Necrotic enteritis affects bone growth and bone microstructure in non-selected conventional and modern meat-type chicken strains

Abstract

Modern meat-type chickens have achieved maximum growth efficiency through successful genetic improvements over the past few decades, but bone health has not been enhanced along with it. Necrotic enteritis (NE), an intestinal disease primarily caused by Clostridium perfringens, can disrupt mineral uptake and can be lethal to immunocompromised chickens, and it remains an important issue in poultry production. To understand the relationship between NE challenge and bone health, a study was conducted using a 2 × 3 factorial arrangement, consisting of two chicken strains: (1) the non-selected conventional-type Athens Canadian Random Bred (ACRB) and (2) Cobb 500, and three NE challenge groups: a non-challenged control and two NE challenge models. Two different doses of Eimeria maxima oocysts (NE1, 2,500 oocysts; NE2, 12,500 oocysts) were inoculated on d 14 and followed by 1 × 10⁹C. perfringens on d 18. On d 14, a total of 216 male ACRB and 216 Cobb 500 chickens were allocated to each cage. The Cobb 500 group had significantly increased body weight (BW) and d 14 to 25 BW gain (BWG) compared to all ACRB chickens, and the NE2 group had decreased BWG compared to the other groups in Cobb 500 (P < 0.05). The ACRB chickens had decreased most of body composition measurements, femoral mineral apposition rate, femoral tissue volume (TV), bone volume (BV), and bone mineral content (BMC) compared to the Cobb 500 (P < 0.01). However, Cobb 500 chickens had decreased femoral bone mineral density and cortical bone volume ratio (BVR), and increased cortical porosity compared to the ACRB (P < 0.05). Necrotic enteritis challenge decreased total tissue weight, BMC, BMC ratio, bone area, and femoral TV and BV compared to the non-challenged group (P < 0.05) regardless of chicken strains. In conclusion, the negative effects of NE infection on bone development occur regardless of chicken strains, and modern meat-type chickens may experience further deterioration of bone health due to increased femoral cortical porosity and decreased BVR.

Introduction

Genetic selection and improvements in chickens over the past half-century have dramatically increased the growth rate, feed efficiency, and breast muscle yield of broilers, while significantly reducing slaughter age (Hartcher and Lum, 2020). However, these genetic improvements, which primarily focus on production efficiency, have led to numerous adverse effects, such as cardiovascular diseases, compromised immunity, increased mortality, and skeletal deformities (Cheema et al., 2003; Kalmar et al., 2013; Collins et al., 2014). In particular, leg deformities in fast-growing modern broilers are diverse, including tibial dyschondroplasia, bacterial chondronecrosis with osteomyelitis, and rickets, which can cause chronic pain and lead to lameness and deteriorated walking ability (Dibner et al., 2007; Wideman, 2016; Choppa and Kim, 2023). Lameness can reduce uniformity by limiting feed consumption and can lead to additional skeletal disorders, skin lesions, and increased culling rates, resulting in significant economic losses for poultry production (Ruiz-Feria et al., 2014; Alharbi et al., 2024).

Concerns regarding leg bone health in fast-growing modern broilers have been continuously raised, and it has been reported that high porosity and low mineralization of broiler bones due to rapid growth can reduce bone mineral density and bone disorder risk (Williams et al., 2004; Rawlinson et al., 2009). In addition, several studies using different chicken strains have observed changes in broiler growth and leg bone health, revealing that slow-growing chickens have increased bone health indicators, such as breaking strength, mineral density, and rigidity per body weight, compared to fast-growing chickens (Shim et al., 2012; Oikeh, 2019; Harash et al., 2020). However, it has also been reported that differences in bone growth may be more influenced by feed consumption and mineral utilization than by species differences (Williams et al., 2004; Pratt and Cooper, 2018). Thus, it may be necessary to understand the relationship between bone development and the factors that may hinder bone growth across various species.

Necrotic enteritis (NE) caused by Clostridium perfringens deteriorates overall intestinal health and triggers an immune response, leading to the consumption of large amounts of energy, which significantly reduces growth performance in chickens (Zhao et al., 2022; Goo et al., 2023a; Moore, 2024). Among the various C. perfringens types, the development of NE by C. perfringens type G strain, which produces the NE B-like toxin (NetB), can seriously impair intestinal health in chickens under appropriate predisposing factors (Keyburn et al., 2008; Lee et al., 2011). In particular, subclinical NE does not cause acute death but results in reduced growth performance and feed efficiency, which may ultimately have a significant adverse impact on poultry production profits (Van Immerseel et al., 2004; Timbermont et al., 2011; Prescott et al., 2016).

Necrotic enteritis may cause deterioration of intestinal health and reduce feed consumption and nutrient digestibility, thereby making it difficult to provide sufficient nutrients and minerals for proper bone growth in chickens (Zanu et al., 2020a; Goo et al., 2023a). In addition, excessive immune response in the intestine due to NE challenge may interfere with bone metabolic processes and skeletal homeostasis (Tomczyk-Warunek et al., 2021; Sharma et al., 2023b). Previous studies have reported that NE challenge can reduce serum calcium and phosphorus levels, decrease bone mineral concentration and length, and weaken the breaking strength of the femur and tibiotarsus in broilers (Zanu et al., 2020b, 2021). Tomczyk-Warunek et al. (2021) also reported that NE challenge decreased bone mineral density, bone stiffness, and calcium concentrations in the chicken tibiotarsus. Furthermore, the NE challenge reduced overall bone mineral content in whole-body Dual Energy X-ray Absorptiometry (DEXA) scans compared to the non-challenged group (Goo et al., 2025).

Previous studies have reported that NE infection significantly negatively impacts both conventional and modern meat-type chicken strains and deteriorates overall growth performance and intestinal health, indicating that NE infection remains an important issue to address (Froebel et al., 2024; Goo et al., 2024a). However, few studies examined the association between NE challenge and bone health and bone microstructure in conventional and modern meat-type broilers. Specifically, there is a lack of research investigating the effects of decades of genetic improvement in chickens on bone growth and development using non-selected conventional-type Athens Canadian Random Bred (ACRB). The reduced weight gain and feed efficiency caused by NE infection, along with leg problems due to deteriorated bone health, can lead to substantial economic losses in poultry production (Talaty et al., 2009; Skinner et al., 2010; Wade and Keyburn, 2015). Therefore, it is necessary to investigate how genetic improvement in broilers relates to these issues. The hypothesis of the current study was that NE challenge has negative effects on bone growth and microstructure in genetically non-selected conventional-type ACRB and modern meat-type Cobb 500 chickens. Therefore, the objective of the current study was to investigate the effects of NE challenge on growth performance, body mineral composition, femoral mineral apposition rate, femoral bone mineral density, femoral bone volume ratio, cortical porosity, and trabecular development on d 25 in the ACRB and Cobb 500 broilers.

Materials and methods

Chickens, experimental design, and necrotic enteritis model

The current experiment was approved by the Institutional Animal Care and Use Committee (A2021 12-012). The experiment was conducted at the Poultry Research Center (PRC) at the University of Georgia. The current study was conducted up to d 25 to observe the effect on bone growth by setting up the same treatment groups as previously reported by Goo et al. (2024a). Briefly, six groups were set up with a 2 × 3 factorial arrangement, with six replicates of twelve chickens per cage. Two main effects were chicken strains (ACRB and Cobb 500) and NE challenge models (two different NE challenge models and a non-challenged group). The six groups in the current study were as follows: (1) non-challenged (NC) ACRB; (2) NC Cobb 500; (3) ACRB with NE challenge model 1 (NE1, 2,500 Eimeria maxima oocyst challenge on d 14, followed by 1 × 10⁹ C. perfringens on d 18); (4) Cobb 500 with NE1; (5) ACRB with NE challenge model 2 (NE2, 12,500 E. maxima on d 14, followed by 1 × 10⁹ C. perfringens on d 18); and (6) Cobb 500 with NE2. On d 14, a total of 216 ACRB chicken and 216 Cobb 500 broilers were allotted into 6 treatments (n = 6) with similar body weight (BW) within each strain and raised until d 25. The average BW of ACRB and Cobb 500 was 113 ± 0.5 g and 436 ± 1.0 g, respectively. The two-phase corn-soybean meal-based mash diets were formulated (21 and 24% CP) following the previously reported NE challenge studies by Goo et al. (2023a, 2024a), and the diet formulations are presented in Table 1. Both chicken strains were raised in nipple-installed 4-layer battery cages (102 × 36 × 41 cm). Feed and water were provided ad libitum throughout the experimental period. Animal husbandry followed the Cobb Broiler Management Guide (2021). The ACRB eggs were obtained from artificially inseminated ACRB breeders at the University of Georgia. The ACRB eggs were then incubated for approximately 21.5 days at the PRC hatchery. The egg incubation proceeded as follows: 18 days at 37.8°C (100℉) and 53% relative humidity (RH) in NMC 2000 incubators, and approximately 3.5 days at 36.9°C (98.5℉) and 65% RH in NMC 2000 hatchers (NatureForm Incubator Co., Jacksonville, FL). For the NE challenge models, the E. maxima and C. perfringens coinfection model was used according to the previously reported study by Goo et al. (2023a). Briefly, 1 mL of sporulated E. maxima oocyst (2,500 and 12,500 oocysts for NE1 and NE2, respectively) mixture was orally administered to the chickens in the NE challenge groups on d 14, followed by 1 mL of 1 × 10⁹ NetB positive C. perfringens Del-1 on d 18 (Goo et al., 2023b). On each challenge day, phosphate-buffered saline (PBS) was administered to the NC groups. On 11 days post inoculation of E. maxima (dpi), BW, relative BW (RBW), and feed intake (FI) were measured. Relative BW was calculated by setting the NC group of each strain to 100%. In addition, from d 14 to 25, BW gain (BWG) and feed conversion ratio (FCR) were calculated.

Table 1.

Diet composition of the current study (as-fed basis, %).

	d 14 to 18	d 19 to 25
Ingredients, %
Corn, grain	59.85	53.86
Soybean meal, 48%	31.52	39.54
Soybean oil	2.20	3.69
Dicalcium phosphate	1.94	1.24
Sand	1.89	-
Limestone	1.27	0.99
Salt	0.35	0.35
DL-Methionine	0.34	0.15
L-Lysine	0.31	-
L-Threonine	0.15	-
Mineral premix1	0.08	0.08
Vitamin premix2	0.10	0.10
Total	100.0	100.0
Calculated value, %
ME, kcal/g	3,000	3,100
Crude protein	21.1	24.0
Total Ca	0.99	0.76
Available P	0.50	0.38
dLysine3	1.22	1.19
dMethionine	0.64	0.49
dTSAA	0.91	0.80
dThreonine	0.83	0.81
dArginine	1.28	1.52
dValine	0.98	1.15
dTryptophan	0.23	0.28

¹Mineral premix provided the following per kg of diet: Mn, 100.5 mg; Zn, 80.3 mg; Ca, 24 mg; Mg, 20.1 mg; Fe, 19.7 mg; Cu, 3 mg; I, 0.75 mg; Se, 0.30 mg.

²Vitamin premix provided the following per kg of diet: vitamin A, 3,527 IU; vitamin D₃, 1,400 IU; vitamin E, 19.4 IU; niacin, 20.28 mg; D-pantothenic acid, 5.47 mg; riboflavin, 3.53 mg; vitamin B₆, 1.46 mg; menadione, 1.10 mg; thiamin, 0.97 mg; folic acid, 0.57 mg; biotin, 0.08 mg; vitamin B₁₂, 0.01 mg.

³Digestible amino acids.

Body composition using DEXA

On d 25 (11 dpi), two chickens with average BW per cage were euthanized by cervical dislocation for body composition analysis using DEXA. All chickens were first measured for BW and then placed without overlapping on the DEXA scanner (GE Lunar Prodigy, GE Healthcare, Chicago, IL). To prevent measurement error due to posture differences in DEXA scans, all chickens maintained the same prone position before scanning. The scanning range was set as small animals (196.6 × 62.0 cm), and the exposure factors were set as follows: voltage (76 kV), current (0.15 mA), time (26 min 37 s), and dose (1.8 μGy), following a previously reported setup by Goo et al. (2024b). Immediately after 2-dimensional (2D) DEXA scanning, data were measured by setting a region of interest (ROI) for each chicken. The data on bone mineral density (BMD), bone mineral content (BMC), bone area (BA), total tissue weight, lean weight, lean percentage, fat weight, and fat percentage were measured based on calibration data prior to chicken scanning (Paneru et al., 2025). The BMC ratio (BMCR) was additionally calculated (BMC / Carcass weight × 100), considering the significant difference in body size between ACRB and Cobb 500, following a previously reported method by Goo and Kim (2025).

Femur mineral apposition rate

By using the calcein (fluorescent dye) labeling method, the femur mineral apposition rate (MAR) was measured following previously reported methods by Shi et al. (2024). In brief, calcein (C₃₀H₂₆N₂O₁₃; Cat. No. C0875, Sigma-Aldrich, St. Louis, MO) solution was prepared based on the chickens’ BW (20 mg/kg of BW) and dissolved in a 1 M NaOH solution and distilled water. On the first injection day (d 18), 200 μL of dissolved calcein solution was intraperitoneally injected into chickens. On the second injection day (d 22), the calcein solution was prepared and injected in the same way. On d 25 (11 dpi), chickens were euthanized by cervical dislocation, and the right femurs were collected and kept in 70% ethanol for further analyses. To determine the femur MAR, approximately 0.5 to 1.0 mm of bone slices were cut from the mid-section of the femur diaphysis by a circular saw (Ryobi, Anderson, SC) and then fixed to glass slides using toluene solution (Cat. No. 179965, Sigma-Aldrich, St. Louis, MO; Sharma et al., 2023a). After drying the glass slides for 24 h, images were captured using a fluorescent microscope (BZ-X810, Keyence, Osaka, Japan), and the distance between the two calcein labels was determined. The distance between the two calcein labels was measured based on the narrowest portion of each image, and an example of the MAR measurement is shown in Fig. 1.

Fig. 1.

Effects of necrotic enteritis challenge on femur mineral apposition rate (MAR) in ACRB and Cobb 500 on d 25 (11 dpi). (A) MAR example of non-challenged ACRB; (B) MAR example of non-challenged Cobb 500; (C) The result figure of femur MAR on d 25. Cobb 500 had significantly increased MAR compared to the ACRB (P < 0.05). No significant interaction was observed. Each bar indicates the standard error of the mean (n = 6). Calcein solutions were intraperitoneally injected on d 18 and 22. Mineral apposition rate was measured between two fluorescent points, marked by white arrows in the figure. Abbreviations: dpi, days post inoculation of E. maxima on d 14; NC, non-challenged control; NE1, 2,500 E. maxima oocysts challenge on d 14 and followed by C. perfringens 1 × 10⁹ on d 18; NE2, 12,500 E. maxima oocysts challenge on d 14 and followed by C. perfringens 1 × 10⁹ on d 18; ACRB, Athens Canadian Random Bred.

Femur microstructure using MicroCT

Femur samples were taken from the same chickens for body composition analysis using DEXA on d 25 (11 dpi). In brief, one chicken was selected after DEXA scanning, and the right femur was taken and and kept at -20°C after muscle tissues were removed. Before scanning, femur samples were thawed at room temperature for up to 6 h and scanned using the Skyscan X-ray Microtomography (MicroCT; Bruker MicroCT, Billerica, MA) for 3-dimensional (3D) bone imaging. The MicroCT scan setting in the current study was used following a previously reported method by Sharma et al. (2023a). In brief, the thawed femur sample was tightly wrapped in 2-layer cheesecloth and held in a 50 mL centrifuge tube. The tube containing the femur sample was then mounted on the scanning stage of the MicroCT scanner. Before starting the sample scanning, fat-field correction and alignment tests were performed based on the MicroCT Manual (Bruker MicroCT, Billerica, MA). The X-ray source and scanning settings were set to 80 kV (voltage), 125 μA (current), 25 μm (pixel size), 0.6° (rotation angle), and 4 images captured per rotation, and a 0.5 mm aluminum filter was applied to reduce the beam hardening effect, following a previously reported setting by Liu et al. (2024) with slight modifications. The scanned images were then reconstructed and straightened using N-Recon and Data Viewer Software, respectively (Bruker MicroCT, Billerica, MA). The dynamic range for all femur samples was set at 0 to 0.03. The VOI was selected using CTAn Software (Bruker MicroCT, Billerica, MA) on the distal metaphysis region of the femur (200 slides, 5 mm). An example of VOI selection in the femur in the current study is shown in Fig. 2. Then, selected 3D model images were separated into cortical and trabecular bone using custom processes following a previously reported method by Chen and Kim (2020). Two solid-state phantoms made of calcium hydroxyapatite were used for calibration and data calculation. The different parameters of the VOI, cortical bone, and trabecular bone were measured as previously defined by Bouxsein et al. (2010). Femoral BMC, BMD, tissue volume (TV), bone volume (BV), bone surface (BS), bone volume ratio (BVR), BS ratio (BSR), BS density (BSD), closed pore number (CPN), closed pore volume (CPV), closed pore surface (CPS), closed porosity ratio (CPR), open pore space volume (OPV), open porosity ratio (OPR), total pore space volume (TPV), total porosity ratio (TPR), trabecular thickness (TbT), trabecular separation (TbS), trabecular number (TbN), trabecular pattern factor (TbPF), trabecular connectivity (TbC), TbC density (TbCD), and structure model index (SMI) were obtained after MicroCT scanning.

Fig. 2.

Effects of necrotic enteritis challenge on femur microstructural architecture in ACRB and Cobb 500 on d 25 (11 dpi). Figures of the selection of the volume of interest (VOI) for cortical and trabecular bone analysis in ACRB and Cobb 500 were presented. The femur VOI was selected on the distal metaphysis region (200 slides, 5 mm total). (A) Non-challenged ACRB; (B) Non-challenged Cobb 500; (C) ACRB with 2,500 E. maxima challenge on d 14 and followed by 1 × 10⁹C. perfringens on d 18; (D) Cobb 500 with 2,500 E. maxima challenge on d 14 and followed by 1 × 10⁹C. perfringens on d 18; (E) ACRB with 12,500 E. maxima challenge on d 14 and followed by 1 × 10⁹C. perfringens on d 18; (F) Cobb 500 with 12,500 E. maxima challenge on d 14 and followed by 1 × 10⁹C. perfringens on d 18. The yellow color represents the cortical bone area, and the red color represents the trabecular bone area. The magnification of the figures of ACRB were adjusted for comparison in the similar size as the figure of Cobb 500. Abbreviations: ACRB, Athens Canadian Random Bred; VOI, volume of interest.

Statistical analysis

All statistical analyses in the current experiment were conducted using RStudio software (R Version 4.2.2, RStudio PBC, Boston, MA), and figures were created using GraphPad Prism software (GraphPad Prism 5.0, GraphPad Software Inc., San Diego, CA). Normal distribution and homoscedasticity of the data were first tested before ANOVA was performed. All data were analyzed using a 2 × 3 factorial arrangement of treatments with a 2-way ANOVA. The interaction between the two main effects and the difference in each main effect were analyzed. Tukey’s honestly significant difference post hoc test was used to determine the differences among the NE challenge groups (NC, NE1, and NE2), while Student’s t-test was used to determine the difference between chicken strains (ACRB and Cobb 500) if the P-value of ANOVA was less than 0.05.

Results

Growth performance

Interactions were observed between the chicken strains and the NE models on d 25 BW and d 14 to 25 BWG (Table 2). Cobb 500 had higher BW and BWG compared to the ACRB (P < 0.05). No significant difference was observed among the ACRB groups, whereas in Cobb 500 groups, the NE2 group had lower BW at d 25 (11 dpi) and BWG (d 14 to 25) compared to the NC and NE1 groups (P < 0.05). Cobb 500 group had higher FI (d 14 to 25) and feed efficiency compared to the ACRB group (P < 0.001). The NE2 group had poorer FCR compared to the NC and NE1 groups (P < 0.01). The NE2 group also had reduced RBW at d 25 compared to the NC and NE1 groups (P < 0.001). There was no significant difference in all growth performance measurements between the NC and NE1 groups.

Table 2.

Effects of necrotic enteritis challenge on growth performance in ACRB and Cobb 500 from d 14 to 25.

		d 25		d 14 to 25 (0 to 11 dpi2)
		BW, g	RBW1, %	BWG, g	FI, g	FCR
Strain	NE challenge3
ACRB	NC	218c	100.0	104.2c	445	4.31
	NE1	211c	96.7	98.0c	424	4.38
	NE2	195c	89.5	82.5c	427	5.24
Cobb 500	NC	1,221a	100.0	784.2a	1,239	1.59
	NE1	1,169a	95.8	733.5a	1,253	1.72
	NE2	1,096b	89.8	658.2b	1,221	1.86
SEM (n = 6)		15.8	2.11	15.96	27.0	0.190
Main effect
Strain
ACRB		208	95.4	94.9	432b	4.64a
Cobb 500		1,162	95.2	725.3	1,238a	1.72b
SEM (n = 18)		9.1	1.22	9.22	15.6	0.108
NE challenge
NC		719	100.0a	444.2	842	2.95b
NE1		690	96.3a	415.8	839	3.05b
NE2		646	89.7b	370.3	824	3.55a
SEM (n = 12)		11.1	1.49	11.3	19.1	0.134
P-value
Strain × NE challenge		0.017	0.956	0.010	0.755	0.122
Strain		<0.001	0.908	<0.001	<0.001	<0.001
NE challenge		<0.001	<0.001	<0.001	0.784	0.008

^a-cMeans in the same column with different superscripts are statistically different (P < 0.05).

¹Each NC group was set to 100% relative body weight.

²Days post inoculation of E. maxima on d 14.

³NE challenge: NC, non-challenged control; NE1, 2,500 E. maxima oocysts challenge on d 14 and followed by C. perfringens 1 × 10⁹ on d 18; NE2, 12,500 E. maxima oocysts challenge on d 14 and followed by C. perfringens 1 × 10⁹ on d 18.

Body composition using DEXA

No interaction between the chicken strains and the NE models was observed in all body composition parameters (Table 3). The ACRB group had lower total tissue weight, lean weight, fat weight, fat ratio, BMD, BMC, BMCR, and BA at d 25 compared to the Cobb 500 group (P < 0.01), whereas lean ratio was increased compared to the Cobb group (P < 0.01). The NE2 group had lower total tissue weight, BMC, BMCR, and BA compared to the NC group (P < 0.05). However, there were no significant differences in all body composition parameters between the NC and NE1 groups.

Table 3.

Effects of necrotic enteritis challenge on body composition in ACRB and Cobb 500 on d 25 (11 dpi).

		Body composition1 on d 25 (11 dpi2)
		Total tissue weight, g	Lean, g	Lean ratio, %	Fat, g	Fat ratio, %	BMD, mg/cm2	BMC, g	BMCR, %	BA, cm2
Strain	NE challenge3
ACRB	NC	220	201	91.2	19.1	8.8	100.2	2.7	1.21	26.7
	NE1	195	179	92.0	15.7	8.0	95.8	2.0	1.05	21.3
	NE2	178	167	93.9	10.8	6.1	92.0	1.8	1.01	19.8
Cobb 500	NC	1,204	1,057	88.0	147.0	12.0	155.3	17.9	1.49	115.0
	NE1	1,185	1,070	90.3	115.1	9.7	157.8	17.9	1.51	113.5
	NE2	1,114	1,000	89.6	114.3	10.4	160.5	15.9	1.43	99.3
SEM (n = 6)		24.0	22.5	1.23	11.29	1.23	2.61	0.45	0.048	3.06
Main effect
Strain
ACRB		198b	183b	92.3a	15.2b	7.7b	96.0b	2.2b	1.09b	22.6b
Cobb 500		1,168a	1,042a	89.3b	125.5a	10.7a	157.9a	17.2a	1.48a	109.3a
SEM (n = 18)		13.9	13.0	0.71	6.52	0.71	1.51	0.26	0.028	1.77
NE challenge
NC		712a	629	89.6	83.1	10.4	127.8	10.3a	1.35a	70.8a
NE1		690ab	625	91.1	65.4	8.9	126.8	10.0ab	1.28ab	67.5ab
NE2		646b	584	91.7	62.5	8.3	126.3	8.9b	1.22b	59.6b
SEM (n = 12)		17.0	15.9	0.87	7.98	0.87	1.85	0.32	0.034	2.18
P-value
Strain × NE challenge		0.470	0.430	0.585	0.406	0.585	0.052	0.163	0.165	0.118
Strain		<0.001	<0.001	0.005	<0.001	0.005	<0.001	<0.001	<0.001	<0.001
NE challenge		0.031	0.101	0.214	0.161	0.214	0.846	0.009	0.046	0.003

^a,bMeans in the same row with different superscripts are statistically different (P < 0.05).

¹BMD, bone mineral density; BMC, bone mineral content; BMCR, bone mineral content ratio (BMC/total tissue weight × 100); BA, bone area.

²Days post inoculation of E. maxima on d 14.

³NE challenge: NC, non-challenged control; NE1, 2,500 E. maxima oocysts challenge on d 14 and followed by C. perfringens 1 × 10⁹ on d 18; NE2, 12,500 E. maxima oocysts challenge on d 14 and followed by C. perfringens 1 × 10⁹ on d 18.

Femur mineral apposition rate (MAR)

No interaction was observed between the chicken strains and the NE models in femur MAR on d 25 (Fig. 1). The ACRB group had lower femur MAR compared to the Cobb 500 group (P < 0.001). However, no significant differences were observed in femur MAR among all NE challenge groups.

Femur microstructure using MicroCT

No interaction between the chicken strains and the NE models was observed in all total VOI of femur measurements (Table 4). The ACRB group had lower femoral BMC, TV, BV, BS, BSR, and BSD compared to the Cobb 500 group (P < 0.001). NE2 group had lower femoral TV and BV compared to the NC group (P < 0.05). However, there were no significant differences in all total VOI of femur measurements between the NC and NE1 groups.

Table 4.

Effects of necrotic enteritis challenge on total volume of interest of femur microstructure in ACRB and Cobb 500 on d 25 (11 dpi).

		Total volume of interest of femur1 on d 25 (11 dpi2)
		BMC, mg	TV, mm3	BV, mm3	BS, cm2	BVR, %	BSR, mm-1	BSD, mm-1
Strain	NE challenge3
ACRB	NC	10.7	100.0	30.2	2.9	30.4	9.5	2.9
	NE1	10.6	88.7	29.6	2.9	33.3	9.6	3.2
	NE2	8.1	96.8	28.3	2.8	29.2	9.9	2.9
Cobb 500	NC	36.8	422.0	125.8	14.2	29.9	11.3	3.4
	NE1	34.0	382.5	121.0	13.1	31.5	11.1	3.4
	NE2	30.7	371.9	108.3	12.2	29.4	11.3	3.3
SEM (n = 6)		2.95	10.94	3.51	0.51	1.54	0.42	0.13
Main effect
Strain
ACRB		9.8b	95.2b	29.4b	2.8b	31.0	9.7b	3.0b
Cobb 500		33.8a	392.1a	118.4a	13.2a	30.3	11.2a	3.4a
SEM (n = 18)		1.70	6.14	3.04	0.28	0.89	0.24	0.07
NE challenge
NC		23.8	261.0a	78.0a	8.5	30.1	10.4	3.1
NE1		22.3	235.6ab	75.3ab	8.0	32.4	10.4	3.3
NE2		19.4	234.3b	68.3b	7.5	29.3	10.6	3.1
SEM (n = 12)		2.09	7.73	2.38	0.36	1.09	0.30	0.09
P-value
Strain × NE challenge		0.827	0.108	0.080	0.184	0.808	0.883	0.735
Strain		<0.001	<0.001	<0.001	<0.001	0.566	<0.001	<0.001
NE challenge		0.317	0.033	0.025	0.135	0.126	0.840	0.086

^a,bMeans in the same row with different superscripts are statistically different (P < 0.05).

¹BMC, bone mineral content; TV, tissue volume; BV, bone volume; BS, bone surface; BVR, bone volume ratio (BV/TV × 100); BSR, bone surface ratio (BS/BV); BSD, bone surface density (BS/TV).

²Days post inoculation of E. maxima on d 14.

³NE challenge: NC, non-challenged control; NE1, 2,500 E. maxima oocysts challenge on d 14 and followed by C. perfringens 1 × 10⁹ on d 18; NE2, 12,500 E. maxima oocysts challenge on d 14 and followed by C. perfringens 1 × 10⁹ on d 18.

No interaction between the chicken strains and the NE models was observed in all femoral BMD measurements (Table 5). The ACRB group had higher femoral BMD in total VOI, cortical BMD, and trabecular BMD compared to the Cobb 500 group (P < 0.05). No significant differences were observed in all BMD measurements among all NE challenge groups.

Table 5.

Effects of necrotic enteritis challenge on femoral bone mineral density in ACRB and Cobb 500 on d 25 (11 dpi).

		BMD of femur1 on d 25 (11 dpi2), mg/cm3
		Total VOI	Cortical bone	Trabecular bone
Strain	NE challenge3
ACRB	NC	108.5	424.6	62.3
	NE1	119.6	435.2	63.5
	NE2	83.7	422.1	70.7
Cobb 500	NC	87.4	341.2	52.6
	NE1	88.2	331.7	61.6
	NE2	83.3	345.4	48.9
SEM (n = 6)		10.45	10.09	6.79
Main effect
Strain
ACRB		104.0a	427.3a	65.5a
Cobb 500		86.3b	339.4b	54.4b
SEM (n = 18)		6.03	5.66	3.81
NE challenge
NC		98.0	382.9	57.5
NE1		103.9	383.4	62.5
NE2		83.5	383.7	59.8
SEM (n = 12)		7.39	7.14	4.80
P-value
Strain × NE challenge		0.317	0.370	0.318
Strain		0.046	<0.001	0.049
NE challenge		0.141	0.996	0.748

^a,bMeans in the same row with different superscripts are statistically different (P < 0.05).

¹BMD, bone mineral density (BMC/TV); VOI, volume of interest.

²Days post inoculation of E. maxima on d 14.

³NE challenge: NC, non-challenged control; NE1, 2,500 E. maxima oocysts challenge on d 14 and followed by C. perfringens 1 × 10⁹ on d 18; NE2, 12,500 E. maxima oocysts challenge on d 14 and followed by C. perfringens 1 × 10⁹ on d 18.

No interaction was observed between the chicken strains and the NE models in all femur cortical bone microstructure measurements on d 25 (Table 6, Table 7). Similar to total VOI results, the ACRB group had lower cortical BMC, TV, BV, and BS compared to the Cobb group (P < 0.001). However, the ACRB group had higher cortical BVR and BSD compared to the Cobb 500 group (P < 0.05). In the pore and porosity microstructure result, the ACRB group had lower all cortical pore and porosity parameters, including CPN, CPV, CPS, CPR, OPV, OPR, TPV, and TPR compared to the Cobb 500 group (P < 0.001). However, no significant differences were observed in all cortical microstructure and pore and porosity measurements among all NE challenge groups.

Table 6.

Effects of necrotic enteritis challenge on femoral cortical bone microstructure in ACRB and Cobb 500 on d 25 (11 dpi).

		Cortical bone of femur1 on d 25 (11 dpi2)
		BMC, mg	TV, mm3	BV, mm3	BS, cm2	BVR, %	BSR, mm-1	BSD, mm-1
Strain	NE challenge3
ACRB	NC	13.2	31.1	28.0	2.3	89.9	8.3	7.5
	NE1	13.5	30.8	27.3	1.9	88.6	8.3	7.3
	NE2	12.5	29.6	26.2	2.3	89.1	8.9	8.0
Cobb 500	NC	44.4	130.4	103.0	9.0	79.1	8.7	6.9
	NE1	43.8	132.3	106.3	9.1	80.4	8.6	6.9
	NE2	39.4	114.0	92.4	8.2	81.1	8.9	7.2
SEM (n = 6)		1.97	4.77	3.77	0.47	1.04	0.41	0.32
Main effect
Strain
ACRB		13.1b	30.5b	27.2b	2.2b	89.2a	8.5	7.6a
Cobb 500		42.5a	125.6a	100.6a	8.8a	80.2b	8.7	7.0b
SEM (n = 18)		1.03	2.67	2.12	0.27	0.58	0.23	0.18
NE challenge
NC		28.8	80.8	65.5	5.7	84.5	8.5	7.2
NE1		28.7	81.5	66.8	5.5	84.5	8.4	7.1
NE2		26.0	71.8	59.3	5.2	85.1	8.9	7.6
SEM (n = 12)		1.28	3.37	2.67	0.33	0.73	0.29	0.22
P-value
Strain × NE challenge		0.435	0.147	0.210	0.325	0.309	0.823	0.869
Strain		<0.001	<0.001	<0.001	<0.001	<0.001	0.499	0.025
NE challenge		0.193	0.079	0.108	0.652	0.810	0.472	0.290

^a,bMeans in the same row with different superscripts are statistically different (P < 0.05).

¹BMC, bone mineral content; TV, tissue volume; BV, bone volume; BS, bone surface; BVR, bone volume ratio (BV/TV × 100); BSR, bone surface ratio (BS/BV); BSD, bone surface density (BS/TV).

²Days post inoculation of E. maxima on d 14.

³NE challenge: NC, non-challenged control; NE1, 2,500 E. maxima oocysts challenge on d 14 and followed by C. perfringens 1 × 10⁹ on d 18; NE2, 12,500 E. maxima oocysts challenge on d 14 and followed by C. perfringens 1 × 10⁹ on d 18.

Table 7.

Effects of necrotic enteritis challenge on femoral cortical bone pore and porosity microstructure in ACRB and Cobb 500 on d 25 (11 dpi).

		Cortical bone of femur1 on d 25 (11 dpi2)
		CPN, n	CPV, mm3	CPS, mm2	CPR, %	OPV, mm3	OPR, %	TPV, mm3	TPR, %
Strain	NE challenge3
ACRB	NC	45	0.018	1.19	0.06	3.1	10.0	3.2	10.1
	NE1	35	0.020	1.15	0.07	3.5	11.3	3.5	11.4
	NE2	41	0.020	1.28	0.07	3.3	10.9	3.3	10.9
Cobb 500	NC	558	0.266	16.02	0.26	27.2	20.8	27.4	21.0
	NE1	561	0.244	16.16	0.23	25.9	19.6	26.2	19.7
	NE2	494	0.253	15.81	0.27	21.4	18.8	21.7	19.0
SEM (n = 6)		37.9	0.025	1.242	0.025	1.37	1.05	1.38	1.06
Main effect
Strain
ACRB		40b	0.019b	1.21b	0.07b	3.3b	10.7b	3.3b	10.8b
Cobb 500		538a	0.254a	16.00a	0.25a	24.8a	19.7a	25.1a	19.9a
SEM (n = 18)		21.3	0.014	0.697	0.014	0.77	0.59	0.78	0.59
NE challenge
NC		301	0.142	8.60	0.16	15.2	15.4	15.3	15.5
NE1		298	0.132	8.66	0.15	14.7	15.4	14.8	15.6
NE2		267	0.137	8.54	0.17	12.4	14.8	12.5	15.0
SEM (n = 12)		26.8	0.017	0.841	0.018	0.97	0.74	0.98	0.75
P-value
Strain × NE challenge		0.575	0.872	0.979	0.639	0.086	0.304	0.090	0.328
Strain		<0.001	<0.001	<0.001	<0.001	<0.001	<0.001	<0.001	<0.001
NE challenge		0.596	0.915	0.996	0.738	0.097	0.809	0.101	0.810

^a,bMeans in the same row with different superscripts are statistically different (P < 0.05).

¹CPN, closed pore number; CPV, closed pore volume; CPS, closed pore surface; CPR, closed porosity ratio (CPV/Bone volume × 100); OPV, open pore space volume; OPR, open porosity ratio (OPV/Tissue volume × 100; BVR + OPR = 100.0); TPV, total pore space volume (CPV + OPV); TRP, total porosity ratio (TPV/Tissue volume × 100).

²Days post inoculation of E. maxima on d 14.

³NE challenge: NC, non-challenged control; NE1, 2,500 E. maxima oocysts challenge on d 14 and followed by C. perfringens 1 × 10⁹ on d 18; NE2, 12,500 E. maxima oocysts challenge on d 14 and followed by C. perfringens 1 × 10⁹ on d 18.

No interaction was observed between the chicken strains and the NE models in all femur trabecular bone microstructure measurements on d 25 (Table 8, Table 9). The ACRB group had lower femur trabecular BMC, TV, BV, BS, BVR, and BSD compared to the Cobb 500 group (P < 0.001). Both NE challenge groups (NE1 and NE2) had decreased trabecular TV compared to the NC group (P < 0.01). In the trabecular development and porosity microstructure measurements, the ACRB group had lower TbS, TbN, TbC, and TbCD compared to the Cobb 500 group (P < 0.001), whereas TbPF, SMI, and TPR were higher than those of the Cobb 500 group (P < 0.01). However, no significant differences were observed in all trabecular development and porosity microstructure measurements among all NE challenge groups.

Table 8.

Effects of necrotic enteritis challenge on femoral trabecular bone microstructure using in ACRB and Cobb 500 on d 25 (11 dpi).

		Trabecular bone of femur1 on d 25 (11 dpi2)
		BMC, mg	TV, mm3	BV, mm3	BS, cm2	BVR, %	BSR, mm-1	BSD, mm-1
Strain	NE challenge3
ACRB	NC	4.0	63.2	1.7	0.61	2.8	36.0	0.98
	NE1	3.3	52.9	1.7	0.65	3.2	39.2	1.23
	NE2	3.8	54.6	1.6	0.57	2.9	36.0	1.04
Cobb 500	NC	14.3	272.6	14.0	5.31	5.1	39.2	1.94
	NE1	14.0	228.5	11.8	4.37	5.2	37.1	1.91
	NE2	11.0	228.1	11.9	4.30	5.3	36.2	1.89
SEM (n = 6)		1.29	8.10	0.88	0.279	0.34	2.94	0.119
Main effect
Strain
ACRB		3.7b	56.9b	1.7b	0.61b	3.0b	37.1	1.08b
Cobb 500		13.1a	243.1a	12.6a	4.66a	5.2a	37.5	1.92a
SEM (n = 18)		0.72	4.54	0.49	0.156	0.19	1.65	0.067
NE challenge
NC		9.2	167.9a	7.9	2.96	3.9	37.6	1.46
NE1		8.7	140.7b	6.8	2.51	4.2	38.2	1.57
NE2		7.4	141.3b	6.8	2.41	4.1	36.1	1.47
SEM (n = 12)		0.91	5.73	0.62	0.197	0.24	2.08	0.084
P-value
Strain × NE challenge		0.318	0.058	0.421	0.143	0.769	0.665	0.474
Strain		<0.001	<0.001	<0.001	<0.001	<0.001	0.848	<0.001
NE challenge		0.362	0.003	0.360	0.142	0.741	0.759	0.575

^a,bMeans in the same row with different superscripts are statistically different (P < 0.05).

¹BMC, bone mineral content; TV, tissue volume; BV, bone volume; BS, bone surface; BVR, bone volume ratio (BV/TV × 100); BSR, bone surface ratio (BS/BV); BSD, bone surface density (BS/TV).

²Days post inoculation of E. maxima on d 14.

³NE challenge: NC, non-challenged control; NE1, 2,500 E. maxima oocysts challenge on d 14 and followed by C. perfringens 1 × 10⁹ on d 18; NE2, 12,500 E. maxima oocysts challenge on d 14 and followed by C. perfringens 1 × 10⁹ on d 18.

Table 9.

Effects of necrotic enteritis challenge on femoral trabecular bone development and porosity microstructure in ACRB and Cobb 500 on d 25 (11 dpi).

		Trabecular bone of femur1 on d 25 (11 dpi2)
		TbT, μm	TbS, mm	TbN, mm-1	TbPF, mm-1	TbC, n	TbCD, mm-3	SMI, 0 to 4	TPR, %
Strain	NE challenge3
ACRB	NC	108.8	1.75	0.21	17.1	272	4.4	2.73	97.9
	NE1	104.2	1.47	0.28	17.0	299	5.7	2.70	97.3
	NE2	150.2	1.79	0.18	16.1	232	4.2	3.10	97.0
Cobb 500	NC	115.4	2.15	0.44	12.6	2,278	8.3	2.42	94.9
	NE1	117.1	2.19	0.45	13.1	1,821	8.0	2.46	94.9
	NE2	121.3	2.33	0.46	11.3	1,969	8.7	2.53	94.5
SEM (n = 6)		16.24	0.099	0.037	0.84	134.9	0.66	0.145	0.35
Main effect
Strain
ACRB		121.1	1.67b	0.22b	16.7a	268b	4.8b	2.84a	97.4a
Cobb 500		118.0	2.22a	0.45a	12.3b	2,023a	8.3a	2.47b	94.8b
SEM (n = 18)		9.11	0.055	0.021	0.47	75.7	0.37	0.082	0.20
NE challenge
NC		112.1	1.95	0.33	14.9	1,275	6.4	2.58	96.4
NE1		110.7	1.83	0.37	15.0	1,060	6.8	2.58	96.1
NE2		135.8	2.06	0.32	13.7	1,101	6.4	2.82	95.7
SEM (n = 12)		11.48	0.070	0.026	0.59	95.4	0.46	0.103	0.25
P-value
Strain × NE challenge		0.367	0.255	0.341	0.835	0.205	0.225	0.464	0.715
Strain		0.810	<0.001	<0.001	<0.001	<0.001	<0.001	0.003	<0.001
NE challenge		0.216	0.074	0.372	0.205	0.247	0.742	0.163	0.158

^a,bMeans in the same row with different superscripts are statistically different (P < 0.05).

¹TbT, trabecular thickness; TbS, trabecular separation; TbN, trabecular number; TbPF, trabecular pattern factor; TbC, trabecular connectivity; TbCD, trabecular connectivity density (TbC/Tissue volume); SMI, structure model index (< 0 = concave, 0 = plate, 3 = rod, and 4 = sphere); TRP, total porosity ratio (Total pore space volume/Tissue volume × 100).

²Days post inoculation of E. maxima on d 14.

³NE challenge: NC, non-challenged control; NE1, 2,500 E. maxima oocysts challenge on d 14 and followed by C. perfringens 1 × 10⁹ on d 18; NE2, 12,500 E. maxima oocysts challenge on d 14 and followed by C. perfringens 1 × 10⁹ on d 18.

Discussion

The current study was conducted to determine how NE infection negatively affects bone growth and microstructure in two different chicken strains, ACRB and Cobb 500. Growth performance was measured to assess the approximate BW difference between the groups prior to bone microstructure analysis. Similar to our previous ACRB in NE challenge study (Goo et al., 2024a), interactions in final BW and BWG were observed, likely due to a significant body size difference between the ACRB and Cobb 500 strains (Collins et al., 2014). This may have been expressed as if there were no statistical differences, even though the BW showed a difference by NE infection, due to the very small size of ACRB. Therefore, no interaction was observed when BW was converted to a relative parameter (RBW); only chickens in the NE2 group (12,500 E. maxima oocysts + C. perfringens) had significantly lower RBW in both chicken strains in the current study. Differences between the two chicken strains were also observed in FI and FCR, with the NE2 model showing significantly decreased feed efficiency compared to the other groups. Notably, feed consumption from d 14 to 25 was not significantly reduced in any NE challenge models. This may be because the subclinical NE model used in the current study resulted in a decrease in BWG and feed efficiency without causing significant mortality or a reduction in feed consumption (Skinner et al., 2010; Shojadoost et al., 2012). In addition, compensatory feed consumption may have occurred in the NE-challenged chickens after the acute challenge period around 6 to 7 dpi (Goo et al., 2023a, 2024a).

Significant weight differences between ACRB and Cobb 500 were confirmed by body composition analysis using DEXA on d 25, and we investigated how the NE challenge altered body composition and body mineral content. The Cobb 500 chickens showed significantly higher total tissue weight, lean weight, fat weight, BMD, BMC, BMCR, and BA compared to the ACRB (Table 3). This result was caused by a significant difference in BW between ACRB and Cobb 500 (Collins et al., 2014; Goo et al., 2024a). Conversely, the lean ratio of Cobb 500 was lower than that of ACRB. This discrepancy may arises because the lean ratio analyzed by DEXA in the current study does not account only for ingestible lean meat; it includes all unremoved parts of the carcass, such as viscera, feathers, head, feet, skin, and wings. Consequently, the relatively high weights of these components in ACRB may have resulted in an increased lean ratio (Collins et al., 2014). Necrotic enteritis infection (NE2) significantly reduced both the total tissue weight of the chickens as well as their mineral content in the body in the current study. Bone mineral content represents the total amount of minerals measured by DEXA but is generally highly influenced by BW in chickens. Therefore, to more accurately determine changes in mineral amounts, weight factors must be calculated and provided together with BMC (Goo et al., 2025). In the current study, the NE-challenged groups (NE2) had reduced both BMC and BMCR, indicating that NE infection contributed not only to weight loss but also to decreased body mineral levels in the chickens. The decrease in BMC levels in the NE challenge group (NE2) may be that the overall deterioration of intestinal health due to NE infection affects normal nutrient absorption and metabolism, therefore reducing mineral digestibility in broilers (Zanu et al., 2020a; Goo et al., 2023a).

To investigate how NE infection affects bone growth in two different chicken strains, femoral MAR was measured, and MicroCT was utilized to evaluate changes in femoral microstructures. The femur cortical MAR analysis using calcein labeling was used in parallel with MicroCT analysis in the current study, as bone growth by osteoblasts can be visually and intuitively observed within particular periods (Fig. 1; Sharma et al., 2023a; Shi et al., 2024). The femoral MAR showed a significant difference, with values approximately twice as large in the Cobb 500 compared to the ACRB. This result indicates that the cortical bone of the femur in the Cobb 500 is growing at a faster rate compared to the ACRB. Although the NE challenge numerically reduced femoral MAR, it did not result in a significant difference. While few studies have measured the relationship between NE challenge and femoral MAR, the result of the current study was contrary to those of previous Eimeria challenge studies, which significantly reduced femoral MAR (Sharma et al., 2023a; Shi et al., 2024). These differences may be due to previous experiments using laying hens as experimental animals or differences in the level of Eimeria infection. Notably, the effect of the NE challenge on femoral MAR was similar in both chicken strains, ACRB and Cobb 500. This suggests that the negative effects of NE infection on femoral growth are not significantly different between modern meat-type chickens and the genetically non-selected conventional type ACRB.

To measure the femoral microstructure, the metaphysis of the femur was analyzed by dividing it into three different parts using 3D MicroCT scanning, including total VOI, cortical bone, and trabecular bone (Fig. 2; Chen et al., 2020). In the total VOI of the femur, Cobb 500 had significantly increased most of the measurements, including BMC, TV, BV, BS, BSR, and BSD, compared to the ACRB (Table 4). This reflects the difference in femur size, as the weight difference between the two chicken strains at the time of sampling on d 25 was approximately 5.6 times (ACRB: 208 g and Cobb 500: 1,162 g). The NE challenge (NE2) significantly reduced femoral TV and BV in the total VOI. Currently, few studies have directly compared the relationship between the NE challenge and the volume (TV or BV) of long bones such as the femur, tibiotarsus, and humerus. Studies related to NE infection and the length or width of long bones have been conducted previously; however, no reduction in bone growth due to the NE challenge has been confirmed (Zanu et al., 2020b, 2021; Tomczyk-Warunek et al., 2021). In addition, TV and BV measurements in the current experiment were analyzed by finely slicing specific distal metaphysis sections of the femur (Fig. 2), thus it is difficult to interpret those parameters in connection with previous measurement methods such as femoral length, width, diameter, area, or weight. Instead, it can be explained in association with the same measurements between Eimeria infection and bone volume metrics, and it has been reported that the deterioration of intestinal health due to coccidiosis may affect mineral utilization and inhibiting osteoblast activity, resulting in reduced growth in broilers and reduced femoral TV and BV (Tompkins et al., 2023a; Liu et al., 2024; Lopes et al., 2024). Therefore, low femoral TV and BV in NE challenged group in the current study may be attributed to inadequate bone growth resulting from deterioration of intestinal health similar to the previous coccidiosis studies.

Interestingly, despite significant size differences between ACRB and Cobb 500 chickens, ACRB exhibited increased femoral BMD in total VOI than Cobb 500 (Table 5). Bone mineral density is a biophysical parameter that represents the mineral content (mg) per unit area (cm²) and is an important indicator that can indirectly validate bone strength and integrity (Shao et al., 2019). Contrary to the absolute indicators, such as BMC, TV, and BV, increased femoral BMD in ACRB may have been caused by the slower growth rate of ACRB compared to the Cobb 500, which gives more time for thorough mineral deposition in the bones and makes more denser bones in ACRB. Previous studies have reported that as tibiotarsus and femur size increase with age, BMD levels may not absolutely increase but rather decrease in broilers (Talaty et al., 2009; Charuta et al., 2013; Damaziak et al., 2019). In addition, it has been reported that tibiotarsus BMC and bone strength increase with age in broilers, whereas tibiotarsus BMD can decrease during the fast-growing period (14 and 21 days of age; Shao et al., 2019). As the bone size increases with age, not only does the area of cortical bone increase but also the area of trabecular bone (Kim et al., 2012; Sharma et al., 2023b). This indicates that the measurement of long bone BMD in broilers may be determined by the ratio of cortical bone to trabecular bone, and increased bone size and weight may lead to decreased BMD in broilers. On the other hand, in the current study, the femoral BMD was analyzed separately in cortical bone and trabecular bone, and the femoral BMD of ACRB was higher than that of Cobb 500 in any area, further indicating that the differences in femoral BMD levels are likely due to the differences in growth rates of two chicken strains. Shim et al. (2012) reported that slow-growing chickens had higher BW-corrected tibiotarsus mineral density compared to fast-growing chickens. Other studies have reported that the high growth rate of fast-growing chickens can be accompanied by increased porosity for bone growth with decreased mineralization, which can lead to lower bone density and an increased risk for bone disorders (Williams et al., 2004; Rawlinson et al., 2009). In other words, the current study again confirmed that there are further needs for bone health improvement in modern broiler chickens along with continuously improvement of meat-yield and feed efficiency.

Similar results to the total VOI of the femur were also observed in cortical bone microstructure analysis, and in particular, the quantitative indicators, including BMC, TV, BV, and BS, showed significant differences due to the femur size between the ACRB and Cobb 500 (Table 6). On the other hand, the relative parameters, BVR and BSD, were significantly higher in the ACRB than the Cobb 500 chickens. Particularly, the Cobb 500 had no significantly higher parameters compared to ACRB chickens in any relative indicators in the femoral cortical bone microstructure analysis. This outcome can be explained in connection with the high femoral BMD of ACRB. Bone volume ratio and BSD are indices representing the mineralized bone segment (BV) and mineralized internal surface area (BS) per total TV, which are assessed within the specific metaphysis slide of the cortical bone, respectively (Adams et al., 2022; Liu et al., 2024). The lower cortical BVR and BSD of the Cobb 500 indicate that the degree of mineralization in the femur is lower than that of the ACRB, which is also related to the lower femoral BMD of the Cobb 500 in the current study. Previous studies have reported that if the BVR value of long bones is low, BMD can also be low (Tompkins et al., 2022; Sharma et al., 2023a; Shi et al., 2023; Liu et al., 2024). The decrease in cortical BVR also indicates that the femoral cortical porosity has increased in the Cobb 500 (Williams et al., 2004; Cooper et al., 2016). This is because there is an absolute correlation between BVR and OPR (open porosity ratio), which can be verified by reversing the calculation of BVR (BV/TV) to obtain OPR values (TV/BV; BVR + OPR = 100%; Adams et al., 2022).

The Cobb 500 had significantly increased all femoral cortical bone closed and open porosity measurements compared to the ACRB (Table 7). The increased porosity parameters of Cobb 500 chickens are closely related to the low cortical BVR mentioned above. Cortical porosity is important not only because of its role in providing physical and mechanical support abilities but also in bone growth and remodeling processes (Cooper et al., 2016). The porosity of the cortical bone is closely linked to the trabecular cavity and plays a key role in the connection of osteocytes, which serve as a mediator of dynamic reactions such as cortical bone remodeling (Cooper et al., 2016). In other words, the high porosity parameters of Cobb 500 chickens in the current study are the main characteristic of fast-growing chickens (Williams et al., 2004; Rawlinson et al., 2009), possibly because they require a more porous space due to significantly higher growth rates and higher mineral metabolism than ACRB. On the other hand, increased cortical porosity from a mechanical perspective is associated with weak mechanical tensile, compressive strength, and elastic modulus, resulting in the inability to support significant loads (Currey, 2004; Augat and Schorlemmer, 2006; MacNeil and Boyd, 2007). This, in turn, indicates that the developmental mechanisms for cortical bone porosity in modern-type chickens may act as a double-edged sword of high growth rate and low bone strength.

Similar to the results of femoral total VOI and cortical bone microstructure, the quantitative parameters of femoral trabecular bone microstructure measurements, including BMC, TV, BV, and BS, were significantly higher in Cobb 500 chickens than in ACRB (Table 8). This difference was likely caused by variations in bone size between the two chicken strains, and in particular, the trabecular bone results were almost similar to the total VOI results rather than the cortical bone microstructure results. The fact that the results of the trabecular bone microstructure analysis were similar to the total VOI can also be confirmed by the reduction of the femoral TV in NE-challenged groups. This may be because the selection area of the femur in the current experiment was distal metaphysis, and the trabecular bone is the most pronounced part of the metaphysis of the femur (Cooper et al., 2016). The lower trabecular TV in the NE-challenged group compared to the NC may be due to growth retardation which is caused by decreased mineral (nutrient) digestibility, reduced feed consumption, and lower feed efficiency resulting from deteriorated intestinal health under NE-challenged conditions (Goo et al., 2023a; Lopes et al., 2024). It is also possible that the inflammatory response generated by NE coinfection model (Eimeria + C. perfringens) may have inhibited the activity of osteoblasts and increased the activity of osteoclasts in the femur, thereby impairing proper bone formation and increasing resorption in broilers (Tompkins et al., 2023a; Shi et al., 2024). Body weight is the most important factor in bone parameter changes, but the analysis using MicroCT did not necessarily lead to a decrease in TV (Tompkins et al., 2023a; Liu et al., 2024; Lopes et al., 2024; Shi et al., 2024). This may occur because bone sampling was performed (6 dpi) before the challenge factors causing severe weight loss interfered with bone growth in chickens, and a positive relationship was observed between BW and TV when the sampling time was delayed to 9 dpi (Liu et al., 2024; Lopes et al., 2024).

Feed intake is an important parameter for bone health, and previous studies have reported that bone mineralization and tibiotarsus porosity can be significantly influenced by feed consumption levels rather than by strain differences (Williams et al., 2004). In addition, Tompkins et al. (2023a) reported that the reduction of feed consumption in broilers resulted in the reduction of femoral total BMC and trabecular BMD, similar to chickens in the Eimeria challenge groups. In the current study, the FI of the NE2 group was significantly reduced compared to the NC group during the maximal NE challenge period from 5 to 7 dpi (ACRB, -25% FI compared to the NC; Cobb 500, -12% FI compared to the NC; data not shown). Although the NE-challenged chickens recovered their FI thereafter, the reduced feed consumption during the maximal challenge period (5 to 7 dpi) may have contributed to lower femoral trabecular TV on d 25 (11 dpi). Conversely, most measurements of femoral cortical and trabecular microstructure were not significantly affected, except for femoral TV and BV of the total VOI. Consequently, it is possible that the recovery of FI in the NE challenge groups did not produce significant differences in the femoral trabecular parameters. The deterioration of intestinal health caused by the NE challenge not only reduces nutrient intake and nutrient digestion efficiency but may also inhibit osteoblast activity and stimulate osteoclast activity due to inflammatory reactions and oxidative stress, which can hinder proper bone growth (Choppa et al., 2023; Tompkins et al., 2023b). Further studies are needed to explore the specific association between NE infection, bone growth, and nutrient intake.

The measurement of femoral trabecular bone development and porosity microstructure also showed no significant difference by NE challenge, which is similar to the cortical bone porosity measurements in the current study. On the other hand, although significant differences were found between the ACRB and Cobb 500 chickens in most measurements, the significant BW differences between the two chicken strains did not show consistent differences in the trabecular developmental microstructure (Table 9). Cobb 500 chickens showed increased TbS, TbN, TbC, and TbCD compared to the ACRB, whereas ACRB had increased TbPF, SMI, and TPR compared to the Cobb 500. Femoral trabecular bone is very porous, and due to its large surface area, bone remodeling and mineral metabolism are more active than in cortical bone (Sharma et al., 2023b). Harash et al. (2020) compared Ross with a dual-purpose strain (Lohmann Dual), and Ross chickens showed significantly higher BW than the dual-purpose strain after 7 days, with increased tibiotarsus TbN and decreased TbS; however, TbT showed little difference between the two chicken strains. Additionally, Oláh et al. (2023) reported that the deterioration of bone health, such as knee osteoarthritis in animals, may initially lead to subchondral trabecular loss and degeneration, which can reduce TbN and TbCD, while increasing TbS and TbPF. Inadequate growth in situations such as Eimeria challenge and feed restriction in broilers can also affect long bone development, resulting in changes in the trabecular microstructure. Furthermore, Eimeria challenge caused significant weight loss in broilers and decreased femoral TbN and TbCD, but increased TbT, TbS, and TbPF (Liu et al., 2024; Lopes et al., 2024). In addition, Tompkins et al. (2023a) reported that inhibition of bone growth due to Eimeria challenge or restriction of feed consumption can reduce femoral TbN. Thus, increased levels of femoral TbN and TbCD and decreased TbPF levels in Cobb 500 chickens may suggest that more active trabecular bone growth and metabolism were achieved compared to the ACRB, which may support the finding that Cobb 500 exhibited increased trabecular BVR. Femoral trabecular SMI was also higher in ACRB than in Cobb 500 chickens, which may indicate more perforation and decreased trabecular quality (Akhter et al., 2007), and has also been reported to increase in Eimeria-challenged chickens (Tompkins et al., 2022; Sharma et al., 2023a). On the other hand, as opposed to the high SMI in the ACRB, TbS levels were decreased, and TbT levels also showed no significant difference between the two chicken strains. Because previous studies have reported that SMI itself may be defective in interpreting various shapes of curved trabecular bones, further analysis is needed of indicators more related to trabecular bone development (Salmon et al., 2015; Felder et al., 2021; Xiong et al., 2023). Therefore, differences in femoral trabecular parameters in the current study may be attributed to unique differences in two chicken strains highlighted by different growth rates.

In conclusion, the modern meat-type, fast-growing Cobb 500 chickens showed a superior growth rate and efficiency compared to the non-selected conventional-type ACRB. The Cobb 500 showed significantly higher feed consumption and feed efficiency, in Cobb 500, resulting in increased total tissue weight, lean weight, BMC, femoral MAR, femoral TV, and femoral BV compared to the ACRB. However, the Cobb 500 also experienced a decrease in its lean ratio due to increased fat accumulation and showed significantly lower BMD in all sections of the femur. This can be attributed to the low BVR of cortical bone and increased porosity parameters, suggesting that the higher bone growth rate and mineral metabolism due to the increased growth rate of the Cobb 500 may compromise bone strength, adversely affecting bone health. The NE challenge significantly lower weight gain, feed efficiency, total tissue weight, BMD, BMCR, and femoral TV and BV. However, no significant differences were observed in most other parameters, including femoral MAR, BVR, BMD, cortical porosities, and trabecular developments, regardless of chicken strain. This result may be due to the recovery of feed consumption during the NE challenge recovery phase; however, further research is required for better understanding of the detailed relationships of strain differences, growth rates, and bone development in broilers under enteric disease conditions to maintain efficient growth and improve bone health, promoting sustainable poultry production.

Disclosures

The authors declare no conflict of interest regarding the publication of the current research paper. The authors affirm that this study was conducted without any commercial or financial relationships that might pose a potential conflict of interest.