Different methionine to cysteine supplementation ratios altered bone quality of broilers with or without Eimeria challenge assessed by dual energy X-ray absorptiometry and microtomography

Abstract

Despite the acknowledged significance of nutrition in bone development, effects of methionine (Met) and cysteine (Cys) on bone quality remain under-researched, particularly during Eimeria challenge. We investigated the effects of different supplemental Met to Cys ratios (MCR) on bone quality of broilers under Eimeria challenge. A total of 720 fourteen-day old Cobb500 broilers were allocated into a 5 × 2 factorial arrangement. Five diets with Met and Cys supplemented at MCR of 100:0, 75:25, 50:50, 25:75, and 0:100 were fed to the birds with or without Eimeria challenge. Body composition was measured by dual energy x-ray absorptiometry, and the femur bone characteristics were assessed by microtomography. Data were analyzed by two-way ANOVA and orthogonal polynomial contrast. The results reaffirmed the detrimental effects of Eimeria challenge on bone quality. On 9 d post inoculation (DPI), significant interaction effects were found for whole body bone mineral content (BMC), lean tissue weight, and body weight (P < 0.05); in the nonchallenged group (NCG), these parameters linearly decreased as MCR decreased (P < 0.05). In the challenged group (CG), body weight and lean tissue weight were unaffected by MCR, and BMC linearly increased as MCR decreased (P < 0.05). For the cortical bone of femoral metaphysis on 6 DPI, bone mineral density (BMD) linearly increased as MCR decreased (P < 0.05). Bone volume to tissue volume ratio (BV/TV) in the CG linearly increased as MCR decreased (P < 0.05). On 9 DPI, BMC and TV linearly increased as MCR decreased (P < 0.05) in the NCG. BMD and BV/TV changed quadratically as MCR decreased (P < 0.05). For the trabecular bone of femoral metaphysis on 9 DPI, BV/TV, and trabecular number linearly increased as MCR decreased (P < 0.05) in the NCG. For the femoral diaphysis, BV, TV, BMC on 6 DPI, and BMD on 9 DPI linearly increased as MCR decreased (P < 0.05). In conclusion, this study showed that both Eimeria challenge and varying supplemental MCR could influence bone quality of broilers.

INTRODUCTION

One of the main goals of the broiler industry is to ensure the optimal growth and well-being of the broiler chickens to maximize the productivity of the birds (Hafez and Attia, 2020). Bone health is crucial for broilers, as it supports their rapid growth rate and ensures efficient mobility (Bradshaw et al., 2002; Fleming, 2008). A robust skeletal structure is also vital for minimizing the risk of leg disorders and fractures (Kierończyk et al., 2017). Maintaining optimal bone health in broilers is thus essential for ensuring the welfare and performance of the broiler birds (Kim et al., 2007; Kolakshyapati et al., 2019).

Coccidiosis, caused by the protozoan parasite of the genus Eimeria, has long been a threat to the broiler production (Dalloul and Lillehoj, 2006; Mesa-Pineda et al., 2021). Eimeria spp. damage the integrity of the intestinal lining of the broilers, leading to anorexia, reduced body weight gain, and even mortality (Yadav et al., 2020; Choi et al., 2023; Liu et al., 2023). Moreover, coccidiosis presents considerable risks to the bone health of birds by suppressing the bone growth rate and impairing bone quality (Fetterer et al., 2013b; Sakkas et al., 2019; Sharma et al., 2023b). Recent studies have highlighted that these risks can be attributed to the disruption of bone homeostasis resulting from the oxidative stress induced by coccidiosis (Tompkins et al., 2023a; Tompkins et al., 2023b).

Nutrients play a crucial role in maintaining the bone health of broilers. Numerous studies have explored the effects of minerals, such as calcium and phosphorus (David et al., 2023), as well as the impact of other nutrient supplementations such as vitamin D, vitamin K, and phytase on the bone development and growth of broilers (Kim et al., 2011; Santos et al., 2019; Adhikari et al., 2020; Shi et al., 2023). However, few studies have been carried out to investigate the effects of functional amino acids on the development and quality of broiler bones.

Methionine (Met) is considered as the first limiting amino acid in broilers fed corn soybean meal based diets (Bunchasak, 2009; Attia et al., 2020) and maintains the normal growth and functions of the broilers (Fagundes et al., 2020; Liu et al., 2022). Cysteine (Cys) is considered a semi-essential amino acid since it can be converted from Met (Castro and Kim, 2020). Both Met and Cys are classified as sulfur amino acids (SAA) that possess potent antioxidant capacity due to their participation in glutathione synthesis (Bianchi et al., 2000; Atmaca, 2004; Bin et al., 2017). Several studies have also demonstrated that both SAA can directly scavenge reactive oxygen species (Elias et al., 2005; Moskovitz, 2005; Miura et al., 2014) further contributing to their antioxidant capabilities. It has been proposed that the detrimental effects of coccidiosis on bone quality could be partially attributed to the oxidative stress induced by the infection (Tompkins et al., 2023a; Tompkins et al., 2023b). This stress increases the osteoclast activity, leading to increased bone resorption and disruption in the balance of bone remodeling, thus contributing to the deterioration of bone quality (Banfi et al., 2008). Therefore, supplementation of SAA might be beneficial for bone health. Multiple studies have shown that dietary SAA supplementations could improve the oxidative status of broilers under challenged conditions (Liu et al., 2019; Magnuson et al., 2020).

Despite the beneficial effects of Met, over supplementation of Met could lead to undesired accumulation of homocysteine (Hcy) (Finkelstein and Martin, 1986; Finkelstein, 1998; Yang et al., 2020), which has been correlated with compromised bone health and increased fracture rate (Behera et al., 2017; Kumar et al., 2017). The NRC recommended a Met: Cys ratio (MCR) of 53: 47 for the broilers (NRC, 1994), Cobb and Aviagen recommended MCR of 56: 44 (Cobb-Vantress, 2018a) and 52: 48 (Aviagen, 2022) respectively. Previous studies also suggested the optimal MCR of 50: 50 for broilers (Powell et al., 2011; Kahn et al., 2015). Under common production practices, only Met is supplemented to meet the SAA requirement that is higher than the recommendation levels of Met without any negative impact on bone traits. However, in birds challenged with Eimeria, the induced oxidative stress and inflammation could potentially amplify the adverse consequences of Hcy accumulation, thereby further compromising bone health. Known that Cys also possesses potent antioxidant capacity without contributing to Hcy accumulation (Atmaca, 2004; Otasevic and Korac, 2016). Partially substituting Met supplementation with Cys in diets of broilers challenged by Eimeria might potentially improve the bone health of the birds. As this topic has been scarcely explored in previous research, the current study was conducted to investigate the effects of different supplemental Met: Cys ratios on the bone characteristics of broilers under Eimeria challenge.

MATERIAL AND METHODS

All the animal experiment procedures used in this study were approved by the Institutional Animal Care and Use Committee of the University of Georgia (A2021 12-012).

Experimental Design and Birds Husbandry

A total of 720 male Cobb500 broilers, aged 14 d, were assigned randomly into ten treatment groups, utilizing a 2 × 5 factorial arrangement. The birds were provided with 5 treatment diets, either with or without an Eimeria challenge. Each treatment consisted of 6 replicates with 12 birds per replicate. The birds were reared in battery cages with feed and water provided ad libitum. Temperature and lighting conditions were regulated according to the Cobb 500 Broiler Management Guide (Cobb-Vantress, 2018b).

Five treatment diets were fed to the birds from day 14 to 23. The diets were supplemented with crystalline forms of Met and Cys in various ratios, with the prerequisite that the sum of supplemented Met and Cys remained unchanged. The concentrations of other nutrients and the overall levels of sulfur amino acids (Met + Cys) were maintained consistently across all treatments, following the breeder recommendation (Cobb-Vantress, 2018a). The control diets were formulated to meet the SAA requirement through only Met supplementation (MET100), and the subsequent diets had a gradual replacement of Met supplementation with Cys at levels of 25, 50, 75, and 100%. These diets were labeled as MET75, MET50, MET25, and MET0, respectively. Detailed information regarding the feed ingredients and the chemical composition of the diets is presented in Table 1.

Table 1.

Feedstuffs and chemical compositions (g/kg) of experimental diets.

Ingredients	Starter	Grower (Supplemented Met: Cys ratio of TSAA)1
		100:0	75:25	50:50	25:75	0:100
Treatment Label		MET100	MET75	MET50	MET25	MET0
Corn	601	628	628	628	628	628
Soybean meal	347	312	312	312	312	312
Soybean Oil	13.1	18.6	18.6	18.6	18.6	18.6
Common Salt	3.50	3.5	3.5	3.5	3.5	3.5
Limestone	11.7	11.0	11.0	11.0	11.0	11.0
Dicalcium Phosphate	15.8	14.5	14.5	14.5	14.5	14.5
Vitamin Premix2	1.00	1.00	1.00	1.00	1.00	1.00
Mineral Premix3	0.80	0.80	0.80	0.80	0.80	0.80
Methionine	3.00	2.90 (100%)	2.18 (75%)	1.45 (50%)	0.72 (75%)	0.00 (0%)
Cysteine	0.00	0.00 (0%)	0.72 (25%)	1.45 (50%)	2.18 (25%)	2.90 (100%)
L-Lysine HCl	2.00	1.90	1.90	1.90	1.90	1.90
L-Threonine	0.90	0.40	0.40	0.40	0.40	0.40
Titanium Dioxide	0.00	3.00	3.00	3.00	3.00	3.00
Sand	0.00	2.00	2.00	2.00	2.00	2.00
Total	1000	1000	1000	1000	1000	1000
Calculated Nutrients (g/kg) and Energy
ME, kcal/kg	3010	3060	3060	3060	3060	3060
Protein	21.3	20.0	20.0	20.0	20.0	20.0
Dig Lys	1.22	1.12	1.12	1.12	1.12	1.12
Dig Met4	0.62	0.57 (67%)	0.50 (59%)	0.43 (50%)	0.35 (41%)	0.28 (33%)
Dig Cys	0.29	0.28 (33%)	0.35 (41%)	0.43 (50%)	0.50 (59%)	0.57 (67%)
Dig TSAA	0.91	0.85	0.85	0.85	0.85	0.85
Ca	0.90	0.84	0.84	0.84	0.84	0.84
Available P.	0.45	0.42	0.42	0.42	0.42	0.42
Analyzed Values (%)
Protein	21.1	20.6	19.6	20.1	19.4	19.8
Lysine	1.37	1.25	1.32	1.23	1.28	1.29
Methionine	0.61	0.65 (67%)	0.55 (58%)	0.52 (55%)	0.45 (46%)	0.37 (39%)
Cysteine	0.35	0.32 (33%)	0.40 (42%)	0.43 (45%)	0.53 (54%)	0.58 (61%)
TSAA	0.96	0.97	0.94	0.95	0.97	0.95

¹Five grower treatment diets with supplemental methionine and cysteine at different ratios when total sulfur amino acids remained the same (Met: Cys at 100: 0; 75; 25; 50: 50; 25: 75; 0: 100%) were fed to the birds.

²Vitamin Premix: Supplemented per kg of diet: thiamin mononitrate, 2.4 mg; nicotinic acid, 44 mg; riboflavin, 4.4 mg; D-Ca pantothenate, 12 mg; vitamin B12 (cobalamin), 12.0 g; pyridoxine HCl, 4.7 mg; D-biotin, 0.11 mg; folic acid, 5.5 mg; menadione sodium bisulfite complex, 3.34 mg; choline chloride, 220 mg; cholecalciferol, 27.5 g; transretinyl acetate, 1,892 g; α tocopheryl acetate, 11 mg; ethoxyquin, 125 mg.

³Mineral Premix: Supplemented as per kg of diet: manganese (MnSO₄.H₂O), 60 mg; iron (FeSO₄.7H₂O), 30 mg; zinc (ZnO), 50 mg; copper (CuSO₄.5H₂O), 5 mg; iodine (ethylene diaminedihydroiodide), 0.15 mg; selenium (NaSeO₃), 0.3 mg.

⁴Values in the parentheses indicate the ratios of total Met and Cys of TSAA.

On day 14, the birds in the challenge groups (CG) were orally inoculated with mixed Eimeria spp., containing 25,000 sporulated oocysts of E. maxima, 25,000 sporulated oocysts of E. tenella, and 125,000 sporulated oocysts of E. acervuline. On the other hand, the non-challenge groups (NCG) were sham-challenged by oral inoculation of water.

Dual-energy X-ray Absorptiometry for Body Composition Analysis

On 9 d post inoculation (DPI), 1 bird per cage was euthanized by cervical dislocation and scanned by a GE whole-body dual-energy X-ray absorptiometry (DEXA) scanner (GE Healthcare, Chicago, IL) for body composition analysis, following the method described by Chen et al. (2020). The sample birds were positioned chest up on the scanner. The scanning mode was set for small animals. Subsequent to scanning, the region of interest was specified for each individual bird for the measurements of whole-body bone density, mineral content, fat percentage, and lean tissue weight.

Microtomography (Micro-CT) for Microstructural Analysis of the Femur Bone

Femur bones were collected from 1 bird per cage on 6 and 9 DPI. The bones were wrapped in wet cheesecloth after removal of the soft tissues. The samples were stored at −20°C until further analysis. Prior to scanning, the samples were fully thawed and then held in a low-density 50 mL tube to prevent any random movement during the scanning. The samples were scanned by the Skyscan Micro-CT scanner (Skyscan 1275; Bruker MicroCT, Billerica, MA) for 3-dimensional image acquisition. The X-ray source was set to 75 kV and 133 μA. A 0.5 mm aluminum filter was used to reduce beam hardening. The pixel size was fixed at 25 μm. The 180 ^o scanning was applied with a rotation angle of 0.4°, and 4 images were captured per rotation. The captured 2D images were then reconstructed by the N-Recon program (Bruker MicroCT) to generate a 3D model. The resulting model was straightened using the Data Viewer program (Bruker MicroCT) and subsequently transferred to CTAn program (Bruker MicroCT) for the region of interest (ROI) selection. The distal metaphysis of 300 slides (7.5 mm in height) and the diaphysis of 200 slides (5 mm in height) were selected for analysis (Figure 1). The 3D model was then processed for cortical and trabecular bone separation according to previously described procedures (Chen and Kim, 2020; Shi et al., 2023), and the parameters listed and described (Sharma et al., 2023b) in Table 2 were measured. Two phantoms of known density (0.25 and 0.75 g/cm³) made from calcium hydroxyapatite were scanned for calibration of bone mineral density (BMD).

Figure 1.

The region of interest (ROI) selection for the analysis. The same ROI was selected for all the samples. For the metaphysis, 300 slides (7.5 mm) were selected and for the diaphysis, 200 slides (5 mm) were selected.

Table 2.

Definition and description of microtomography measured parameters.

Parameters (Abbreviation)	Description	Standard unit
Bone mineral density (BMD)	Measure the bone mineral content per unit of volume	g/cm3
Bone mineral content (BMC)	Measure the bone mineral content of the tissue. The value is calculated by BMD × TV	g
Tissue volume (TV)	The volume of the entire region of interest, including pores and cavity inside the bone	mm3
Bone volume (BV)	Volume of the bone segments	mm3
Bone volume fraction (BV/TV)	Ratio of the bone volume to the tissue volume	%
Number of closed pores (NCP)	Number of closed pores within the bone segments
Volume of closed pores (VCP)	Total volume of the closed pores	mm3
Closed pore percentage (CPP)	The volume of closed pores as a percentage of the bone volume	%
Trabecular number (TBN)	The average number of trabeculae per unit length	1/mm
Trabecular thickness (TBT)	Mean thickness of trabeculae, measured using 3D methods	mm
Trabecular separation (TBS)	Mean distance between trabeculae, measured using 3D methods	mm
Connectivity density (CD)	A measure of the degree of connectivity of trabeculae normalized by tissue volume	1/mm3

Statistical Analysis

The PROC GLM program of SAS software (SAS Institute Inc., Cary, NC) was used for the statistical analysis of the obtained data. The data were analyzed by two-way ANOVA. The Tukey's honestly significant difference test was applied to separate means when significant differences were detected. Orthogonal polynomial contrasts were used to assess linear and quadratic trends between the different MCRs and measured parameters. Statistical significance was set at P ≤ 0.05.

RESULTS

Body Composition Analyzed by Dual-energy X-ray Absorptiometry

Eimeria challenge significantly decreased the BMD, fat percentage, and fat weight of the birds (P < 0.001) as shown in Table 3. Significant diet by challenge interaction effects were observed in total body weight of the scanned bird (BW) (P = 0.002), bone mineral content (BMC), and lean tissue weight (P = 0.013) (Figure 2). The BW linearly or quadratically decreased as MCR decreased (P < 0.001) in the NCG. However, in the CG, BW was not affected by MCR. The lean tissue weight linearly or quadratically decreased as MCR decreased in the NCG (P < 0.001 or P = 0.002). However, it was not affected by MCR in the CG. The BMC linearly decreased as MCR decreased in the NCG (P = 0.012). On the contrary, the BMC linearly increased as MCR decreased in the CG (P = 0.002).

Table 3.

Effect of different supplemental methionine to cysteine ratios and Eimeria challenge on the body composition of the broilers on 9 DPI.

Items1		BMD	Fat%	Fat Weight
Challenge
NCG (-)		0.147a	12.3a	108b
CG (+)		0.133b	10.2b	71.8a
Met: Cys
100: 0		0.136	11.1	90.2
75: 25		0.142	11.5	99.6
60: 40		0.136	10.7	84.5
25: 75		0.143	11.2	90.8
0: 100		0.140	11.8	84.1
Interaction
-	100: 0	0.144	11.8	108
-	75: 25	0.149	12.1	119
-	60: 40	0.146	12.2	105
-	25: 75	0.147	12.3	110
-	0: 100	0.146	13.2	96.9
+	100: 0	0.128	10.3	72.4
+	75: 25	0.134	10.9	80.3
+	60: 40	0.128	9.47	63.5
+	25: 75	0.138	10.4	71.6
+	0: 100	0.134	11.8	71.3
P value
Challenge		<0.001	<0.001	<0.001
Diet		0.214	0.803	0.408
Challenge × Diet		0.689	0.821	0.907
P trend
Linear		0.231	0.541	0.292
Linear at NCG		0.798	0.265	0.270
Linear at CG		0.151	0.798	0.697
Quadratic		0.423	0.569	0.651
Quadratic at NCG		0.466	0.749	0.369
Quadratic at CG		0.689	0.626	0.794
SEM		0.003	0.62	8.83

^a-bMeans within a column lacking a common superscript differ (P ≤ 0.05).

¹DPI, days post inoculation; BMD; bone mineral density (g/cm³); Fat weight (g).

²NCG, non-challenged group; CG, challenged group.

³MET100, Met and Cys supplemented at the ratio of 100:0; MET75, Met and Cys supplemented at the ratio of 75:25; MET50, Met and Cys supplemented at the ratio of 50:50; MET25, Met and Cys supplemented at the ratio of 25:75; MET0, Met and Cys supplemented at the ratio of 0:100.

Figure 2.

Body composition analyzed by the Dual-energy X-ray absorptiometry. MET100, Met: Cys = 100: 0; MET75, Met: Cys = 75:25; MET50, Met: Cys = 50:50; MET25, Met: Cys = 25:75; MET0, Met: Cys = 0:100. The error bars represent the SEM values. Bars without a common letter differ significantly. (A), P-values: P-interaction < 0.001, P-linear at NCG < 0.001, P-linear at CG = 0.360. P-quadratic at NCG < 0.001, P-quadratic at CG = 0.879. (B), P-values: P-interaction = 0.013, P-linear at NCG < 0.001, P-linear at CG = 0.637, P-quadratic at NCG = 0.002, P-quadratic at CG = 0.749. (B), P-values: P-interaction = 0.013, P-linear at NCG = 0.012, P-linear at CG = 0.002.

Femur Bone Microstructure Analyzed by Microtomography

Metaphysis Cortical Bone. On 6 and 9 DPI, Eimeria challenge significantly decreased BMC, tissue volume (TV), bone volume (BV), number of closed pores (NCP), volume of closed pores (VCP), and closed pore percentage (CPP) (P < 0.001) (Table 4). On 6 DPI, a significant diet effect was observed for the BMD where it linearly decreased as MCR decreased (P = 0.028) as shown in Figure 3. A significant diet by challenge interaction effect was observed for the bone volume fraction (BV/TV) (P = 0.036) as it linearly increased as MCR decreased only in the CG (P = 0.045) but not in the NCG. On 9 DPI, the BMD and BV/TV were higher in the CG than the NCG (P < 0.001) (Figure 4). Furthermore, the BMD and BV/TV changed quadratically as MCR decreased (P = 0.016 and 0.010). The BMC and TV in the NCG linearly increased as MCR decreased (P = 0.032).

Table 4.

Effect of different supplemental methionine to cysteine ratios and Eimeria challenge on the femoral metaphysis cortical bone quality.

		6 DPI						9 DPI
Items1		BMC	TV	BV	NCP	VCP	CPP	BV	NCP	VCP	CPP
Challenge2
NCG (-)		0.068a	151a	123a	739a	0.334a	0.268a	160a	784a	0.441a	0.283a
CG (+)		0.051b	115b	94.1b	546b	0.188b	0.196b	116b	418b	0.250b	0.211b
Diet3
MET100		0.055	124	99.9	619	0.259	0.240	132	566	0.326	0.242
MET75		0.061	137	110	748	0.280	0.252	139	592	0.323	0.223
MET50		0.060	129	108	649	0.290	0.257	135	607	0.381	0.272
MET25		0.061	138	112	629	0.255	0.219	145	674	0.369	0.248
MET0		0.062	137	112	569	0.221	0.193	139	568	0.329	0.227
Interaction
-	MET100	0.062	141	113	512	0.332	0.062	146	706	0.397	0.266
-	MET75	0.070	155	127	680	0.342	0.070	167	826	0.443	0.264
-	MET50	0.070	151	125	511	0.401	0.070	152	724	0.435	0.279
-	MET25	0.070	159	127	556	0.322	0.070	170	894	0.481	0.283
-	MET0	0.068	150	122	471	0.273	0.068	163	770	0.448	0.273
+	MET100	0.048	111	89.3	727	0.186	0.048	118	426	0.256	0.217
+	MET75	0.051	119	93.4	816	0.218	0.051	111	359	0.204	0.182
+	MET50	0.050	107	91.2	786	0.180	0.050	118	489	0.326	0.264
+	MET25	0.052	113	94.6	701	0.187	0.052	119	453	0.256	0.213
+	MET0	0.057	124	102	668	0.169	0.057	114	366	0.210	0.181
P value
Challenge		<0.001	<0.001	<0.001	<0.001	<0.001	<0.001	<0.001	<0.001	<0.001	0.001
Diet		0.476	0.531	0.569	0.296	0.541	0.223	0.338	0.444	0.625	0.431
Challenge × Diet		0.816	0.707	0.809	0.921	0.698	0.674	0.169	0.293	0.514	0.660
P trend
Linear		0.105	0.261	0.160	0.251	0.306	0.075	0.182	0.557	0.635	0.943
Linear at NCG		0.334	0.427	0.398	0.396	0.330	0.166	0.077	0.354	0.368	0.723
Linear at CG		0.179	0.424	0.247	0.437	0.638	0.249	0.947	0.902	0.794	0.635
Quadratic		0.594	0.767	0.553	0.187	0.183	0.153	0.512	0.223	0.260	0.299
Quadratic at NCG		0.230	0.254	0.210	0.348	0.127	0.202	0.389	0.384	0.560	0.798
Quadratic at CG		0.637	0.456	0.660	0.349	0.732	0.455	0.960	0.389	0.307	0.219
SEM		0.004	8.08	6.91	84.5	0.042	0.031	7.90	63.2	0.048	0.029

^a-bMeans within a column lacking a common superscript differ (P ≤ 0.05).

¹DPI, days post inoculation; BMC; bone mineral density (g); TV, tissue volume (mm³); BV, bone volume (mm³); NCP, number of closed pores; VCP, volume of closed pores (mm³); CPP, closed pore porosity.

²NCG, non-challenged group; CG, challenged group.

³MET100, Met and Cys supplemented at the ratio of 100:0; MET75, Met and Cys supplemented at the ratio of 75:25; MET50, Met and Cys supplemented at the ratio of 50:50; MET25, Met and Cys supplemented at the ratio of 25:75; MET0, Met and Cys supplemented at the ratio of 0:100.

Figure 3.

Femoral metaphysis cortical bone mineral density and bone volume fraction analyzed by microtomography on 6 DPI. MET100, Met: Cys = 100: 0; MET75, Met: Cys = 75:25; MET50, Met: Cys = 50:50; MET25, Met: Cys = 25:75; MET0, Met: Cys = 0:100. The error bars represent the SEM values. Bars without a common letter differ significantly. (A), P-values: P-diet = 0.040, P-linear = 0.028. (B), P-values: P-interaction = 0.036, P-linear at NCG = 0.681, P-linear at CG = 0.045.

Figure 4.

Femoral metaphysis cortical bone mineral density and bone volume fraction analyzed by microtomography on 9 DPI. MET100, Met: Cys = 100: 0; MET75, Met: Cys = 75:25; MET50, Met: Cys = 50:50; MET25, Met: Cys = 25:75; MET0, Met: Cys = 0:100. The error bars represent the SEM values. Bars without a common letter differ significantly. The asterisk (*) denoted significant difference. (A), P-values: P-challenge < 0.001, P-diet = 0.036, P-quadratic = 0.01. (B), P-values: P-challenge < 0.001, P-quadratic = 0.010. (C), P-value: P-challenge < 0.001, P-linear at NCG = 0.032. (D), P-value: P-challenge < 0.001, P-linear at NCG = 0.032.

Metaphysis Trabecular Bone. On 6 DPI, Eimeria challenge significantly decreased BV/TV, trabecular number (TBN), and connectivity density (CD) (P < 0.001), as well as BMD and BV (P = 0.017 and 0.027), whereas it significantly increased trabecular thickness (TBT) (P = 0.042) and trabecular separation (TBS) (P < 0.001) as shown in Table 5. On 9 DPI, Eimeria challenge significantly decreased BV, BV/TV, and TBN (P < 0.01), whereas it increased TBT and TBS (P < 0.001). On 9 DPI, in the NCG, the BV/TV linearly increased as MCR decreased (P = 0.040) and the TBN linearly increased as MCR decreased (P = 0.020) (Figure 5).

Table 5.

Micro-CT femoral metaphysis trabecular bone .

		6 DPI									9 DPI
Items1		BMD	BMC	TV	BV	BV/TV	TBT	TBS	TBN	CD	BMD	BMC	TV	BV	TBT	TBS	CD
Challenge2
NCG (-)		0.066a	0.013	206	10.6a	5.31a	0.130b	1.88b	0.429a	11.0a	0.082	0.023	274.3	14.4a	0.126b	2.28b	8.66a
CG (+)		0.042b	0.010	219	8.89b	3.99b	0.147a	2.25a	0.265b	7.12b	0.082	0.022	272.0	12.1b	0.167a	2.63a	6.46b
Diet3
MET100		0.061	0.014	226	10.3	4.58	0.142	2.05	0.345	8.79	0.077	0.021	274.7	12.7	0.150	2.55	6.90
MET75		0.052	0.011	119	9.27	4.65	0.141	2.14	0.335	9.24	0.087	0.025	283.0	13.2	0.137	2.39	7.60
MET50		0.055	0.011	208	9.51	4.64	0.131	2.05	0.361	8.96	0.062	0.017	263.5	12.7	0.163	2.52	7.67
MET25		0.055	0.012	213	9.46	4.44	0.145	2.03	0.333	8.95	0.093	0.026	274.4	14.4	0.136	2.34	8.50
MET0		0.046	0.009	217	10.2	4.93	0.133	2.03	0.360	9.36	0.090	0.024	269.6	13.1	0.148	2.47	6.99
Interaction
-	MET100	0.065	0.014	214	10.8	5.06	0.123	1.88	0.429	10.9	0.061	0.018	281.1	13.3	0.139	2.46	7.37
-	MET75	0.055	0.011	201	10.5	5.35	0.140	1.96	0.394	10.8	0.082	0.023	275.7	13.9	0.114	2.31	8.66
-	MET50	0.069	0.013	197	10.7	5.49	0.126	1.91	0.442	10.9	0.070	0.020	273.6	13.9	0.133	2.33	8.71
-	MET25	0.073	0.014	199	9.99	5.03	0.121	1.87	0.431	11.0	0.096	0.026	270.5	15.5	0.118	2.06	9.66
-	MET0	0.067	0.013	219	11.2	5.59	0.139	1.79	0.449	11.4	0.100	0.028	269.8	15.4	0.126	2.22	8.95
+	MET100	0.057	0.014	237	9.98	4.18	0.158	2.19	0.276	7.05	0.092	0.024	268.4	12.2	0.162	2.65	6.42
+	MET75	0.049	0.010	197	8.00	3.95	0.141	2.33	0.276	7.67	0.092	0.027	290.2	12.6	0.160	2.48	6.54
+	MET50	0.042	0.010	218	8.36	3.79	0.136	2.19	0.280	7.01	0.054	0.014	253.5	11.5	0.194	2.70	6.63
+	MET25	0.037	0.009	229	8.82	3.73	0.175	2.21	0.216	6.45	0.090	0.025	278.4	13.3	0.154	2.62	7.35
+	MET0	0.025	0.005	214	9.25	4.27	0.127	2.32	0.271	7.33	0.080	0.021	269.4	11.0	0.166	2.68	5.35
P value
Challenge		0.017	0.106	0.150	0.027	<0.001	0.042	<0.001	<0.001	<0.001	0.998	0.838	0.801	0.002	<0.001	<0.001	<0.001
Diet		0.865	0.560	0.433	0.841	0.893	0.718	0.894	0.887	0.961	0.338	0.361	0.684	0.545	0.244	0.366	0.149
Challenge × Diet		0.654	0.672	0.621	0.942	0.963	0.117	0.862	0.857	0.953	0.515	0.575	0.685	0.610	0.692	0.384	0.418
P trend
Linear		0.389	0.211	0.932	0.730	0.770	0.765	0.830	0.975	0.882	0.361	0.491	0.541	0.406	0.738	0.326	0.346
Linear at NCG		0.613	0.878	0.858	0.434	0.672	0.803	0.497	0.592	0.666	0.082	0.141	0.531	0.111	0.639	0.061	0.058
Linear at CG		0.137	0.089	0.759	0.929	0.987	0.506	0.711	0.612	0.817	0.630	0.601	0.817	0.634	0.995	0.578	0.520
Quadratic		0.930	0.783	0.142	0.429	0.567	0.914	0.723	0.702	0.750	0.501	0.612	0.907	0.694	0.827	0.451	0.060
Quadratic at NCG		0.973	0.657	0.180	0.338	0.983	0.828	0.486	0.777	0.791	0.936	0.948	0.871	0.959	0.537	0.468	0.219
Quadratic at CG		0.932	0.992	0.460	0.657	0.402	0.712	0.847	0.797	0.854	0.369	0.501	0.999	0.538	0.345	0.737	0.144
SEM		0.015	0.003	14.0	1.33	0.53	0.02	0.14	0.04	0.88	0.016	0.005	13.4	1.10	0.01	0.12	0.66

^a-bMeans within a column lacking a common superscript differ (P ≤ 0.05).

¹DPI, days post inoculation; BMD, bone mineral density (g/cm³); BMC, bone mineral density (g); TV, tissue volume (mm³); BV, bone volume (mm³); TBT, trabecular thickness (mm); TBS, trabecular separation (mm); TBN, trabecular number; CD, connectivity density (1/mm).

²NCG, non-challenged group; CG, challenged group.

³MET100, Met and Cys supplemented at the ratio of 100:0; MET75, Met and Cys supplemented at the ratio of 75:25; MET50, Met and Cys supplemented at the ratio of 50:50; MET25, Met and Cys supplemented at the ratio of 25:75; MET0, Met and Cys supplemented at the ratio of 0:100.

Figure 5.

Femoral metaphysis trabecular bone volume fraction and trabecular number analyzed by microtomography on 9 DPI. MET100, Met: Cys = 100: 0; MET75, Met: Cys = 75:25; MET50, Met: Cys = 50:50; MET25, Met: Cys = 25:75; MET0, Met: Cys = 0:100. The error bars represent the SEM values. The asterisk (*) denoted significant difference. (A), P-values: P-challenge = 0.001, P-linear at NCG = 0.040. (B), P-values: P-challenge < 0.001, P-linear at NCG = 0.020.

Metaphysis Total Bone. On 6 DPI, Eimeria challenge significantly decreased BMD, BMC, BV, and BV/TV (P < 0.001) (Table 6). On 9 DPI, Eimeria challenge significantly decreased BMC, TV, BV (P < 0.001), and BMD (P = 0.003). Also, a significant interaction effect was observed in BV/TV (P = 0.021). On 9 DPI, in the NCG, the BMD, BMC, and BV/TV linearly increased as MCR decreased (P = 0.017, 0.017, and 0.025) (Figure 6).

Table 6.

Micro CT femoral metaphysis total bone.

		6 DPI					9 DPI
Items1		BMD	BMC	TV	BV	BV/TV	TV	BV
Challenge2
NCG (-)		0.219a	0.081a	371	137a	37.1a	483a	178a
CG (+)		0.153b	0.055b	361	106b	29.3b	423b	132b
Diet3
MET100		0.179	0.066	371	115	31.0	446	149
MET75		0.185	0.067	365	123	33.7	467	156
MET50		0.192	0.068	352	121	34.3	439	151
MET25		0.188	0.070	368	124	33.6	464	163
MET0		0.185	0.070	376	126	33.5	448	156
Interaction
-	MET100	0.208	0.077	368	127	34.5	471	164
-	MET75	0.219	0.081	372	141	38.1	498	185
-	MET50	0.224	0.080	361	139	38.8	472	170
-	MET25	0.228	0.084	371	141	38.0	491	190
-	MET0	0.216	0.082	384	138	36.1	482	184
+	MET100	0.151	0.056	374	103	27.5	421	134
+	MET75	0.151	0.054	358	105	29.3	436	127
+	MET50	0.160	0.055	343	102	29.7	407	133
+	MET25	0.148	0.055	365	107	29.1	437	136
+	MET0	0.155	0.057	367	114	30.9	414	129
P value
Challenge		<0.001	<0.001	0.340	<0.001	<0.001	<0.001	<0.001
Diet		0.955	0.979	0.639	0.704	0.501	0.433	0.243
Challenge × Diet		0.967	0.963	0.949	0.854	0.803	0.982	0.174
P trend
Linear		0.672	0.530	0.725	0.200	0.266	0.993	0.156
Linear at NCG		0.643	0.493	0.544	0.408	0.632	0.786	0.050
Linear at CG		0.896	0.845	0.904	0.319	0.269	0.785	0.986
Quadratic		0.522	0.989	0.190	0.773	0.183	0.628	0.481
Quadratic at NCG		0.442	0.722	0.510	0.325	0.087	0.667	0.416
Quadratic at CG		0.897	0.732	0.227	0.550	0.883	0.800	0.862
SEM		0.017	0.006	16.2	7.94	1.90	17.3	6.80

^a-bMeans within a column lacking a common superscript differ (P ≤ 0.05).

¹DPI, days post inoculation; BMD, bone mineral density (g/cm³); BMC, bone mineral density (g); TV, tissue volume (mm³); BV, bone volume (mm³).

²NCG, non-challenged group; CG, challenged group.

³MET100, Met and Cys supplemented at the ratio of 100:0; MET75, Met and Cys supplemented at the ratio of 75:25; MET50, Met and Cys supplemented at the ratio of 50:50; MET25, Met and Cys supplemented at the ratio of 25:75; MET0, Met and Cys supplemented at the ratio of 0:100.

Figure 6.

Femoral metaphysis total bone mineral density, bone mineral content and bone volume fraction analyzed by microtomography on 9 DPI. MET100, Met: Cys = 100: 0; MET75, Met: Cys = 75:25; MET50, Met: Cys = 50:50; MET25, Met: Cys = 25:75; MET0, Met: Cys = 0:100. The error bars represent the SEM values. Bars without a common letter differ significantly. The asterisk (*) denoted significant difference. (A), P-values: P-challenge = 0.003, P-linear at NCG = 0.017. (B), P-values: P-challenge < 0.001, P-linear at NCG = 0.017. (C), P-values: P-challenge < 0.001, P-interaction = 0.021, P-linear at NCG = 0.025, P-linear at CG = 0.771.

Diaphysis Cortical Bone. On 6 DPI, Eimeria challenge significantly decreased BMD, BV/TV (P < 0.01), and BMC (P = 0.034), whereas it increased NCP, VCP, and CPP (P < 0.001) (Table 7). On 9 DPI, Eimeria challenge significantly decreased BMC, TV, BV, NCP (P < 0.001), and VCP (P = 0.026), whereas it increased BMD and BV/TV (P < 0.001). On 6 DPI, the BMC, TV, and BV linearly increased as MCR decreased (P = 0.011, 0.027, and 0.024) as shown in Figure 7. On 9 DPI, the BMD linearly increased as MCR decreased (P = 0.003)

Table 7.

Micro-CT femoral diaphysis cortical bone.

		6 DPI					9 DPI
Items1		BMD	BV/TV	NCP	VCP	CPP	BMC	TV	BV	BV/TV	NCP	VCP	CPP
Challenge2
NCG (-)		0.586a	97.0a	192b	0.074b	0.075b	0.071a	126a	121a	96.0b	245a	0.157a	0.126
CG (+)		0.548b	95.4b	336a	0.153a	0.180a	0.054b	87.5b	85.5b	97.9a	62.9b	0.088b	0.089
Diet3
MET100		0.557	96.0	277	0.135	0.158	0.060	107	102	97.2	158	0.109	0.105
MET75		0.555	95.2	271	0.107	0.129	0.065	105	108	96.8	169	0.113	0.099
MET50		0.576	97.2	265	0.108	0.119	0.061	106	102	96.4	172	0.150	0.132
MET25		0.566	96.0	288	0.111	0.123	0.062	112	102	97.0	152	0.115	0.105
MET0		0.579	96.4	218	0.110	0.113	0.064	105	104	97.3	134	0.123	0.101
Interaction
-	MET100	0.578	96.7	171	0.087	0.095	0.068	121	116	96.3	239	0.124	0.101
-	MET75	0.577	97.6	148	0.053	0.056	0.078	139	133	95.7	279	0.170	0.122
-	MET50	0.598	97.5	184	0.070	0.071	0.069	125	119	95.5	279	0.204	0.172
-	MET25	0.589	96.9	233	0.059	0.059	0.070	124	119	95.9	245	0.151	0.124
-	MET0	0.586	96.5	213	0.104	0.096	0.072	122	118	96.7	184	0.142	0.119
+	MET100	0.539	95.4	366	0.175	0.211	0.053	88.6	86.9	98.0	76.5	0.092	0.109
+	MET75	0.534	93.2	394	0.161	0.202	0.052	84.6	82.9	98.0	59.3	0.066	0.079
+	MET50	0.555	96.9	361	0.153	0.177	0.053	87.0	84.5	97.2	65.7	0.104	0.092
+	MET25	0.538	94.9	355	0.164	0.187	0.054	86.4	84.4	98.3	41.6	0.079	0.086
+	MET0	0.571	96.4	223	0.116	0.127	0.056	91.5	89.6	97.9	71.2	0.104	0.080
P value
Challenge		<0.001	0.002	<0.001	<0.001	<0.001	<0.001	<0.001	<0.001	<0.001	<0.001	0.026	0.122
Diet		0.461	0.257	0.772	0.958	0.861	0.615	0.784	0.785	0.271	0.823	0.887	0.915
Challenge×Diet		0.801	0.074	0.362	0.627	0.636	0.557	0.346	0.371	0.715	0.712	0.907	0.857
P trend
Linear		0.179	0.474	0.563	0.623	0.307	0.651	0.844	0.865	0.577	0.371	0.802	0.939
Linear at NCG		0.581	0.664	0.390	0.723	0.974	0.964	0.517	0.547	0.510	0.276	0.911	0.753
Linear at CG		0.173	0.142	0.091	0.273	0.128	0.553	0.711	0.717	0.894	0.840	0.809	0.692
Quadratic		0.944	0.711	0.395	0.658	0.773	0.984	0.721	0.870	0.067	0.434	0.533	0.553
Quadratic at NCG		0.559	0.291	0.935	0.321	0.398	0.705	0.257	0.332	0.058	0.127	0.275	0.294
Quadratic at CG		0.487	0.575	0.203	0.698	0.647	0.685	0.526	0.458	0.482	0.695	0.826	0.856
SEM		0.016	0.75	61.9	0.040	0.040	0.003	5.53	5.13	0.46	41.5	0.048	0.040

^a-bMeans within a column lacking a common superscript differ (P ≤ 0.05).

¹DPI, days post inoculation; BMD, bone mineral density (g/cm³); BMC, bone mineral density (g); TV, tissue volume (mm³); BV, bone volume (mm³); NCP, number of closed pores; VCP, volume of closed pores (mm³); CPP, closed pore porosity.

²NCG, non-challenged group; CG, challenged group.

³MET100, Met and Cys supplemented at the ratio of 100:0; MET75, Met and Cys supplemented at the ratio of 75:25; MET50, Met and Cys supplemented at the ratio of 50:50; MET25, Met and Cys supplemented at the ratio of 25:75; MET0, Met and Cys supplemented at the ratio of 0:100.

Figure 7.

Femoral diaphysis cortical tissue volume, bone volume, bone mineral content on 6 DPI and bone mineral density on 9 DPI analyzed by microtomography. MET100, Met: Cys = 100: 0; MET75, Met: Cys = 75:25; MET50, Met: Cys = 50:50; MET25, Met: Cys = 25:75; MET0, Met: Cys = 0:100. The error bars represent the SEM values. Bars without a common letter differ significantly. (A), P-values: P-linear = 0.027. (B), P-values: P-linear = 0.024. (C), P-values: P-linear = 0.011. (D), P-values: P-diet = 0.011, P-linear = 0.003.

DISCUSSION

The skeletal system serves as the foundation for the structure of the birds to support their body weight and ensure proper movement and activity levels (Bradshaw et al., 2002). While coccidiosis related damage in birds is often linked to intestinal health, its impact on bone health remains understudied. Additionally, while the roles of nutrients like calcium and phosphorus in bone development is well-documented (Bonjour, 2011), limited research has explored the effects of SAA on broiler bone health. The current study utilized X- ray scanning techniques to assess several bone health related parameters of broilers under Eimeria challenge and fed different dietary supplementation levels of MCRs, shedding light on this vital aspect of broiler welfare and performance.

The decreased BMC, fat, lean, and total body weight caused by coccidia infection obtained from the whole body DEXA scanning were correlated with results from previous studies (Fetterer et al., 2013a; Sharma et al., 2023b). These findings clearly demonstrated the harmful impact of coccidiosis on nutrient depositions, including minerals, fat, and protein in broilers. As our previously reported findings from this present study indicated (Liu et al., 2024), the Eimeria challenge significantly induced anorexia and compromised nutrient absorption and intestinal integrity. Similar adverse effects have been documented in other previous studies (Castro et al., 2020a; Teng et al., 2020; Teng et al., 2021), potentially resulting in alterations to the body composition of the broilers. Besides the decreased BMC, the birds in CG also had lower BMD, which further confirmed the negative effects of coccidiosis on bone health (Fetterer et al., 2013a; Tompkins et al., 2022). In addition to the decreased calcium and phosphorus absorption (Sharma et al., 2022; Sharma et al., 2023a), as reported in our previous manuscript (Liu et al., 2024), the Eimeria challenge in the current study also resulted in increased liver malondialdehyde, decreased serum antioxidant capacity, and altered antioxidant enzymes activities, indicating an elevated oxidative stress, which could lead to increased osteoclast activity, and subsequently increased bone resorption (Banfi et al., 2008). While this deterioration in bone quality could be attributed to the reduced nutrient intake caused by the anorexia, Tompkins et al. (2023a) reported in their experiment that, despite receiving an equivalent amount of feed to the Eimeria-infected broilers, the pair-fed group exhibited significantly better bone quality compared to the Eimeria challenge group. They further indicated that the pair-fed group had lower oxidative stress and osteoclast activity compared to the challenge group. These results suggested that this disruption in the balance of bone remodeling caused by oxidative stress would further contribute to the deterioration of bone quality (Tompkins et al., 2022; Tompkins et al., 2023b). The challenged birds exhibited a lower fat percentage, or in other words, a higher lean percentage, compared to the non-challenged birds. Previous research has demonstrated that as animal growth and gain weight, protein was accreted prior to fat (Trenkle and Marple, 1983; Schumacher et al., 2022). Consequently, lighter animals, such as the challenged birds in the current study, tended to have lower fat composition as they are still prioritizing the deposition of protein (Cabel and Waldroup, 1990). It would be interesting to conduct further studies to investigate the changes in body composition of broilers under Eimeria challenge to further confirm this hypothesis.

As for the effects of MCR on the whole body compositions, decreased MCR linearly decreased lean tissue weight and BW in the NCG which corresponds with previous studies (Jariyahatthakij et al., 2018; Liu et al., 2022) suggesting the importance of Met in body growth cannot be replaced by Cys. While in the CG, partially substituting Met with Cys did not affect the BW and lean tissue weight. This might be caused by the anorexia and decreased nutrient absorption leading to the similar Met available to the birds when they are challenged (Amerah and Ravindran, 2015; Castro et al., 2020b; Teng et al., 2023). More detailed results and discussion on the effects of MCR on growth performance of broilers under Eimeria challenge can be found in the previously published paper (Liu et al., 2024). Interestingly, we observed that in the CG, the reduced MCR linearly increased BMC of the birds, whereas in the NCG, BMC linearly decreased as MCR decreased. This observation could suggest that Eimeria challenge affected the metabolism of SAA in the broilers, leading to the opposite responses to the changes of MCR on BMC. However, when we considered the effects of MCR on the body weight of the DEXA scanned birds, the changes in BMC correlated with that in BW in the NCG whereas the BMC increased despite the absence of difference in BW of the challenged birds. This indicated that the decreased MCR might have a positive impact on the bone quality of Eimeria challenged birds.

To further investigate the impacts of Eimeria challenge and MCRs on bone quality, 3D structure characteristics of the metaphysis and diaphysis of the femur bone were assessed by Micro-CT scanning. The diaphysis predominantly consists of cortical bone while the metaphysis is characterized by trabecular bone surrounded by cortical bone (Clarke, 2008). Cortical bone is important for providing strength and protection to bones, the cortical porosity is considered to be correlated with mechanical properties of cortical bone and affecting bone strength and risk of fracture (Cooper et al., 2016). Trabecular bone is a highly porous and spongy form of bone tissue that is organized into a network of interconnected rods and plates called trabeculae. Trabecular bone is important for bone strength, flexibility, and providing structural support (Weinstein and Hutson, 1987). A decrease in trabecular number associated with increase in trabecular separation and thickness are usually correlated with bone loss and deterioration of bone quality (Alberich-Bayarri et al., 2008; Ozan et al., 2017). Through Micro-CT, a bone separation process was employed, allowing us to distinguish cortical bone from trabecular bone. This enabled independent analysis of their specific characteristics, such as cortical porosity and trabecular structure (Chen and Kim, 2020; White et al., 2023). Integrating this information with BMD and BMC could provide a comprehensive understanding of the morphological and architectural aspects of bone quality.

This study further confirmed the negative impacts of coccidia challenge on the bone quality which was evident by the reduction in TV, BV, and BV/TV of both femoral metaphysis and diaphysis. The decrease in BMD, BMC, and trabecular number and the higher trabecular separation and thickness further signified a deterioration in bone quality. Similar results were reported in previous studies (Tompkins et al., 2022; Sharma et al., 2023b). Notably, the challenged birds exhibited lower pore numbers, pore volume, and porosity. One possible explanation for this observation could be the substantial decrease in BV, leading to limited space for pore formation. In contrast, in the diaphysis cortical bone on 6 DPI where BV did not significantly differ, challenged birds displayed significantly higher porosity and pore numbers.

More interestingly, the Micro-CT results demonstrated that as MCR decreased, the BMD and BMC in the cortical bone and total ROI linearly increased. Moreover, the trabecular number in the trabecular bone and the BV/TV in cortical, trabecular bone and total ROI linearly increased. These results again suggested that the substitution of dietary Met supplementation with Cys improved bone quality of the broiler birds. As mentioned earlier, the metabolism of Met is closely related to the synthesis of Hcy. Homocysteine has been considered as a risk factor for bone disease development (Herrmann et al., 2005; Behera et al., 2017) due to its modulatory effects on bone remodeling. It has been shown that Hcy could increase the activity of osteoclasts whereas decrease the activity of osteoblasts. Additional findings from previous studies further revealed that Hcy could decrease blood flow in bone, elevate matrix metalloproteinases that degrade extracellular bone matrix, and disrupt normal synthesis of collagen crosslinking (Townsend et al., 2004; Herrmann et al., 2007; Vacek et al., 2013). Extensive research has shown that excessive Met would lead to the accumulation of Hcy (Ditscheid et al., 2005; Xie et al., 2007; Yang et al., 2020; Liu and Kim, 2023). Since broiler chickens are selected for rapid growth rates which challenges the correct bone mineralization making them more prone to bone disorders (Raehtz et al., 2018; Sanchez-Rodriguez et al., 2019), they might be more sensitive to changes in Hcy levels. The replacement of Met with Cys might improve the bone quality via a reduction in Hcy production. Furthermore, it has been reported that reduced Cys availability in humans could lead to reduced collagen formation and affect bone strength and density (Baines et al., 2007). Another study showed that lower plasma Cys was significantly associated with low BMD (Elshorbagy et al., 2009). Unfortunately, few studies have been conducted to investigate the effects of Met or Cys on bone health in broiler chickens. While most human and mice studies on Met restriction on bone health drastically reduced dietary Met without providing Cys (Huang et al., 2014; Ouattara et al., 2016). To unravel the mechanisms underlying the alterations in bone quality resulting from changes in the dietary Met to Cys ratios, more studies are needed. Increased exploration in this area would contribute valuable insights into the specific effects of these amino acids on bone health in the context of poultry physiology.

In summary, the Eimeria challenge had a significant impact on the body composition of broilers, resulting in reduced BW, lean tissue weight, BMD, and BMC. Additionally, coccidiosis-infected birds exhibited a decline in femur bone quality, as indicated by decreased femoral BMD, BMC, and trabeculae, along with increased cortical bone porosity. Notably, the study also uncovered that decreasing the supplemental methionine to cysteine ratios could lead to an improvement in femoral bone quality for both Eimeria challenged and non-challenged broiler chickens. This improvement was reflected in the linear enhancement of BMD, BMC, and BV in both cortical and trabecular bones, as well as an increase in trabecular numbers. These results suggested that adjusting the methionine to cysteine ratio in broiler diets could potentially improve the bone health, highlighting the importance of optimizing dietary strategies for enhanced skeletal development and overall performance in poultry nutrition.