Feed costs alone represent over 70% of total production expenses in poultry farming, underscoring the critical importance of improving feed efficiency (Akintunde et al., 2024; Dinani et al., 2021). Efficient feed utilization is pivotal in reducing production costs, environmental impact and resource use (Rashid et al., 2020). Feed conversion ratio (FCR), defined as the amount of feed consumed per unit of body weight gain, remains a widely used metric for feed efficiency, where lower values signify higher efficiency (Singh et al., 2018). An alternative metric, Residual Feed Intake (RFI), introduced by Koch et al. (1963), accounts for the difference between observed and predicted feed intake, adjusted for maintenance and production. RFI is favored for its linear properties, avoiding biases associated with ratio-based measures. Berry and Crowley (2012) proposed residual intake and body weight gain (RIG), a novel trait combining feed consumption and growth performance, offering a comprehensive measure of feed efficiency.
       
The Colored Synthetic Male Line (CSML) is a specialized parent line of colored broilers developed in 1995 under the All India Coordinated Research Programme (AICRP) at the ICAR-Central Avian Research Institute (CARI), Izatnagar, India, a premier institute in poultry science. The CSML was established from a synthetic base population developed during 1989-1990. The origins of this population date back to 1972, when Cornish, Plymouth Rock and New Hampshire breeds were included in a genetic improvement programme. This programme initially focused on improving the 10^th week body weight through mass selection, which was later refined to emphasize body weights primarily at the 8^th and then to 6^th week of age (CARI Annual Report- 2017).  From 2001, selection of these lines focused for higher 5^th week body weight. So, as a consequence of long-term selection (near about 40 years), the population has reached the plateau for production with reduced genetic variability. Hence, the current study was carried out on the same population with the prime objective to improve the FCR for development of feed efficient line.
       
The CSML is notable for its exceptional growth performance, feed efficiency and adaptability. CSML exhibits a multi-colored plumage and brown eggshells, closely resembling native desi birds, making it highly desirable for crossbreeding to develop indigenous poultry. The mixed-sex average body weight of CSML at 5 weeks is 1222.63 g, with an FCR of 1.61 and reaches 1623.7 g by six weeks. Farmers highly favor CSML due to its rapid growth, superior feed efficiency and robust immunocompetence, which also command higher market prices. Studies by Thapa (2018) and Vispute (2021) highlight the superiority of CSML birds, with respect to broiler traits, immunological attributes and suitability for rural farming systems, further emphasizing its potential in genetic improvement programs.
               
Advances in selective breeding and dietary optimization have significantly enhanced feed efficiency, enabling increased production with reduced feed requirements (Yi et al., 2015). Improvements in FCR by 0.20-0.25 units can markedly lower production costs and enhance profitability. Molecular regulation of feed efficiency involves neural signals, mitochondrial function, hormonal pathways and nitrogen recycling. Gene expression studies indicate that upregulation of genes associated with efficient cellular energy production contributes to improved feed efficiency and muscle development (Bottje et al., 2012). Integrating genetic, molecular and management approaches to optimize feed efficiency is crucial for sustainable poultry production to meet the growing demand for eggs and meat in India. The current study aimed to integrate genetic basis of RFI with feed efficiency genes like CDx, SGLT1 and GHRL in Chicken.

Population structure and experimental design
 
The study was conducted at the ICAR- Central Avian Research Institute, Izatnagar, under the approval and guidelines of the Institutional Animal Ethics Committee. It involved 100 CSML broiler parent chicks of the 21^st generation, sourced from CARI’s germplasm unit. The chicks were reared under Completely Randomized Design for a 12-week period (April-June, 2024).
       
From 0 to 2 weeks, chicks were group-fed on deep litter, then housed individually in metal cages from 3 to 12 weeks of age. Two experimental diets-chicks (0-4 weeks) and grower (5-12 weeks) were formulated according to ICAR (Mandal, 2013) standards and essential micronutrients were incorporated to ensure balanced nutrition. A measured quantity of feed was offered daily in mash form, with care taken to minimize feed wastage. Daily feed intake and leftover feed were recorded to determine feed consumption, while body weight was measured biweekly using a digital weighing scale (±0.10 g). The body weight gain (BWG) and weekly feed consumption were then determined.
       
Feed efficiency traits were calculated at 2-week intervals (2-4 weeks, 4-6 weeks, 6-8 weeks, 8-10 weeks and 10-12 weeks) as well as for the overall study period (2-12 weeks). The FCR, RFI and Residual Body Weight Gain (RG) and RIG was calculated as follows.

 
RFI: The difference between the actual and expected feed intake was referred to as residual feed intake (based on regression). The RFI was calculated using linear regression, as suggested by Yi et al. (2015) for poultry, using the equations below:
 

RFI= TFI-{b0+b1MMW+b2BWG}

 
Where,
TFI= Total feed intake.
MMW= Metabolic mid weight=
[(Body weight at start +Body weight at end)/2]^0.75.
BWG= Body weight gain.
b0 , b1 , b2= Regression coefficients.
 

RG= WG-{b0 +b3 MMW+b4 TFI}

 
Where,
WG= Weight gain.
TFI= Total feed intake.
MMW= Metabolic Mid Weight= [(Body weight at start + Body weight at end)/2]^0.75.
b0= Intercept.
b3= Partial regression coefficients of MMW.
b4= Partial regression coefficient of TFI.
 
Residual intake and body weight gain (RIG)
 
It is a linear combination of RFI and RG.
 

RIG= RG - RFI

 
On the basis of the overall performance of all four feed efficiency traits (FCR, RFI, RG and RIG), birds were ranked and divided into two categories: low and high feed efficiency. Six birds from each category were selected for gene expression analysis.
 
Gene expression analysis
 
At 12 weeks of age, 200 mg jejunum tissue samples were aseptically collected and preserved in RNA Later (QIAGEN). RNA was extracted using the TRIzol method and quantified with a NanoDrop spectrophotometer. cDNA was synthesized using the HiGenoMB kit (Hi-Media). Gene-specific oligonucleotide primers are listed in Table 1. Real-time PCR was conducted on a Bio-Rad CFX96 system to assess the expression of feed efficiency-related genes, using GAPDH as the reference. Relative gene expression was analyzed with REST 2009 software.

Body weight
 
The data on BWG, FI and FCR, RFI, RG, RIG of colored broiler parent during 2-12 weeks was presented in the Table 2. Body weights in this study ranged from 150-296 g, 388-966 g, 877-1588 g, 923-1609 g, 1540-2134 g and 1619–2856 g at 2, 4, 6, 8, 10 and 12 weeks, respectively. Similar trends were reported by Ekka et al. (2016), with mean weights of 221.44±24.18 g, 663.42±36.50 g, 1359.89±84.19 g and 1993.55±35.72 g at 2, 4, 6 and 8 weeks in CSML birds. However, the weights observed in the present study were lower than those reported by Vispute (2021) and Thapa (2018) at all corresponding weeks of age. Yousefi et al. (2024) also reported higher weights in Cobb 500 birds-422.4 g, 1501.1 g and 2718.7 g at 2, 4 and 6 weeks. Notably, the 4^th week body weight in this study exceeded that of Arbor Acres (586 g) and Hubbard Classic (508 g) but was comparable to Cobb 500 (730 g) as reported by Nassar et al. (2019).


 
Body weight gain (BWG)
 
The BWG recorded at 2-week intervals showed that the average BWG during the 0-4-week period was higher than the 317 g reported by Thanabalan and Kiarie (2022), but the 5-12-week BWG was lower than their 1837 g value for the 5-19-week growing phase. Nassar et al., (2019) also reported higher BWG during weeks 3-4 in Broiler crossbreds. Compared to our study, Prakash et al., (2017) observed slightly higher BWG in weeks 4 and 5 (239 g and 328 g, respectively), while Ekka et al., (2016) reported higher BWG in CSML birds during weeks 2-4 (441.98 g), 4-6 (696.47 g) and 6-8 (633.66 g). Yousefi et al., (2024) noted substantially greater BWG during weeks 2-4 and 4-6 (1178.74 g and 1217.75 g) in Cobb 500 birds. Similarly, Thapa (2018) reported higher BWG during the 2-4 (572.58 g) and 4-6 week (732.18 g) periods. The cumulative BWG from 0-6 weeks in this study (1025.12 g) was also lower than Vispute (2021) 1266.91 g. These comparisons suggest relatively lower growth performance during the early growth phase in the present study.
 
Feed intake
 
Yousefi et al., (2024) reported feed intake between 900-1400 g in optimized broiler strains, consistent with the 942.91 g recorded at week 6 in this study. Compared to Vispute (2021), who observed 435.48 g (0-3 weeks) and 831.43 g (3-6 weeks), the current study recorded higher intake during weeks 2-4 (961.29 g) and 4-6 (1560.91 g), indicating better early-stage feed intake in CSML birds. At week 8, feed intake peaked at 1738.52 g, exceeding the 1250-1450 g range reported by Nassar et al., (2019), but slightly below the 1650 g noted by Ekka et al., (2016) for CSML birds. Thanabalan and Kiarie (2022) reported intake between 1000-1600 g in broiler breeders, aligning with the present findings, though the sharp peak at week 8 was not observed.
 
Feed efficiency traits
 
During the 2-4 and 4-6-week periods, birds showed relatively efficient FCRs of 2.32 and 2.60, which significantly deteriorated in later stages, rising to 5.16, 3.98 and 4.77 during the 6-8, 8-10 and 10-12 week period respectively. Prakash et al., (2017) reported a similar FCR of 2.32 in week 5, while Ekka et al., (2016) noted a more gradual increase from 1.75 (week 1) to 3.22 (week 8), contrasting with the abrupt spike in this study. Final FCRs reported by Yousefi et al. (2024) for Ross 308, Cobb 500 and Arian (1.67-2.2) and those by Thapa (2018) and Vispute (2021) (ranging from 1.45 to 1.95) were all better than values observed here. Overall, the current study revealed better and more variable FCRs, particularly in later stages, indicating lower feed efficiency likely influenced by genetic, environmental and managemental factors.
       
On the basis of data on FI, BWG and MMW for 2-12 weeks the RFI and RG were calculated using coefficient of regression equation (Supplementary Table 1 and 2). Significantly (P<0.05) better RFI was observed during the 2-4 weeks period and it declines as age progresses. The RG reported during 10-12 were better than other periods. There is no significant difference in RG, RIG reported in the current study. Significantly better (P<0.05) RIG was observed in 10-12-week period.




 
Ranking of CSML birds
 
Based on overall data (2-12 weeks) for FCR, RFI, RG and RIG, the birds were ranked and grouped into high feed efficient (top 20%) and low feed efficient (bottom 20%) using a combined index (Supplementary Table 3  and 4).




 
Production traits of selected birds during 2-12 weeks
 
In this study, HFE CSML birds showed higher body weight gain, lower feed intake and better FCR than LFE birds (Table 3), consistent with findings by Willems et al. (2013); Zampiga et al., (2021) attributed lower feed intake in efficient birds to better digestive efficiency and nutrient allocation for improved growth rate. As noted by Havenstein et al., (1994), FCR tends to worsen with age due to rising maintenance energy needs. Birds with low RFI values had significantly lower feed intake and better FCR than high RFI birds, making RFI a valuable selection trait. Selecting for low RFI not only reduces feed costs but also minimizes nitrogen waste and environmental impact (Bezerra et al., 2013; Fathi et al., 2021). When grouped by RG, high-efficiency birds gained more weight with less feed. Similarly, birds with high RIG values showed both lower feed intake and higher weight gain, highlighting the advantage of combining RFI and RG. RIG closely represents the ideal of low feed intake with high growth and has moderate correlations with body weight gain. However, excessive emphasis on growth in selection indices may negatively affect conformation traits like hip and leg structure, as well as footpad and breast skin health potentially impairing mobility and welfare.


 
Expression of feed efficiency-related genes in CSML birds
 
Differential expression analysis of growth-related genes in intestinal tissue of High and Low Feed Efficient (HFE and LFE) CSML birds revealed significant variations (Table 4).


       
SGLT1 is a primary mediator of glucose absorption in the small intestine (Hediger and Rhoads, 1994) and its higher expression indicates better assimilation efficiency. In this study, SGLT1 was upregulated in high feed efficiency (HFE) CSML birds, aligning with findings by Bottje et al., (2012); Bottje and Carstens (2009) and Mott et al., (2008), who linked elevated SGLT1 expression to improved nutrient uptake and growth.
       
Ghrelin plays a role in regulating growth hormone (GH) release (Nagaya et al., 2001) and exists in two molecular forms-acyl ghrelin (active) and des-acyl ghrelin (inactive)-in both gastric tissues and circulation (Ariyasu et al., 2001). Ghrelin has been identified in several species, including mammals and birds (Wada et al., 2003). Ghrelin-immunoreactive cells are found in parts of the chicken hypothalamus and administration of human ghrelin has been shown to elevate plasma GH levels in young chickens (Ahmed and Harvey, 2002). Ghrelin’s active form can suppress feed intake (Saito et al., 2002), consistent with our findings and previous studies showing a genetic link between RFI and feed intake (Prakash et al., 2017).
       
The CDx gene, which regulates intestinal development and maintenance (Geyra et al., 2002), showed lower expression in HFE birds. Similarly, Shinde et al., (2018) observed reduced CDx gene expression in chicks deprived of feed for 6, 24 and 36 hours, suggesting that intestinal stress diminishes CDx expression. This may indicate a more efficient gastrointestinal system or reduced reliance on CDx due to optimized gut function or microbiota in HFE birds.

Based on findings of the study it is confirmed that selecting parent birds based on a combination of multiple feed efficiency traits-such as RFI, FCR, RG and RIG is more efficient, both in terms of growth performance and economic viability, compared to selection based on a single trait alone. High feed-efficient CSML birds consistently exhibited enhanced feed efficiency traits, including lower feed intake, better FCR and higher body weight gain, leading to substantial cost savings in poultry production. Integrating these feed efficiency traits with molecular data, such as the up regulation of nutrient absorption-related genes and growth regulation genes provides a more extensive approach for improving selection and breeding strategies. This multi-trait selection approach not only optimizes growth performance but also reduces feed costs and environmental hazard by minimizing nitrogenous waste. Future research should explore genetic polymorphisms and gut microbiome influences the advance cost-effective and sustainable poultry production.

This study was financially supported by All India Coordinated Research Project on Poultry Breeding (AICRP-PB) with the necessary facilities. The authors would also wish to acknowledge the Director, ICAR-CARI for their kind support to carry out this research work.
 
Disclaimers
The views and conclusions expressed in this article are solely those of the authors and do not necessarily represent the views of their affiliated institutions. The authors are responsible for the accuracy and completeness of the information provided, but do not accept any liability for any direct or indirect losses resulting from the use of this content.
 
Informed consent
 
All animal procedures for experiments were approved by the Committee of Experimental Animal care and handling techniques were approved by the University of Animal Care Committee.

The authors declare that there are no conflicts of interest regarding the publication of this article. No funding or sponsorship influenced the design of the study, data collection, analysis, decision to publish, or preparation of the manuscript.