Effect on growth performance, carcass traits, and myostatin gene expression in Aseel chicken fed varied levels of dietary protein in isocaloric energy diets

A study was conducted to assess the effect of feeding different crude protein (CP) levels with isocaloric metabolizable energy (ME) diets on growth performance, carcass traits, and myostatin (MSTN) gene expression of Aseel chicken during 0 to 16 weeks of age. A total of two hundred and ten day-old Aseel chickens were randomly allotted to seven dietary treatment groups. Each group had thirty chicks distributed into three replicates of ten chicks in each. Experimental diets were formulated to have varying levels of CP, viz. 18.5, 19.0, 19.5, 20.0, 20.5, 21.0, and 21.5%, with isocaloric energy of 2800 kcal ME/kg diets of mash feed fed to birds in a completely randomized design. Different CP levels had a significant effect (P < 0.05) on the body weight gain (BWG) of Aseel chicken. At the end of 16 weeks of age, the group fed 21% CP gained 223.53 g more than the lowest CP (18.5%)–fed group. The different CP levels did not significantly (P > 0.05) influenced the feed intake of all treatment groups, but numerically highest feed intake was observed in the lowest CP (18.5%)–fed group. However, significant differences in feed efficiency (FE) appeared from the 13th week only with the 21.0% CP–fed group showing the best FE until the 16th week (3.86 to 4.06). The maximum dressing % (70.61) was observed by the 21% CP–fed group. The CP 21% diet down-regulated the MSTN gene expression in breast muscle tissue to 0.07 folds when compared to the diet of CP 20%. The best economical coordinates for maximum performance for Aseel chicken appeared to be CP of 21% and ME of 2800 kcal/kg to achieve the best FE of 3.86 at the earliest age of 13 weeks. In conclusion, 21% CP in an isocaloric diet of 2800 kcal ME/kg, in Aseel chickens, would be optimum to improve the growth performance at maximum in terms of BWG and FE up to 16 weeks of age.


Introduction
Native chickens are imperative poultry species that form a vital part of the farming systems in rural areas of many developing and tropical countries like India. Native chickens are vital for the livelihood of many farmers having limited resources (FAO 2013). In India, there is great scope for native chicken rearing to bridge the gap between the supply and demand of poultry meat (Manoj Kumar et al. 2019). According to FAO projections, by 2050, there will be an increase in demand for meat and egg protein by 73% compared to 2011 (Dridi et al. 2015). With 25% of the total poultry population being native chickens in India, it hence implies that the productivity of native chickens for food security and adequate nutrition needs improvement for the increasing population.
Native chickens are being reared as rural backyard poultry and their genetic potential has not been fully explored (Padhi, 2016). Systematic studies to determine the precise nutrient requirements of native chicken breeds are warranted (Haunshi and Rajkumar, 2020). Aseel, the predominant indigenous poultry breed, is being widely grown in India, for meat purposes, in deep litter, with renewed interest in the consumption of native chickens for the delicacy of meat. A few earlier detailed works are found on Aseel's performance; however, they were either in the juvenile stage of 0-8 weeks (Haunshi, et al. 2012), 0-12 weeks (Mandal et al. 2016) or laying performance (Haunshi, et al. 2011 andMandal et al. 2016).
The molecular basis studies seem to be a promising way to understand the dietary nutrient utilization and even may help to suggest recommendations for diet formulation. The myostatin (MSTN) gene, a member of the transforming growth factor-beta superfamily, is a negative regulator of skeletal muscle growth (Xianghai et al. 2007). The elevations of MSTN expression in broilers fed on protein-reduced diets are reflected in decreased muscle yield and have a negative influence on muscle hypertrophy and hyperplasia (Yang et al. 2013). Liu et al. (2016) demonstrated that a slow-growing Wuding chicken has a greater breast muscle myostatin expression than fast-growing broilers. Thus, the MSTN gene has an important role in the regulation of growth performance and muscle mass in chickens. The current study is aimed to assess the growth performance, carcass traits, and expression profile of the MSTN gene in the Aseel chicken up to 16 weeks of age in response to varying dietary protein levels with a hypothesis that high plane of nutrition positively enhances the growth while downregulate the MSTN gene.

Location of the experiment
The present experiment was carried out from August to November in an experimental poultry shed of the Department of Animal Nutrition, Veterinary College and Research Institute, Namakkal, Tamil Nadu Veterinary and Animal Sciences University (TANUVAS), Tamil Nadu, India (78°9′41.11′′E, 11°9′41′′N). The mean maximum temperature was observed from 35.3 to 31.6°C during the experiment period.

Experimental birds, diets, and design
A total of 210 one-day-old Aseel breed chickens were wingbanded, weighed individually, and randomly distributed into seven groups. Each group had 30 chicks distributed into three replicates with ten chicks of equal sex in each. It was noted that there was no significant difference in initial body weight among the seven treatment groups. Chicks were reared in a deep litter system from 0 to 16 weeks of age. Experimental mash form of maize-soybean meal-based diets was formulated with seven levels of CP (18.5%, 19.0%, 19.5%, 20.0%, 20.5%, 21.0%, and 21.5%) with an isocaloric energy level of 2800 kcal ME/kg diets (Table 1) fed to the birds belonging to seven treatment groups in a completely randomized design. Feed and water were provided ad libitum for all treatment groups during the experimental period. Standard management and health care practices were followed uniformly for all the seven treatment groups.
Mortality of birds was documented as and when it occurred. The present study was conducted with the approval of the Institutional Animal Ethics Committee.

Growth performance
Data on body weights were recorded on day-old chickens, at a weekly interval of individual birds, and body weight gain was calculated for each treatment group. Weekly feed intake was recorded from 1 to 16 weeks of age and feed efficiency (gain per gram of feed intake) and protein efficiency ratio (gain per gram of protein intake) were also derived for each treatment group. Weekly body weight gain was plotted against the corresponding feed efficiency to arrive at the coordinates to identify the best economic traits (BWG and FE) of Aseel chickens.

Carcass traits
At the end of 16 weeks, four birds of equal sex ratio (two males and two females) close to the mean body weight of each pen were selected (twelve birds per treatment group) and slaughtered after 4 h of feed deprivation to evaluate carcass traits. Pre-slaughter weight, weight after bleeding, eviscerated carcass weight, and weight of the liver, heart, and abdominal fat were measured and expressed as percent live body weight.

Quantitative real-time PCR analysis
At the end of 16 weeks of age, breast muscle samples from six birds of equal sex ratio per treatment group were harvested for gene expression studies. The work surfaces, collection area, and equipment were decontaminated with RNase @ ZAP solution. Collected samples were immediately snap-frozen in liquid nitrogen and stored at − 80°C for subsequent analysis. The total RNA was extracted from breast muscle samples by using the TRIzol @ method (Rio et al. 2010). The RNA purity and concentrations were determined at 260/280 nm using a nanodrop spectrophotometer (Thermo Fisher Scientific, USA) and the RNA integrity was assessed by non-denaturing agarose gel electrophoresis (Wilfinger et al. 1997). The first-strand cDNA was synthesized using the iScript™ cDNA synthesis kit (Bio-Rad, USA) according to the manufacturer's protocol.
The real-time (RT) PCR was performed using the SYBR Green method. Real-time PCR primers (Table 2) were designed to amplify the target gene (MSTN) relative to endogenous control of the housekeeping gene (GAPDH). The reaction mixture (20 µL) contains 10 µL SYBR Green Supermix (Bio-Rad, USA), 0.2 µM concentration of 3′ and 5′ gene-specific primers, and 2.0 µL of cDNA template. The real-time thermal cycler (Illumina Real-time Machine, USA) was performed following the cycling conditions (5 min at 95°C, then 30 cycles of 95°C for 30 s, annealing temperature 60°C for 30 s, and extension for 1 min at 72°C).  The relative gene expression was performed between the target gene (MSTN) after normalization with the housekeeping gene (GAPDH). The Cq (cycle quantification) / Ct (Cycle threshold) values were recorded for each gene expression assayed in qRT-PCR using the SYBR green chemistry. All the Cq values were the mean of six samples tested. The expression profile of the gene (fold increase/decrease) was calculated using the formula stated by Pfaffl (2001). The comparison of the effect of increase and decrease in CP levels was made with the basal protein of 20.0%.

Statistical analysis
Data collected from various parameters were subjected to analysis of variance procedures appropriate for a completely randomized design using the SPSS software (version 26.0). The means are presented with their standard error of means and the means of different experimental groups were tested for statistical significance by Duncan's multiple range test (Duncan 1955). Correlation coefficients were analyzed using bivariate correlation analysis between gene expression and body weight gain, carcass weight, and dressing %.

Body weight gain
The effect of varying protein diets on weekly mean cumulative body weight gain (0-4, 0-8, 0-12, and 0-16 weeks) is presented in Table 3. The BWG (g) in Aseel chickens at the end of 16 weeks of the feeding experiment was 1061.32, 1135.17, 1121.51, 1201.18, 1178.73, 1284.85, and 1210.63 for 18.5, 19.0 , 19.5, 20.0, 20.5, 21.0, and 21.5% CP-fed groups, respectively. Though the difference in BWG was seen among the groups particularly the male and female Aseel chicken, it was notable after the 6th week onwards, and it was significant from 15 weeks only in favor of higher CP levels (above 20%). It amounted to a maximum body weight gain of 223.53g by the group with CP of 21% over the group with CP of 18.5% (a relative increase of 17.40%); the value was significantly different over all the lower CP groups except for the mid-value group of 20.0% CP. It could also be seen from the average daily gain (ADG) data (Table 3) that the 21.0% CP group recorded a maximum ADG of 11.47g.

Feed intake
The effect of varying protein diets on mean cumulative feed intake of Aseel chicken (0-4, 0-8, 0-12, and 0-16 weeks) is presented in Table 3. The feed intake among all the treatment groups did not differ significantly (P > 0.05) but considering the length of period of the experiment studied, the difference in average feed intake per bird per day ranged narrow (45.60 to 48.60g). Moreover, there was no specific trend between the quantum of the feed intake and the CP levels attempted in the present study in Aseel chickens.

Feed efficiency and protein efficiency ratio
The effect of varying protein diets on cumulative mean FE of Aseel chicken (0-4, 0-8, 0-12, and 0-16 weeks) is presented in Table 3. A significant differences (P < 0.05) appeared from the 13th week only with the 21.0% CP-fed group showing the best FE until the 16th week (3.86 to 4.06).
In the present study, the protein efficiency ratio (PER) at the end of the 16 weeks was significantly (P < 0.001) influenced by changing the CP level. It showed an increasing trend with an increase in protein level, up to 21% (1.05 to 1.25). However, it was evident that 21.5% CP diet did not improve the FE and PER values in native chickens with iso-energy of 2800 kcal ME/kg (FE, 4.48 and PER, 1.04).

Economical coordinates
The best FE of 3.86 was achieved with a maximum body weight gain (887.61 g, 21% CP) at the end of the 13th week which were the best coordinates in terms of CP (21.0%) and FE for Aseel chickens (Fig. 1). Though the 21% CP-fed group continued to perform best until the end of the experimental period of 16 weeks, FE increased from 3.86 to 4.06. Hence, the best economical coordinates for the excellent performance of the Aseel type of native chicken appeared to be CP of 21% among various levels attempted in the present trial on a constant ME value of 2800 kcal/kg, by the end of the 13th week, which could be a valid finding for marketing of birds to get maximum returns.

Carcass traits
The effects of varying protein diets on carcass traits of Aseel chicken are presented in Table 4. The dressing percent values were significantly (P < 0.05) affected due to varied CP diets. The highest dressing percent was found in the higher CP diet-fed groups (21 and 21.5%). The weights of other carcass traits were not influenced significantly (P > 0.05) due to varied protein diets in Aseel chickens.

Relative expression of myostatin mRNA
The relative fold changes in myostatin (MSTN) mRNA expression in the breast muscle tissue of Aseel chicken are presented in Table 5 and Fig. 2. The higher CP (21%) diet down-regulated the MSTN gene expression in breast muscle tissue to 0.07 folds when compared to the 20% CP diet. It is noticed that the relative expression of MSTN mRNA was down-regulated in high protein-fed groups whereas low protein-fed groups were up-regulated.

Correlation between body weight gain, carcass weight, dressing percent, and myostatin mRNA expression
The results of the study on the correlation between the MSTN gene expression and BWG, carcass weight, and dressing % are presented in Table 6. The expression of the MSTN gene was negatively correlated with BWG, carcass weight, and dressing percent of Aseel chickens fed with varied dietary protein diets (0-16 weeks of age). the protein level above the recommended level (20%) for commercial layer chicks increased body weight gain linearly up to the CP level of 21.0% but not beyond. The increase in body weight could be due to better assimilation of protein from the diet utilizing the available energy that was kept constant (isocaloric) at 2800 kcal/kg of feed. The varying crude protein diets were formulated by changing the levels of soybean meal. At the same time, the level of essential digestible amino acids (lysine and methionine) was maintained equally in all treatment groups (Table 1). However, the birds registered a decrease in body weight gain in the 21.5% CP group (1210.63 g) by a margin of 74.22 g (5.77%) over the CP 21.0% group. Possible reasoning for the reduction in the body weight gain in the group of birds with the highest CP (21.5%) might be due to unutilized excess protein due to the non-availability of energy for this biochemical process as the ME used in this experiment was fixed (2800 kcal/kg of diet) and might be also due to sparing of energy to the process of excretion of nitrogenous compounds (NRC 1994 andBarzegar, et.al., 2019). As observed in an 8-week

Discussion
Dietary protein levels influenced the ability of the native chicken considerably in moderating their body weight gain. It could be seen from this experiment that an increment in growth study on Aseel chicken where Haunshi et al. (2012) reported no significant increase in the BWG by increasing the CP level from 16 to 20% pointing to the slow-growing character of Aseel chickens (10 g/day), this slow-growing character of Aseel could be another possible reason for the birds not showing further weight gain upon extending the protein level beyond 21.0%, in our study. However, the present work showed better BWG at a higher CP level (21.0%) with 11.47 g/day of daily gain, slightly higher than the level reported by Haunshi et al. (2012). Miah et al. (2016) used a single CP level of 23% for an indigenous chick variety with two energy levels of 2800 and 3000 kcal/kg up to 14 weeks and reported a body weight of 758 and 768 g respectively with 7.74 and 7.84 of ADG (g) which were lower than the values recorded in our study. Perween et al. (2016) found 21.0% CP, rather than 19.0%, had a positive effect on body weight (1403.60 g) in fast-growing dual-purpose breed Vanaraja at 8th week which required higher energy of 3000 kcal ME/kg. In another study with an improved chicken Rajasri, Deepak et al. (2017) reported higher BWG when dietary CP was increased from 16 to 20.0% in 8 weeks of age which had similar ME (2800 kcal/kg) followed in our dietary treatments. However, Batool et al. (2018) revealed that Mushki Aseel chicken required 17.29% CP with 1.30% lysine and 2760 kcal ME/kg to show an improved growth performance in body weight gain (372.15 g) from 0 to 6 weeks of age. Though there was variation in the body weight gain in different native chickens reported earlier, it could be noted that 21.0% CP was required for maximum performance in terms of body weight gain in the Aseel type of chicken with 2800 kcal/kg for meat purposes when reared intensively.
The group with the highest CP of 21.5% consumed numerically higher feed (5427.30 g) but resulted in lower body weight gain which may be clarified by a possible explanation that excretion of excess protein is an energyconsuming metabolism which might be a possible reason for the birds to consume extra feed in an attempt to satisfy the energy requirement. Thus, the reason for the lower body weight gain at the end of the experiment could be the direct result of an imbalance in the protein:energy ratio in the feed. However, there was no significant difference in feed intake among all the treatment groups. Such non-significant difference in feed intake due to varying protein levels was also reported earlier in native chickens (Elangovan et al. 2004;Haunshi et al. 2012;Chandra Deo et al. 2014;Liu et al. 2014;Hidayat et al. 2016 andKamble et al. 2019) experimented with protein levels from 12 to 20%. However, Perween et al. (2016) observed a significantly lowered feed intake due to a change in dietary protein levels with 19.0 than 17.0% CP in Vanaraja chicken.
As FE is a mere reflection of the ratio of feed intake (g) to body weight gain (g), it was clearly seen that a decrease in CP below the basal diet of 20.0% resulted in reduced FE (4.33-4.42) while the opposite was true in high CP-fed groups (4.33-4.06); the positive effect was observed only up to CP of 21.0% (4.06). Earlier, Mandal et al. (2016) also reported an efficient feed conversion ratio of 3.55, 3.69, and 3.88 when fed with 20, 18, and 16% CP diet, respectively, to Aseel chicks (0-8 weeks). However, Haunshi et al. (2012) observed protein efficiency was better at 16% followed by 18 and 20% CP during the juvenile phase (0-8 weeks) of Aseel chicks.
In the present study, the PER, at end of the 16 weeks, in Aseel chicken was significantly (P < 0.001) influenced by changing the protein level. It showed an increasing trend with an increase in protein level, but the increase was recorded only up to 21% level (1.05 to 1.25). It was evident that 21.5% CP diet did not improve the FE and PER values in native chickens with 2800 kcal ME/kg (FE, 4.48 and PER, 1.04).
Dressing percent was influenced significantly by dietary protein levels in Aseel chicken. The maximum dressing percent (70.61) was observed in the 21% CP-fed group and the lowest (68.07) was observed in the 19.0% CP group. Research works on the influence of protein level on the dressing yield were scanty in indigenous breeds. However, Singh and Pathak (2016) reported a lower dressing percent (61.97) in the Aseel birds at the age of 6 weeks.
There was not much influence on the protein content of feed on organ and abdominal fat percent. Interestingly, Rabie et al. (2017) reported a significant decrease in abdominal fat percent with increased dietary protein levels (from 18 to 22%) in broiler chickens which are fast-growing meat birds with 57-60 g ADG which happens to be more than 5 times of indigenous breed's ADG.
In the current study, the MSTN gene expression level is inversely proportional to the BWG, carcass weight, and dressing percent in Aseel chickens, as seen from the trend line (Fig. 2). It could be noted that the relative expression of the MSTN gene was higher in the low protein groups and lower in high protein groups. Yang et al. (2009) recorded elevated MSTN gene expression and decreased muscle yield in broilers fed with reduced protein (23 to 18.4% CP) diets and reduced energy (13.4 to 12 MJ ME/kg) levels as well. It might be explained that decreased muscle yield was reflected as a result of the negative influence on muscle hypertrophy and hyperplasia due to protein-reduced diets. The highest body weight gain and lowest MSTN gene expression were observed in the 21.0% protein-fed group which is perfectly corroborating the expression pattern of the MSTN gene. A similar pattern of the MSTN gene expression was documented in the biceps femoris muscle of Indian colored broiler chickens (Saxena et al. 2007) and broilers (Saxena et al., 2020) in response to the dietary protein and energy changes. Likewise, Bhattacharya and Chatterjee (2013) and Yang et al. (2013) also revealed a negative correlation between muscle mass and elevation of MSTN gene expression in broilers.

Conclusion
The results of the present study revealed that varying dietary protein levels had a significant impact on BWG, FE, and PER in Aseel chickens. An increase of CP from 20.0 to 21.0%, not beyond that, on iso-calorie ME value of 2800 kcal/kg feed resulted in an absolute positive change of 6.97%, 8.14%, and 10.63% in BWG, FE, and PER values, respectively. The same combination of CP and ME revealed an economic performance in Aseel chickens with the best FE of 3.86 for a body weight gain of 887.61 g at an early age of 13 weeks. It was documented that the MSTN gene was down-regulated when the dietary protein level was increased to 21.0% from 18.5%. Based on the results obtained from the present study, it would be recommended that 21% CP in an isocaloric diet of 2800 kcal ME/kg, in Aseel chickens, is optimum to improve the growth performance to the maximum in terms of BWG and FE with maximum PER up to 16 weeks of age, and the economic coordinates strikingly suggested that the best returns could occur as early as 13 weeks. Further studies are recommended to ascertain the performance of Aseel chickens by varying dietary metabolizable energy and amino acid levels.