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Carcass Characteristics and Meat Quality of Betong Chicken Fed with Diets Supplemented with Crude Glycerin

Experiments were conducted to evaluate the effect of crude glycerin inclusion in the diets of Betong chicken on the characteristics of their carcasses, internal organs, meat quality, lipid oxidation, and fatty acid profiles. One hundred 1-day-old chicks were raised for 8 weeks. Subsequently, the birds were sexed based on their morphological features, and weighed. Forty-eight male chickens, with comparable body weights, were randomly allotted to receive any of the three experimental diets, containing 0, 50 or 100 g crude glycerin/kg feed, on an as fed basis until they were 20 weeks old. A total of 24 chickens were slaughtered and their carcass characteristics and meat quality were studied. Results showed that carcass characteristics and internal organ parameters were not affected by crude glycerin supplementation (P>0.05). After chilling for 24 h, pH of the meat decreased in all groups (P>0.05), while shear force and cooking loss were not affected (P>0.05). Furthermore, crude glycerin did not affect the parameters such as crude protein, ether extract, ash, moisture and proportions of different fatty acid contents of meat of the Betong chicken (P>0.05). However, breast meat color and lipid oxidation were influenced by crude glycerin in diet (P<0.05). These results suggest that crude glycerin can be used at concentrations up to 10% in Betong chicken diet from 8 to 20 weeks of age. Nevertheless, its effect on breast meat color and lipid oxidation need to be considered.


Introduction
In Thailand, native chicken meat is popular among those consumers who prefers tasty and chewy meat. These chickens are also thought to have better meat quality, with higher protein content and less fat compared to imported broiler chicken (Wattanachant et al., 2004;Makchumpon et al., 2015). Betong chicken, a popular indigenous breed from southern Thailand, is well known for its higher growth rate and greater carcass weight as compared to other native chickens (Gongruttananun and Chotesangasa, 2001). Betong chickens take 16 to 20 weeks to reach the optimum weight of 1.6 to 2 kg (Chatreewong and Waree, 2006). Nevertheless, the cost of production of Betong and other native chickens is high, due to the high cost of feed. Therefore, an alternative feed source is one of the approaches to reduce production cost.
Crude glycerin (CG) is the principal by-product of biodiesel production, and it is used in pharmaceutical and cosmetic industry, livestock feed production, and food industries. However, purification for human need is very costly and not feasible for small and medium scale biodiesel producers (Thompson and He, 2006), particularly in Southeast Asian countries. Thus, the use of CG for livestock feed is probably the cheapest alternative. Several studies have confirmed that CG could substitute corn by 5 to 10% in the chicken feed without compromising growth performance and carcass yield in broiler chicken (Cerrate et al., 2006;Sehu et al., 2012;Topal and Ozdogan, 2013;Urgnani et al., 2014;Freitas et al., 2017;Da Silva et al., 2017;Garcia et al., 2018;Legawa et al., 2018) and meat quail (Arif et al., 2017;Farrapo et al., 2017). Although utilization of CG in broiler nutrition is widely reported, limited information is available regarding the effect of CG on meat quality, particularly in indigenous chicken breeds. Hence, the aim of the present study was to evaluate the effect of CG incorporation in the feed on carcass characteristics and meat quality of Betong chicken. Songkla University. The chemical composition of CG was previously evaluated by Sopian et al. (2018). The feed was based on a corn-soybean meal with 19% CP and 3,000 kcal/kg ME (in mash form) formulated according to Nguyen and Bunchasak (2010) recommendation. All experimental procedures were approved by Institutional Animal Care and Use Committee, Prince of Songkla University (MOE 0521.11/484). The feed composition and fatty acid profile are shown in Table 1 and Table S1 (Supplemental data), respectively.

Carcass Characteristics and Meat Quality
At the end of the rearing period (20 week), from each treatment group, eight chickens with body weight closest to the median, were starved overnight, weighed, and slaughtered according to the Islamic procedure of Thai Agricultural Standard 8400-20078400- (ACFS, 2007. Subsequently, carcass and internal organs were weighed, and chilled carcass and slaughter weights were compared. The pH values and meat color were the mean of the three measurements, at the right breast muscle. The pH was measured using a pH meter (Seven2Go, Mettler-Toledo, Switzerland) at 45 min (pH45 min) and 24 h (pH24 h) post-slaughter. The color was evaluated using a colorimeter (Konica Minolta, Japan) and reported in the complete International Commission on Illumination (CIE) system color for the lightness (L*), redness (a*), and yellowness (b*). The breast meat samples were immediately transferred to and stored at −20℃ for chemical and fatty acid analyses. To determine cooking loss, the left part of each breast muscle sample was cut into small pieces (2.0×1.0×0.5 cm), put in a sealed plastic bag and cooked in a water bath at 80℃ for 10 min, cooled and weighed. The weight before and after cooking were compared for each sample and presented as percent cooking loss. The same meat samples were subjected to shear-force analysis by Texture Analyzer (TA-XT2i, Texture expert Vision 1.17, Stable Micro System, Godalming, Surrey, UK). The operating parameters consisted of a cross-head speed of 2 mm/s and a 50-kg load cell (Wattanachant et al., 2004). The proximate analysis (dry matter, crude protein (CP), ether extract (EE), and ash) was performed according to AOAC (2006). The measurements for each sample were performed in duplicate.
Thiobarbituric acid-reactive substances (TBARS), a byproduct of lipid peroxidation, were estimated following the method described by Buege and Aust (1978). Briefly, 1.5 g of ground meat samples were mixed with 7.5 ml of TBA reagent (0.375% thiobarbituric acid, 15% trichloroacetic acid, and 0.25 N hydrochloric acid) and homogenized at 3000 rpm for 2 min (IKA-T18 Ultra Turrax, IKA ® -werke GmbH & Co. KG, Staufen, Germany). Subsequently, the samples were heated in a boiling water bath for 10 min and cooled under running tap water. These were then centrifuged for 20 min at 3600×g (Hettich-Universal-320R, Andreas Hettich GmbH & Co. KG, Tuttlingen, Germany).
The absorbance of supernatants were measured at 532 nm using UV-spectrophotometer (Evolution 260 Bio, Thermo Fisher Scientific, Waltham, MA, USA) against blank. The results were compared to a standard curve of absorbance of 1,1,3,3-tetramethoxypropane and expressed as mg malonaldehyde (MDA) per kg of meat.
Total lipids were extracted with a mixture of chloroform and methanol (2:1, v/v) according to the method of Folch et al. (1957) and methylated as described by Metcalfe et al. (1966). The fatty acid methyl esters were quantified by gas chromatography (Hewlett-Packard 7890A; Agilent Technologies, Santa Clara, CA) equipped with a flame-ionization detector and a Varian SP-2560 capillary column (100 m length×0.25 mm internal diameter×0.2 μm film thickness). The initial column temperature was 70℃ held for 4 min, increased to 175℃ (13 C/min) held for 27 min, increased to 215℃ (4 C/min) held for 17 min and increased to 240℃ (4℃/min) held for 10 min. Helium was used as the carrier gas at a constant flow rate of 1 mL/min. The temperatures of the injector and detector were 240℃ and 250℃, respectively.

Data Analysis
Individual birds were used as an experimental unit and the data were analyzed using one-way ANOVA with SPSS version 16 (SPSS, 2007). The significance of differences were analyzed further with Tukey test. The statistical significance level was set at P＜0.05 for all analyses. Sopian et al.: Glycerin and Betong Chicken Meat Quality 293

Results
The effect of treatments on live weight, chilled carcass, relative organ weight (breast, drumsticks with thighs, wings, fillet, liver, heart, gizzard, and abdominal fat), and carcass composition (meat, bone, skin, and fat) is shown in Table 2. CG supplementation had no effect (P＞0.05) on all these parameters.
As shown in Table 3, the lowest pH45 min (pH 6.11) was observed (P＜0.05) in samples from birds fed with 5% CG. After 24 h chilling, pH decreased in samples from all treatments and showed no significant difference (P＞0.05) among the three groups. Shear force and cooking loss were not affected (P＞0.05) by CG in the diet. However, breast meat color in term of yellowness, and redness were influenced by CG inclusion (P＜0.05) but not the lightness (P＞0.05).
The highest MDA values, a marker of lipid oxidation, of meat samples were found in CG10 group followed by CG5 and CG0 groups (P＜0.01). The EE, which is the measure of total crude fat, from breast meat of the birds fed with CG had numerically higher values (CG5: 2.01%, CG10: 2.23%) than those fed with control diet (CG0: 1.91%), but these were not statistically significant. The results also showed that CG did not affect CP, ash and moisture contents of the meat (P＞ 0.05). Moreover, the fatty acid composition of the breast meat was not altered by CG except for the stearic acid content, which reduced significantly.

Discussion
The carcass characteristics and parameters of internal organs were not affected by CG supplementation. This could be attributed to the energy content in the CG and proved that CG was suitable as energy source for Betong chicken breed. The CG used in the current study, was previously evaluated to minimize the energy overestimation (Sopian et al., 2018). Therefore, these results were expected, as the feed was formulated to be isonitrogenous and isocaloric diet. These findings are in agreement with the previous studies which have reported that CG inclusion up to 10% had no negative effect on carcass traits (Sehu et al., 2012;Freitas et al., 2017) and on internal organs (Topal and Ozdogan, 2013). Similarly, Arif et al. (2017) and Farrapo et al. (2017) also found that CG did not alter the carcass characteristics and relative organ weight of quail. In contrast, Cerrate et al. (2006) reported several adverse effects on carcass yield and internal organs when 10% CG was used. Legawa et al. (2018) found increased abdominal fat in broilers fed with 5% CG. However, the former authors stressed that reduced carcass yield was related to a feed flow problem that led to reduced feed intake; contrastingly, the latter authors explained that the high abdominal fat content was due to a high content of fat in their CG. Therefore, it is important to evaluate the nutrient content of CG before it is mixed into the feed.
In the present study, the meat quality was not affected by experimental diets, except the pH45 min and meat color. However, pH24 h was not significantly different among samples from different groups. These values were similar to those reported by Bungsrisawat et al. (2018) in Betong chicken (KU line). Shear force and cooking loss were also not affected by CG. These results were consistent with the study using broiler chickens (Urgnani et al., 2014;Garcia et al., 2018;Legawa et al., 2018). However, in the present study, shear force values were greater and cooking loss values were lesser than those reported for 16-week-old Thai indigenous chicken (Wattanachant et al., 2004), and these might be related to the differences in the strain and the age of chickens used in these two studies.
The lower values of yellowness and redness of the meat were observed in CG-fed birds. The reduced meat color may simply be due to the lower corn content in these formulations (5.56% and 10.4% in CG5 and CG10, respectively), as compared to the basal diet. Peng et al. (2017) have reported that poultry diets have a great impact on the color of meat. It is well known that corn contains xanthophylls and other carotenoids that are the primary sources of pigments. This result was consistent with previous findings (Garcia et al., 2018) that in broiler chicken thigh meat yellowness value Journal of Poultry Science, 57 (4) 294 decreased linearly, with increasing levels of mixed semipurified glycerin in diet. However, the same authors also found that breast meat color was not affected by the dietary glycerin content. Similarly, Urgnani et al. (2014) and Legawa et al. (2018) also did not find any significant change in the color of the breast muscles of broilers due to CG in their feed. These observations suggest that inconsistencies may arise because of differences in the genotypes of the birds, as well as difference in the source and levels of CG used in these studies.
To evaluate the oxidative stability of the meat samples, amount of TBARS was measured. The results suggested that compared to basic diet, inclusion of CG in the feed promoted significantly higher levels of MDA in the breast meat, which has higher fat and unsaturated fatty acid contents. Present results support Kovalík et al. (2018), who suggested that the susceptibility of meat to lipid oxidation increases with increasing unsaturated fatty acid contents, and Legawa et al. (2018), who concluded that level of MDA is directly dependent on the amount of fat in the meat. However, the oxidative stability of the meat can be enhanced by including antioxidants in the feed (Marzoni et al., 2014). No significant differences in the other chemical composition were observed among different experimental groups in the present study. The study by Silva et al. (2017) also confirmed that CG in diet did not affect EE, CP, ash and moisture content of the breast meat. Conversely, there are other studies which showed a higher EE content in breast meat (Legawa et al., 2018) and lower EE in thigh meat (Topal and Ozdogan, 2013) of broiler chicken, fed with CG. Authors of these reports proposed that these results were related to the EE content of the diets. However, the difference in the levels of EE in the different experimental diets in this study was insufficient to make a significant effect on the chemical composition of the meat. The breast meat CP was reported to be between 23.11% and 25.13% and EE content between 1.69% and 1.84% in Betong chicken (Makchumpon et al., 2015;Ritchoo et al., 2019). In this study, the CP content was seen to be within that range, but EE was slightly higher, probably due to difference in the age of the birds at slaughter and their diets.
Furthermore, although saturated fatty acid composition of the chicken diets differed significantly, only stearic acid (C18:0) content of the breast meat decreased significantly. The proportions of other saturated, monounsaturated and polyunsaturated fatty acids were not significantly affected by CG. Nevertheless, the saturated fatty acid contents reduced and the unsaturated fatty acid contents increased in the meat of Betong chicken fed with CG. Erol et al. (2009) reported significant decline in the stearic acid content in the egg yolk of quails maintained on feeds supplemented with glycerol. Urgnani et al. (2014) also described that stearic acid content reduced linearly in the thigh meat of broilers, fed with increasing amounts of crude vegetable glycerin. However, these authors also observed a linear increase in the saturated fatty acids (myristic-, palmitic-and stearic-acids) in chicken fed with semi-purified vegetable glycerin. In contrast, Garcia et al. (2018) reported an increasing and decreasing quadratic effect in monounsaturated and polyunsaturated fatty acids, respectively in broilers fed with mixed semi-purified glycerin. The variations in fatty acid profiles in these studies may be related to the sources and levels of CG inclusion in diet and fatty acid profiles of the CG, as well as that of other ingredients of the diet.
In conclusion, the CG can be used at concentrations up to 10% in the diet of Betong chickens at 8 to 20 weeks of age, without any adverse effects on carcass characteristic, chemical composition, and meat quality. However, its effect on breast meat color and lipid oxidation need to be considered.