins can form complexes with fibers, inhibiting the cellulolytic bacteria action and causing lower acetic acid production [44]. Similarly, the antimicrobial activity of saponins present in barbatimão is more evident for Gram-positive bacteria [45]. Inhibition of Gram-positive bacteria in ruminants reduces the proportion of acetate produced in the rumen [46].

The aspartate aminotransferase and alanine aminotransferase enzymes are markers of liver damage and can be identified in the cytoplasm, liver cell mitochondria, cardiac system, and skeletal muscle [42]. Our study's results corroborate the literature reports [47]. This literature indicates that appropriate doses (<5% in dry matter) do not cause adverse effects, even with extensive biotransformation of liver metabolism in mammals and intestinal microbiota. Thus, the extracts used in the present study did not cause liver injury in the lambs.

#### **5. Conclusions**

The use of barbatimão bark extracts may replace lasalocid sodium in the diet of feedlot lambs, with no detrimental effects on performance and metabolic parameters. Therefore, it is suggested that further studies are conducted to evaluate the bioavailability of the biocompounds and the inclusion of higher doses of milled bark and hydroalcoholic extract of dried barbatimão in the diet of feedlot lambs.

**Author Contributions:** Conceptualization, C.R.B., T.F., C.G.S. and F.M.V.J.; methodology, C.R.B., J.C.P., T.F., C.G.S. and F.M.V.J.; software, C.R.B., T.F. and F.M.V.J.; validation, T.F. and F.M.V.J.; formal analysis, T.F., R.A.C., C.G.S. and A.R.D.S.; investigation, C.R.B., J.C.P., R.A.C., A.R.D.S. and M.R.S.; resources, C.R.B., T.F., C.G.S., A.R.D.S. and F.M.V.J.; data curation, C.R.B., T.F., C.G.S., A.R.D.S. and F.M.V.J.; writing—original draft preparation, C.R.B., T.F., C.G.S. and F.M.V.J.; writing—review and editing, C.R.B., T.F., A.R.D.S. and F.M.V.J.; visualization, C.R.B., T.F., C.G.S., A.R.D.S., M.R.S. and F.M.V.J.; supervision, T.F., C.G.S. and F.M.V.J.; project administration, F.M.V.J. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

**Institutional Review Board Statement:** The Animal Use Ethical Committee of the Federal University of Grande Dourados, Brazil, approved the experimental animal procedures (Protocol 032.2020).

**Data Availability Statement:** The data presented in this research are available from the corresponding author.

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

#### **References**


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## *Article* **Meat Quality of Male Layer-Type Chickens Slaughtered at Different Ages**

**Teodora Popova 1,\*, Evgeni Petkov 1, Maya Ignatova 1, Desislava Vlahova-Vangelova 2, Desislav Balev 2, Stefan Dragoev 2, Nikolay Kolev <sup>2</sup> and Krasimir Dimov <sup>3</sup>**


**Abstract:** An experiment with male layer-type chickens of the Lohmann Brown Classic breed was carried out at the Institute of Animal Science-Kostinbrod, Bulgaria, aiming to investigate the effect of age at slaughter on the meat quality. The birds were reared in a controlled microclimate, with an initial stocking density of 22 birds/m2. At five weeks of age, fragmentation of the stocking density was applied, decreasing the number to seven birds/m2. Chickens were slaughtered at five and nine weeks of age at an average live weight of 329 g and 1096 g, respectively. After slaughter, 10 chickens from each age group were subjected to analysis to determine the quality of breast and thigh meat. The results of the study showed that the age affected the meat quality parameters of the male layer-type chickens and its effect differed between the breast and thigh. The chickens slaughtered at nine weeks of age displayed a lower pH but darker meat color (*p* < 0.001) than those slaughtered at five weeks. Furthermore, the older birds showed a significant decrease in the intramuscular fat content in thigh meat (*p* < 0.01) and a tendency for diminishing in breast meat. This decrease corresponded to the lower percentage of monounsaturated fatty acids (MUFA) in the meat of the nine-week-old chickens (*p* < 0.01). On the other hand, the meat of the older chickens displayed a higher content (*p* < 0.01) of polyunsaturated fatty acids (PUFA), especially n-6, leading to a considerably higher n-6/n-3 ratio.

**Keywords:** male layer-type chickens; age; meat quality; fatty acids

#### **1. Introduction**

The increasing demands of consumers regarding the quality of poultry meat produced globally require intensive selection of broilers for rapid growth and low feed intake. The selection according to these indicators has an economic effect but leads to negative changes in the quality of the harvested meat [1]. According to Baldi et al. [2] the major meat quality concerns are associated with abnormalities in breast meat, such as wooden breast, white striping, and spaghetti meat, that affect alone or in combination the meat of fast-growing broilers. These growth-related abnormalities not only impair the appearance of the meat, but also have a detrimental effect on the technological qualities [3]. Hence, the interest in the meat of slow-growing chickens has considerably increased. It has gradually grown in popularity in the market as a product with excellent taste and dietary values [4,5], and in some EU countries its production has increased significantly in the recent years [6].

Previous studies [7,8] found that the growth rate and the feed conversion of male layer-type chickens meet the minimum criteria for slow-growing chickens; even in the first four weeks of rearing their feed intake resembles slow-growing broilers. This reveals possibilities for their utilization and the conversion of a waste product from the production of female layer chickens into a secondary product for an innovative, independent, and economically sustainable niche for producing high-quality poultry meat products. After

**Citation:** Popova, T.; Petkov, E.; Ignatova, M.; Vlahova-Vangelova, D.; Balev, D.; Dragoev, S.; Kolev, N.; Dimov, K. Meat Quality of Male Layer-Type Chickens Slaughtered at Different Ages. *Agriculture* **2023**, *13*, 624. https://doi.org/10.3390/ agriculture13030624

Academic Editors: Arabela Elena Untea, Mihaela Saracila and Petru Alexandru Vlaicu

Received: 10 February 2023 Revised: 1 March 2023 Accepted: 3 March 2023 Published: 5 March 2023

**Copyright:** © 2023 by the authors. 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/).

hatching, the male layer-type chickens are often used in the manufacturing of pet foods or are culled due to their low live weight. This poses both animal welfare and ethical issues [9] provoking sharp criticism in society [10] and made it necessary to explore other ways to use layer-type cockerels. Three methods for utilization have emerged to avoid culling of this type of chicken: in ovo sex determination, use of dual-purpose breeds, and rearing male cockerels for meat. In Bulgaria, research on this topic commenced in 2010; however, studies on the meat quality of these birds are scarce. Our previous studies with male layer-type chickens showed that despite the certain disadvantages of the performance traits of these birds compared to the fast-growing broilers, they also have more positive traits, such as low deposition of abdominal fat and lower intramuscular and subcutaneous fat [8]. We found that the meat of layer-type cockerels slaughtered at 12 weeks of age is not inferior in quality to that obtained from slow-growing chickens; on the contrary, they have a higher protein content, a higher WHC, and lower intramuscular fat [8]. The quality of meat is determined by various factors, among which is the age at slaughter [11]. Dal Bosco et al. [12] compared chickens from different genotypes slaughtered at different ages and showed that the meat of younger chickens was more tender, while the older birds had lighter meat. Li et al. [13] confirmed higher tenderness of the meat in younger chickens and found improved waterholding capacity with increasing age. Age can affect the nutritional profile and healthy value of the meat, which is also determined by its fatty acid profile. In slow-growing chickens slaughtered at different ages [14], a significantly higher content of saturated fatty acids in thigh meat and a higher content of polyunsaturated fatty acids in both the breast and thighs of older chicks was reported. Furthermore, the content of cholesterol in meat was significantly lower in chickens slaughtered at an older age. Determining the optimal age for slaughtering animals and poultry is crucial to obtain the best characteristics in the carcass and meat and the highest nutritional and health values [13,15–17].

With a current scarcity of scientific information on the male layer-type chickens, this research was conducted to provide a better insight into the differences in the meat quality of this type of chickens slaughtered at different age. The data obtained in this study will complement the current scientific knowledge on slow-growing chickens and can also be used as a basis for further research on the processing quality of this type of meat.

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

#### *2.1. Ethical Procedure*

The experimental protocol used in this study, including the animal management and housing, was designed in compliance with the guidelines of the European and Bulgarian legislation regarding the protection of animals used for experimental and other scientific purposes [18]. The protocol was based on the permit for use of animals in experiments No. 227 of the Bulgarian Food Safety Agency (Statement No. 193 of the Bulgarian Animal Ethics Committee, prot.No.18/02.07.2020).

#### *2.2. Experimental Birds and Housing*

The trial involved 800 male layer-type chickens of the Lohmann Brown Classic breed hybrid and was carried out in the experimental poultry farm at the Institute of Animal Science-Kostinbrod, Bulgaria. The 1-day-old chickens were supplied by Bulagro 97 AD. The birds were housed in 5 pens each containing 160 chickens and were reared conventionally until slaughter. The initial stocking density was 22 chickens/m<sup>2</sup> and at 5 weeks of age, this was decreased to 7 birds/m2. The fragmentation of the stocking density of the chickens was undertaken through preliminary weighing of each chicken and differentiation by the live weight. Thus, all the male layer-type chickens with a final body weight of ≤360 g were slaughtered at 5 weeks of age, and the remaining chickens were reared until 9 weeks old. The chickens were reared in deep litter and a controlled microclimate. The lighting regime was 3 h light and 3 h dark which repeated during the 24 h cycle. Feeding was ad libitum with standard broiler feed (Tables 1 and 2) according to the instructions for Ross 308 broilers [19]. Water was provided through gravity drinkers. During the trial period, the live weight of the birds was controlled weekly.


**Table 1.** Composition of the diet of the male layer-type chickens.

<sup>1</sup> Vitamin and mineral premix provided the following per kg of diet: Fe,185 mg; Cu, 25 mg; Zn, 120 mg; Mn, 145 mg; I, 1.70 mg; Se, 0.45, vit. A 10500 IU; vit. D, 3750 IU; vit. E, 45 mg; BHT, 0.10 mg; endo-1,4 beta-xylanase 1500EPU.

#### **Table 2.** Fatty acid composition of the feed.


#### *2.3. Slaughtering Procedure and Sampling*

The chickens were slaughtered at 5 and 9 weeks of age in a commercial poultry abattoir at an average live weight of 329 g and 1096 g, respectively. The birds were stunned, decapitated, and bled. The carcasses were then plucked and eviscerated. Feet and edible viscera (heart, liver, gizzard) were removed in order to obtain the ready-to-cook carcass. The carcasses were then chilled and stored at 4 ◦C for 24 h. After 24 h of chilling, 10 chickens from each age group were randomly selected for meat quality analysis. The breast (pectoralis profundus et superficialis) and thighs of each carcass were collected, skinned, and deboned. One part of the samples was immediately used for analysis of meat technological quality while the other was vacuum-packed and stored frozen for further analysis of the proximate composition.

#### *2.4. Analysis of the Meat Quality*

#### 2.4.1. Measurement of pH and Color

Muscle pH and color were measured at the time of deboning of the breast and thigh cuts. The pH measurements were undertaken using a portable pH meter equipped with a glass electrode. Calibration prior to use at pH 4.0 and 7.0 was performed. The surface color of the breast and thigh muscles was measured by Croma meter CR-410 (Konica Minolta Inc., Osaka, Japan) using CIE values expressed as lightness (L\*), redness (a\*), and yellowness

(b\*). A measuring area of 50 mm, illuminant D65, and 2◦ standard observer were used. The instrument was calibrated using a standard white plate. The measurements of the pH and color were undertaken at 3 locations in the muscles and the results were averaged.

#### 2.4.2. Determination of Water Holding Capacity (WHC)

Water holding capacity measured as free water content (%) was determined according to the filter press method as described by Honikel and Hamm [20].

#### 2.4.3. Texture Analysis

The tenderness of the meat was measured by Warner–Bratzler shear force (WBSF) using a Belle texture analyzer (Agrosta, Serqueux, France). The measurements were undertaken on cooked meat from breast and thigh at the day of carcass analysis. The breast and thigh meat pieces were weighed, placed into plastic bags, sealed, and cooked in a water bath at 80 ◦C until the internal temperature of the meat reached 70 ◦C. The bags were then removed and the meat was left to cool at room temperature for approximately 30 min. The meat pieces were dried to eliminate any water left on the surface. Shear force was evaluated on cores cut from the thickest part of the cooked samples by cutting them perpendicularly to the direction of the fibers [21].

#### 2.4.4. Proximate Analysis

The content of moisture, protein, fat, and ash in the breast and thigh meat was assessed according to the methods of AOAC [22].

#### *2.5. Fatty Acid Profile*

The fatty acid composition of the feed and meat was determined according to the method of Bligh and Dyer [23] with slight modifications [24]. Lipids were extracted from 10 g of the muscle/feed sample and homogenized using a HG-15D homogenizer (Witeg Labortechnik GmbH, Wertheim, Germany) with 10 mL of chloroform and 20 mL of methanol for 30 s. Following this, 10 mL of chloroform and 10 mL of NaCl (1% in distilled water) were added to the mixture and homogenized for 30 s. The samples were centrifuged (4000 rpm for 10 min) and finally the chloroform layer was evaporated. The fatty acids were trans esterified following the procedure described by Domínguez et al. [25] with some modifications: 20 mg of extracted fat dissolved in 1 mL of toluene was mixed with 2 mL of a sodium methoxide (0.5 N) solution, vortexed for 10 s, and allowed to stand for 15 min at room temperature. Then, 4 mL of a H2 SO4 solution (10% of H2 SO4 in methanol) was added, vortexed for 10 s, and left for 5 min before adding 2 mL of saturated sodium bicarbonate solution. Fatty acid methyl esters were extracted as 1 mL of hexane was added to the samples, vortexed for 10 s, and the organic phase was transferred to an appropriate GC vial. Separation and quantification of FAMEs were carried out using a gas chromatograph (CSi 200 series, Cambridge Scientific Instruments Ltd., Ely, UK) equipped with a capillary column (DM-2330:30 m × 0.25 mm × 0.20μm) and hydrogen as a carrier gas. The oven temperature was first set to 160 ◦C for 0.2 min, then raised to 220 ◦C at a rate of 5 ◦C/min and then held for 5 min. The temperatures of the detector and injector were 230 ◦C. Methyl esters were identified through comparison of the retention times of the standards. Fatty acids are presented as percentages of the total amount of the methyl esters (FAME) identified. The amount of each fatty acid was used to calculate the atherogenic (AI) and thrombogenic (TI) indices [26]:

AI = (4 × C14:0 + C16:0)/[MUFA + Σ(n − 6) + Σ(n − 3)]; TI = (C14:0 + C16:0 + C18:0)/[0.5 × MUFA + 0.5 × (n − 6) + 3 × (n − 3) + (n − 3)/(n − 6)]

#### *2.6. Statistical Evaluation*

Results are presented as mean ± SD. Comparisons between the 2 age groups in regard to the meat quality traits and the fatty acid profiles were performed through *t*-test (JMP v.7, SAS Institute Inc. Cary, NC, USA).

#### **3. Results**

*3.1. Effect of Age on the Technological Quality of Meat*

The pH values were significantly lower in the breast (*p* < 0.05) and thighs (*p* < 0.001) of the older chickens (Table 3). In breast the pH ranged from 5.67–5.73, while for the thighs this trait varied from 6.08 to 6.25.


**Table 3.** Quality traits of the breast and thigh meat.

WHC: Water holding capacity; WBSF: Warner–Bratzler shear force. Significance: \* *p* < 0.05; \*\* *p* < 0.01; \*\*\* *p* < 0.001, ns—non-significant.

The color parameters of breast and thigh meat differed between the chickens slaughtered at five and nine weeks of age. The older birds had darker meat with lower L\* (*p* < 0.001) and b\* values (*p* < 0.001). On the other hand, the redness was significantly higher in the breast of the chickens slaughtered at five weeks of age (*p* < 0.01) and tended to be higher in the thighs of the younger birds but without significant difference.

The water holding capacity (Table 3) as expressed by lower percentage of free water was better in the breast of the chickens that were slaughtered at nine weeks of age (*p* < 0.001), while no such difference was observed regarding this trait in the thigh meat. No difference between the age groups was found in regard to the shear force values.

#### *3.2. Proximate Composition*

Difference between the age groups regarding the proximate composition was observed in thighs (Table 4). The intramuscular fat content decreased significantly in the older cockerels (2.72% vs. 3.80%, respectively, for the nine- and five-week-old male layer-type chickens, *p* < 0.01). The chickens slaughtered at nine weeks old also displayed higher ash (*p* < 0.001) and moisture (*p* < 0.05) contents.


**Table 4.** Proximate composition of the breast and thigh meat.

Significance: \* *p* < 0.05; \*\* *p* < 0.01; \*\*\* *p* < 0.001, ns—non-significant.

#### *3.3. Fatty Acid Composition*

The fatty acid composition of the meat of the male layer-type chickens differed between the two groups (Table 5).

**Table 5.** Fatty acid (% FAME) profile of the breast and thigh meat.



**Table 5.** *Cont.*

Significance: \* *p* < 0.05; \*\* *p* < 0.01; \*\*\* *p* < 0.001, ns—non-significant.

The changes in the percentage of the individual fatty acids affected by age did not follow the same pattern in breast and thigh. The breast meat showed significantly higher contents of C17:1, C18:0, C20:4n-6, C22:4n-6, and C22:5n-3 in the older chickens. On the other hand, the breast meat of these birds displayed a lower percentage of C16:1n-7, C17:0, C18:1n-9, C18:3n-3, and C20:5n-3. More fatty acids were affected by the age in the thigh meat. The male layer-type chickens, slaughtered at nine weeks of age had a significantly higher content of C14:0, C14:1, C15:0, C17:0, C18:2n-6, C18:3n-6, C20:2n-6, C20:4n-6, and C22:4n-6, while the percentages of C18:1n-9, C18:3n-3, and C20:5n-3 were lower. In regard to the total amounts of the fatty acids, generally, the meat from older chickens had lower MUFA (*p* < 0.01) and higher PUFA (*p* < 0.01), while the total percentage of SFA remained unaffected (Table 6). The values of n-6/n-3 ratio increased with age in both breast and thighs, while the P/S ratio was higher only in the thigh meat of the older chickens. The values of AI and TI were similar in both age groups.

**Table 6.** Lipid nutritional indices of the breast and thigh meat.


SFA: Saturated fatty acids; MUFA: monounsaturated fatty acids; PUFA: polyunsaturated fatty acids; P/S: poly- /saturated fatty acids; AI: atherogenic index; TI: thrombogenic index. Significance: \*\* *p* < 0.01; \*\*\* *p* < 0.001, ns—non-significant.

#### **4. Discussion**

Meat quality, including poultry meat, is very complex and might be affected by many factors [27]. This study illustrates the effect of age on the physical traits, chemical composition, and fatty acid profile with related lipid nutritional indices in the meat of male layer-type chickens. One of the physical traits that is known to be crucial for meat technological quality and safety is the ultimate pH [28]. The rapid pH decline postmortem and low values of pH are associated with PSE-like conditions, poor WHC, and functionality [29]. On the other hand, higher pH values can decrease the shelf life of the meat making it more susceptible to bacterial spoilage [30]. The pH also depends on the muscle type, which could explain the difference in this trait between the breast and the thigh meat. The values of pH that were observed in this study are within the normal range according to the limits set by Zhang and Barbut [31] for the five-week-old chickens. The effect of age on pH was reported in broilers [32,33] and also in spent hens [34]. In line with our results, these studies showed lower pH in the birds slaughtered at an older age. However, in a previous experiment [35], significantly increased pH values were reported in the breast and thigh meat of male layer-type chickens slaughtered at 12 weeks of age when compared to 5-week-old chicks. Contrary to us, Lichovníková et al. [36] did not find a difference in pH in the breast meat of male layer-type chickens, reporting values similar to ours (5.73–5.77); however, they were significantly higher than the pH in broiler meat.

Water holding capacity is very important for meat functional properties and determines the quality for further meat processing [37]. It was demonstrated that both pH and WHC correlate with meat color [38,39]. The results of the trial revealed a darker color of the breast and thigh meat in the older chickens, despite the lower pH in this age group, which is usually correlated with a lighter meat color [38]. On the other hand, the higher WHC in the older birds in breast meat corresponds to the lower L\*, since the lower percentage of free water decreases the light reflected from meat surface [40]. In addition to the lower L\* values determined in the male chickens slaughtered at the age of nine weeks, this age group also showed lower yellowness (b\*) in the breast and thigh meat. This is in line with the results in broilers [41] and in Da-Heng meat-type chickens [13].

Usually, the ageing of animals is associated with lower tenderness of meat, mainly due to a decrease in the collagen solubility [42]. In poultry, the results of the effect of age on the tenderness of meat are contradictory. In heavy lines of broilers aged 35–63d there was no effect of age at slaughter on the shear force [41]. Recently, it was found that the tenderness of breast and thigh meat of broilers slaughtered at 28 days of age was significantly higher when compared to those at 30, 32, and 34 days of age [43]. However, the authors did not observe differences between age 30 and 34 days. In slow growing chickens, Wang et al. [44], found a significant increase in the shear force of breast and thigh meat between age 63 d and 105 d. On the other hand, they found no effect of age on the shear force in native chickens reared under a semi-free-range system [45]. The values of the shear force reported in our study are similar to those of Choo et al. [46], when comparing egg-type males, white mini broilers, and commercial broilers (Ross 308). As shown in Table 3, the shear force values were higher in the breast, which can be attributed to the higher content of intramuscular fat [47]. Nevertheless, the values of the shear force that were measured in this experiment classify the meat as "very tender" (<3.62 kg) [48].

The proximate composition differs between the groups only for thigh meat. While the intramuscular fat decreased in the older chickens, the moisture and the ash content increased in these birds compared to the five-week-old birds. Decreased intramuscular fat and increased moisture with aging in chickens have been reported in previous studies with slow-growing [14] and male layer-type chickens [35]; however, results on the effect of the slaughtering age on this parameter in poultry have been rather inconsistent. Dal Bosco et al. [12] observed increased lipid content at an older age in the breast meat of various commercial chicken genotypes reared organically and little to no effect on the moisture. When comparing dual-purpose chickens with layer hybrids, Mueller et al. [49] observed decreased intramuscular fat in Lohmann Brown chickens with prolonged age, but not in the other studied hybrids. The intramuscular fat and its composition are important for organoleptic characteristics but also in the health value of meat and meat products [50]. In the present study, despite the different number of individual fatty acids affected by age in breast and thigh meat, in both meat cuts the MUFA was lower in the older birds. This was determined mostly by the decreased percentage of C18:1n-9 in the breast and thigh, and also corresponds with the decreased intramuscular fat in the meat, particularly in thighs. In a previous study on two slow-growing lines slaughtered at 9 and 18 weeks, a similar decrease in MUFA in the chickens slaughtered at an older age was observed [51]. An extensive review [52] has well outlined the useful properties of C18:1n-9 for immunomodulation, treatment, and prevention of cardiovascular and autoimmune diseases, metabolic disorders, skin injuries, and even certain types of cancer. Hence, the decrease in the percentage of C18:1n-9 and MUFA can be a disadvantage for the male layer-type chickens slaughtered at the age of nine weeks, compared to those slaughtered at five weeks old.

On the other hand, this was compensated by the lack of changes in the SFA but a significant increase in the PUFA. It should be noted that the increase in PUFA is mainly at the expense of n-6 PUFA. The major n-6 fatty acid in the poultry meat is C18:2n-6. A significant increase in this fatty acid was found in the thigh meat of the nine-week-old chickens. Since it is essential and derived exclusively from feed, its increase in the meat of older chickens might be explained by the higher feed consumption at this age compared to those at five weeks old [48]. The content of C18:2n-6 in the feed as presented in Table 2 is 55.31%. The increase in this fatty acid was also accompanied by increase in C20:4n-6, both in breast and thigh of the older chickens. In a recent study [53], it was recommended that for normal physiological function, the body requirement for optimal n-6/n-3 ratio is approximately 1–2:1. In this study, the n-6/n-3 ratio is much higher. This indicates a certain imbalance of the fatty acid profile of the male layer-type chickens in regard to PUFA that can be improved through feeding strategies or housing systems with access to pasture [54]. Meat has often been implicated in imbalanced fatty acid intake by consumers due to some meats naturally having a low PUFA to SFA ratio (P/S). Thus a P/S ratio of no less than 0.1 is recommended [55]. In the current study, in the older chickens the P/S ratio was higher than the set limit, ranging from 1.13–1.23 in breast muscle versus 1.00 to 1.20 in thighs.

#### **5. Conclusions**

The results of the study showed a significant effect of age in meat quality parameters of male layer-type chickens that was different in the different meat portions (breast and thigh). Generally, the chickens slaughtered at nine weeks of age displayed a lower pH and darker color. In regard to the nutrient components, older age was associated with a significant decrease in intramuscular fat in thighs and tended to diminish in breast meat. Its reduced content corresponded to the lower MUFA in the nine-week-old layer-type cockerels. On the other hand, the meat of older chickens was richer in PUFA, especially n-6, significantly increasing the n-6/n-3 ratio. As a whole, the meat of nine-week-old male layer-type chickens showed certain disadvantages in regard to the fatty acid profile which opens the possibilities for further studies on different feeding strategies or housing systems to improve this trait.

**Author Contributions:** Conceptualization, T.P., E.P., D.V.-V. and D.B.; methodology, E.P., T.P. and M.I.; formal analysis T.P. and E.P.; investigation, T.P., E.P., D.V.-V. and D.B.; resources, T.P.; data curation, T.P., E.P., M.I., S.D., N.K. and K.D.; writing—original draft preparation, T.P.; writing—T.P., review and editing, T.P., D.V.-V., D.B., S.D., N.K. and K.D., project administration, T.P.; funding acquisition, T.P. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the Bulgarian National Science Fund, Ministry of Education and Science in Bulgaria (Project INOVAMESPRO, Contract No KP-06-N56/10, 12 November 2021).

**Institutional Review Board Statement:** The experimental protocol used in this study was designed in compliance with the guidelines of the European and Bulgarian legislation regarding the protection of animals used for experimental and other scientific purposes (Directive 2010/63; EC, 2010–put into law in Bulgaria with Regulation 20/2012). The protocol was based on the permit for use of animals in experiments No. 227 of the Bulgarian Food Safety Agency (Statement No. 193 of the Bulgarian Animal Ethics Committee, prot.No.18/02.07.2020).

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

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

#### **References**


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