Experimental protocols were approved by the Institutional Animal Care and Use Committee of the National Institute of Animal Science, Rural Development Administration, Korea (NIAS 2021-0519). In this study, WMD1 breeder hens (Figure 1), a crossbreed between Red-Brown KNC males and Korean Rhode Island Red females, were raised from 4 to 16 weeks of age. A total of 240 four-week-old WMD1 breeder pullets with an average weight of 215±5 g were assigned into 20 floor cages (12 birds per cage, 120 cm×60 cm×40 cm; 600 cm² per bird) under environmentally controlled room conditions. Experimental diets were formulated for broiler breeder pullets through starter phase (4 to 6 weeks of age) and grower phase (7 to 16 weeks of age). Dietary groups consisted of four levels: standard metabolizable energy (SME), SME-200, SME-100, and SME+100) at all phases. The SME diet was formulated to meet nutrient requirements (2,800 kcal AME_n/kg) of broiler breeder pullets as described in the Korean Feeding Standard for Poultry in the National Institute of Animal Science [18]. SME-200, SME-100, and SME+100 diets were formulated with 2,600, 2,700, and 2,900 kcal AME_n/kg, respectively. The other nutrient contents, including crude protein (CP), calcium (Ca), and available phosphorus (AP), were formulated to be consistent across all treatments as recommended by Korean Feeding Standard for Poultry in the National Institute of Animal Science [18]. Specifically, diets for 4 to 6-week-old birds contained CP 18.5%, Ca 1.04%, and AP 0.241%, while diets for 7 to 16-week-old birds maintained CP 15.5%, Ca 1.01%, and AP 0.220% (Table 1). The temperature inside the experimental house was initially set at 27°C. It was then gradually decreased by 2°C each week until reaching a final temperature of 21°C. The relative humidity was maintained at a constant level of 50±10% during the whole experiment period. All experimental diets were provided in mash form throughout the trial period. Birds were given free access to feed via rectangle feeders. Fresh drinking water was provided via a nipple drinker system.

To evaluate productive performances, weight gain (WG), feed intake (FI), and feed conversion ratio (FCR) were measured. All birds from the four dietary groups were individually weighed to determine WG every four weeks. The FI was calculated by subtracting the leftover feed from the total feed provided to each treatment group. To determine the FCR, total FI per pen was divided by the total WG per pen. Ages at egg laying were recorded for each pen. After 16 weeks old, birds were fed a commercial diet (2,800 kcal AME_n/kg, 14.0% CP, 1.20% Ca, and 0.45% AP in calculated composition) to assess laying performance. The laying age at first egg was recorded. Eggs were laid for three consecutive days with an egg production of 30% or more, which was recorded as age at 30% egg production.

At 8 and 16 weeks of age, six birds from each dietary group were randomly selected. BW was measured before sacrificing them via exsanguination. After livers were collected and weighed, samples were analyzed to confirm fat content and distribution using a Digital Diagnostic X-Ray unit (Inalyzer, #XRB80N100X4391; Medikors Inc., Seongnam, Korea). The liver fat content was quantified using Dual Energy X-ray Absorptiometry (DXA) with two energy levels, 55 kV and 80 kV. This technique allows for the differentiation of fat and lean tissues by analyzing the differences in X-ray absorption between the two energy beams. The software calculated the liver fat content by comparing the attenuation differences with pre-established calibration standards, ensuring accurate quantification of fat content and distribution within the liver tissue. Liver color parameters (lightness, redness, and yellowness) were measured using a colorimeter (JP/CR-400; Minolta, Osaka, Japan). Abdominal fats of birds were collected meticulously and weighed at 16 weeks of age. The abdominal fat was calculated as a percentage (%) of the BW.

At 16 weeks of age, blood samples were taken from wing veins of six birds selected from each group. The collected blood was centrifuged at 2,000×g and 4°C for 20 minutes, and the supernatant (serum) was separated. Biochemical compositions (total cholesterol [TC], triglyceride [TG], glucose [GLU], total protein [TP], aspartate aminotransferase [AST], alanine aminotransferase [ALT]) of the serum were analyzed using a hematology analyzer (AU480 Chemistry Analyzer; Beckman Coulter Inc., Brea, CA, USA).

All statistical analyses were conducted with one-way analysis of variance (ANOVA) procedure in SAS software version 9.4 (SAS Institute Inc., Cary, NC, USA). Duncan’s multiple range test was employed to assess significant (p<0.05) differences among dietary groups. Results are presented as mean values with pooled standard errors.

Productive performances of WMD1 breeder pullets fed different dietary AME_n levels are summarized in Tables 2. Overall, both BW and WG from 4 to 16 weeks of age were higher (p<0.05) in groups fed dietary SME-100, SME, and SME+100 than in the group fed dietary SME-200. There were no dietary group differences in FI during this experimental period. Pullets fed diets containing SME+100 and SME demonstrated significantly (p<0.05) improved FCR compared to those fed a diet containing SME-200. There was no significant difference in age at first egg laying among groups. However, the age at 30% egg production occurred was significantly (p<0.05) earlier in SME-100, SME, and SME+100 groups than in the SME-200 group.

Figure 2 shows fatty liver status of pullets fed various dietary AME_n levels. Liver images of pullets at 8 weeks old similarly appeared in a blue color among groups (Figure 2A). Fat contents in livers showed similar results (Figure 2B). However, as the dietary AME_n level increases at 16 weeks of age, images of livers appear to have red color or red spots, indicating a higher fat distribution (Figure 2C). Indeed, our results indicated that birds fed SME and SME+100 diets had significantly (p<0.05) higher fat contents in livers compared to those fed the SME-200 diet at 16 weeks of age (Figure 2D).

There were no significant differences in liver color (lightness, redness, yellowness) among dietary groups at 8 weeks of age (Figures 3A–3C). However, as depicted in Figures 3D–3F (16 weeks of age), both the SME and SME+100 groups showed a significant (p<0.05) increase in liver lightness compared to the SME-200 group. Additionally, SME and SME+100 diets significantly (p<0.05) increased liver yellowness in pullets compared to the SME-100 diet. Furthermore, the SME+100 group had higher (p<0.05) abdominal fat (g) and abdominal fat per BW (%) than SME, SME-100, and SME-200 groups (Figure 4).

Table 3 shows results of blood biochemical parameters of birds according to dietary AME_n level. Results revealed no significant difference in blood composition (TC, TG, GLU, TP, AST, and ALT) among groups fed various dietary AME_n levels.

Our results indicated groups fed 2,700 to 2,900 kcal/kg had higher BWs than the group fed 2,600 kcal/kg. These findings were partially similar with those reported by Choo et al [20]. They found that a diet with 2,650 to 2,800 kcal AME_n/kg had no effect on BW of WMD breeder hens aged 20 to 32 weeks. While the energy content in poultry diet typically correlates linearly with BW, it has been observed that levels surpassing birds’ requirements do not significantly influence BW or their digestibility [30,31]. Both BW and dietary energy management for maturation of breeders and laying pullets have emerged as key determinants influencing egg production during the laying phase [22,23,32]. Based on our results of 30% egg production, birds fed SME-100, SME, and SME+100 diets matured approximately 7 days earlier than those fed the SME-200 diet. Consequently, our research suggests that the SME-100 diet with a minimum AME_n level may prove to be efficient in terms of weight management of WMD1 breeder pullets. However, since our study focused on the pullet stage, further research is needed to investigate these effects during the laying period of WMD1 breeder hens, with particular attention to egg production and their economic implications.

Fatty liver disease (FLD) primarily affects poultry, characterized by excessive fat accumulation syndrome within the liver. This syndrome can lead to reduced egg production and egg quality in hens [33,34]. In severe cases, it may result in sudden death of hens due to liver rupture [35,36]. Since breeder and laying hens are typically raised until 70 to 80 weeks old through various rearing phases on poultry farms, it is crucial to regulating nutritional management of their diet from the grower phase onwards to mitigate the potential onset of FLD at laying phase [35,37,38]. In this study, fat content and intracellular fat distribution in the liver of WMD1 breeder pullets showed significant increase in SME and SME+100 groups, which might be related to the dietary energy level reported in previous studies [35,38,39]. Furthermore, the FLD of laying hens is characterized by initially presenting a yellowish color in the pale liver. However, in severe cases, it is marked by enlarged blood vessels and significant hemorrhaging [37,40]. In our results from 16-week-old, we observed higher liver color of lightness and yellowness in groups fed with SME and SME+100 diets compared to those fed with SME-200 and SME-100 diets. These results (fat content and color) may indicate a higher risk of FLD during the laying period of WMD1 breeder hens [35]. Accordingly, we recommend the SME-100 (2,700 kcal/kg) level during the grower phase (4 to 16 weeks) to mitigate the potential onset of FLD during the laying phase (17 weeks~). In our results, abdominal fat (g or %) was the highest in the SME+100 group formulated with the highest AME_n level, which might have partly contributed to the difference in BW. Similarly, Sunder et al [30] have reported that the abdominal fat (%) of broiler breeder pullets (5 to 20 weeks of age) is increased significantly when the dietary energy concentration is increased from 2,080 to 2,860 kcal/kg. Furthermore, recent research studies suggested that a high-energy diet can upregulate the mRNA expression of the apolipoprotein B (APOB), fatty acid-binding protein 3 (FABP3), fatty acid synthase (FASN), and lipoprotein lipase (LPL) genes, which can increase the deposition of intramuscular fat or abdominal fat [41,42]. The major goal of the poultry feed industry is to reduce carcass fatness, mainly abdominal fat, as it is seen as wasted dietary energy with limited economic value [43]. Especially, excessive accumulation of abdominal fat of broiler breeder pullets may adversely affect egg production during the laying phase due to difficulties for recommended BW targets [44]. Our results suggest that dietary energy levels below SME (2,800 kcal/kg) could alleviate abdominal fat accumulation of birds.

We confirmed that increasing energy level in the diet from 2,600 to 2,900 kcal/kg did not affect blood parameters of pullets. Results of blood parameters are supported by previous studies [42,45] showing no significant difference in blood concentration of laying hens (Hy-Line Brown) or Yangzhou geese according to an increase of dietary AME_n level. However, it has been reported that an increase from 2,700 to 2,900 kcal/kg AME_n in a diet for Japanese quails can increase TG and cholesterol concentrations [46]. Blood parameters serve as indicators of overall health status, reflecting physiological, pathological, and nutritional dynamics within the organism [47]. However, concentrations may fluctuate due to various factors including diet, environmental conditions, hormonal influences, gender, and poultry genetics [39,48]. Since no studies have reported blood parameters of KNCs fed different dietary energy levels, further research is required to evaluate blood parameters across diverse KNC breeds.