Evaluating the effects of dietary glutamine on bone geometry, mechanical properties, and mineral composition in laying quail

Abstract

This study investigated the effect of dietary L-glutamine (Gln) supplementation on tibial quality in Japanese quail (Coturnix japonica) during the early and peak laying periods. A total of 288 female quail were assigned to four dietary treatments with Gln added at 0 %, 0.5 %, 1.0 %, or 1.5 % of the diet. Birds were euthanized at 6 and 12 weeks of lay, and tibiae were collected for morphometric, densitometric, mechanical, and mineral composition analyses, as well as X-ray diffraction measurements. Dietary Gln supplementation affected several bone parameters in a Gln level- and age-dependent manner, including a significant increase in bone calcium content (P < 0.001). However, these changes did not consistently translate into improvements in mechanical strength. Bone breaking strength showed no significant main effect of Gln or age, although a significant Gln level × age interaction was observed (P < 0.05), indicating that the response to Gln varied with the stage of lay. The absence of consistent improvement in bone strength despite enhanced mineralization may reflect differences in the distribution or type of mineralized tissue, as medullary bone contributes little to biomechanical performance in laying birds. Overall, these findings suggest that dietary Gln supplementation improves mineralization-related parameters but does not substantially enhance tibial mechanical strength in laying quail. Glutamine may therefore support skeletal calcium buffering during lay without markedly improving resistance of the tibia to mechanical loading.

Introduction

Glutamine (Gln) is the most abundant free amino acid in avian plasma and a key carbon and nitrogen donor for rapidly proliferating tissues. Beyond its classical roles in maintaining gut integrity and immune function, Gln enters osteoblasts, fuels the tricarboxylic-acid cycle, and supports collagen synthesis (Chen and Long, 2018; Durán et al., 2012; Yu et al., 2019). Because energy expenditure and amino acid turnover increase markedly during the early laying cycle, skeletal demand for Gln may increase at the same time that dietary supply becomes limiting.

Most poultry studies have focused on Gln as a gut-protective nutrient under conditions of heat stress or pathogen challenge, reporting improvements in villus height and nutrient absorption (da Silva Junior et al., 2024; EFSA, 2020; He et al., 2021; Tomaszewska et al., 2020). Previous work has also shown that dietary supplementation with 0.5 to 1.5 % Gln improves not only gut morphology but also the fatty acid profile of quail meat (Tomaszewska et al., 2025a, b).

Given the importance of Gln in multiple physiological systems, its potential impact on skeletal health warrants further investigation. Data on long-bone development and biomechanics in quails are far more limited than in other poultry species, despite the critical role of skeletal integrity in animal welfare and production performance. Although quails are widely used as a research model and several studies have examined bone formation during embryonic development and early growth (Hiyama et al., 2019; Kawai et al., 2018; Pourlis et al., 1998), information on bone structure and strength in adult laying quails maintained under farm conditions remains scarce. Recent evidence suggests that dietary Gln at 0.5–1.5 % may affect systemic indicators of mineral metabolism in this species. Tomaszewska et al. (2025b) reported increased plasma alkaline phosphatase (ALP) activity in young laying quails after Gln supplementation, which may reflect altered bone or intestinal metabolism, although ALP is not a bone-specific marker in birds.

Whether similar inclusion levels improve skeletal quality remains unknown, as biomechanical outcomes depend on the relative contribution of mineralizing tissue, particularly medullary versus cortical bone. Although quails share many physiological characteristics with chickens, differences in metabolic rate and nutrient requirements limit direct extrapolation of chicken data to quails. This highlights the need for species-specific studies, since compromised skeletal integrity in laying quails predisposes birds to fractures and locomotor pain, resulting in feed wastage, reduced egg output, and measurable welfare impairment.

This issue is particularly relevant because quails reach peak egg production while long-bone modelling is still ongoing, increasing their susceptibility to early osteoporosis. Therefore, we tested the hypothesis that moderate Gln inclusion (0.5 to 1.5 %) improves tibial mineralisation and mechanical strength during the first 12 wk of lay. Bone geometry, whole-bone mechanical properties, ash composition, and hydroxyapatite crystal characteristics were evaluated using a 2 × 4 factorial design (age × Gln level) to identify dietary Gln levels most supportive of skeletal health.

Materials and methods

Ethical approval

Approval from the Local Ethics Committee for Animal Experiments in Lublin, Poland (resolution No. 40/2023) was obtained before the research commenced.

Birds and experimental diets

The experimental procedures, housing conditions, and husbandry practices were identical to those reported in the companion analyses from this same experiment (Tomaszewska et al., 2025a, b). All measurements were obtained from the same flock, raised under uniform housing and fed the same basal diet, which ensures full comparability across the different physiological endpoints evaluated. Building on the previously published performance and meat-quality data from this cohort, the present study examines the appendicular skeleton and provides a complementary assessment of how moderate dietary Gln influences bone integrity during the early laying cycle.

Briefly, at 6 wk of age, 288 clinically healthy dual-purpose female Japanese quails (Coturnix japonica) were weighed and assigned to four dietary treatments in a completely randomized design (8 replicate cages per treatment; 9 birds per cage). A basal wheat-soybean diet formulated for early-laying quail served as the control (Tomaszewska et al., 2025a). The experimental diets were obtained by supplementing the basal diet with 0.5 %, 1.0 %, or 1.5 % L-Gln (Biomus, Lublin, Poland; purity ≥99.5 %). The ingredient and calculated nutrient composition of the basal diet have been reported previously (Table 1; Tomaszewska et al., 2025a). The feeding period lasted 12 wk (from 7 to 19 wk of age). Amino acids in the basal diet were determined using the official EU method for feed control (Table 2; European Commission, 2009). According to this method, Gln is analyzed together with Glu because Gln is converted to Glu during acid hydrolysis (Fontaine, 2003). The analysed Glu + Gln content in basal diets was 45.7 g/kg, and 50.67 g/kg, 56.7 g/kg, 61.7 g/kg in experimental diets, respectively. Apart from the expected increase in Glu + Gln, the analysed concentrations of the remaining amino acids were comparable among diets and showed no meaningful differences.

Table 1.

Composition and nutrient content of experimental diets, % dry matter (Tomaszewska et al., 2025).

	Diets
Ingredients, %	0 %	0.5 % Gln	1.0 % Gln	1.5 % Gln
Soybean meal	30.40	30.40	30.40	30.40
Wheat	26.65	26.65	26.65	26.65
Corn	14	14	14	14
Triticale	10	10	10	10
Limestone	5.18	5.18	5.18	5.18
Wheat bran	5	5	5	5
Soybean oil	4.30	4.30	4.30	4.30
Monocalcium phosphate	1.95	1.95	1.95	1.95
NaCl	0.36	0.36	0.36	0.36
DL-Methionine	0.08	0.08	0.08	0.08
L-lysine	0.08	0.08	0.08	0.08
Vitamin-mineral premix1	0.5	0.5	0.5	0.5
L-glutamine	0	0.5	1.0	1.5
Kaolin	1.5	1.0	0.5	0.0
Nutrients composition, calculated
Metabolizable energy, kcal/kg	2800
Crude protein	21
Crude fiber	2.954
Arginine	1.396
Lysine	1.15
Methionine	0.384
Methionine +cysteine	0.75
Treonine	0.78
Ca	2.5
Total P	0.858
Available P	0.55
Na	0.16

¹The premix provided per 1 kg of diet: vitamin A, 10,000 IU (retinol); vitamin D3, 2,500 IU (cholecalciferol); vitamin E, 30 IU (α-tocopherol); 6-phytase, 1500 FTU; Endo-1,4-beta-xylanase 2000 IU.

Table 2.

Analysed amino acid content (g/kg) of basal control diet fed during the experimental period.

Item	Supplementation level
	0 %	0.5 % Gln	1.0 % Gln	1.5 % Gln
Ala	7.749
Arg	10.199
Asp + Asn	14.576
Gln + Glu	45.711	50.670	56.698	61.709
His	4.297
Leu	12.918
Lys	9.118
Met	5.498
Thr	6.200
Trp	2.298
Tyr	5.493
Val	7.593

45.7 g/kg, and 50.67 g/kg, 56.7 g/kg, 61.7.

Performance data were recorded daily on a cage basis, as reported previously (Tomaszewska et al., 2025b). Bone collection was carried out after 6 and 12 wk of dietary supplementation (birds 12 and 18 wk of age, respectively). At each time point, one bird per cage (n = 8 birds per treatment per time point) was randomly selected and euthanized by decapitation for tissue collection, as previously described (Tomaszewska et al., 2025a).

Bone collection and pre-testing preparation

Immediately after euthanasia, both tibiae were excised and cleaned of adhering tissues. Morphometric measurements (bone length and mid-shaft diameters) and densitometric indices (the Seedor ratio) were determined for the left tibia using digital calipers and an analytical balance. The relative bone weight (RBW; bone weight-to-body weight ratio) was also calculated. The bones were subsequently wrapped in saline-soaked gauze and stored at −20 °C until further analysis. The storage time did not exceed 8 weeks, and bones were kept moist throughout handling and testing to minimize dehydration related changes in mechanical properties.

Bone mechanical testing

All steps followed the protocol used earlier for long bones (Osiak-Wicha et al., 2023; Tomaszewska et al., 2024) with minor adaptations for the present experiment. Right tibiae were thawed overnight at 7 °C before measurements. The three-point bending test was performed on a Zwick Z010 universal testing machine (Zwick-Roell GmbH & Co., Ulm, Germany) (Tomaszewska et al., 2024). The load was applied to the midpoint of the bone diaphysis until fracture. A fixed span of 20 mm (about 40 % of average total bone length) and a constant load rate of 5 mm min⁻¹ were used. Next, the tibiae were cut at the midpoint of the diaphysis using a diamond bandsaw (MBS 240/E, Proxxon GmbH, Foehren, Germany). Geometric parameters of the bone diaphysis, including the cortical index, cross-sectional area, mean relative wall thickness, and cross-sectional moment of inertia, were determined based on measurements of cortical bone cross-sectional diameters using a digital caliper (Tomaszewska et al., 2024). Finally, on the basis of recorded load-deformation curves and measured diameters, bone mechanical (yield load, fracture load, elastic work, work to facture, and stiffness) and apparent material properties (Young’s modulus, elastic stress, and ultimate stress) were determined using standard beam-theory equations, using Origin software (OriginLab, Northampton, MA, USA) (Tomaszewska et al., 2024).

Bone ash and mineral analysis

The percentage of bone ash was determined after ashing the dried, cartilage cap-free bone samples in a muffle furnace at 500 °C for 12 h. The concentrations of Ca and P in the bone ash were then determined using flame atomic absorption spectrophotometry and colorimetric analysis with ammonium molybdate, respectively.

X-ray diffraction

Prior to analysis, the cartilage caps were removed from the right tibiae, and the bones were finely ground in an agate mortar. Powder X-ray diffraction (XRD) patterns were collected on a Malvern Panalytical Empyrean diffractometer operating in Bragg-Brentano geometry. The instrument was equipped with a Cu Kα X-ray tube (operated at 40 kV and 40 mA), employing Cu Kα radiation (λ_Kα1 = 1.5405980 Å, λ_Kα2 = 1.5444260 Å, Kα₂/Kα₁ ratio = 0.5000). The incident beam path included a focusing X-ray mirror for Cu radiation (W/Si Graded Multilayer, elliptic shape, 0.800° acceptance angle) and 0.04 rad Soller slits. A fixed 20 mm mask was used. The diffracted beam path was equipped with 0.04 rad Soller slits. Data were acquired using a PIXcel3D area detector in scanning mode, with energy discrimination set at 25.0–100.0 %. For data collection, samples were mounted on a metal sample holder and measured in reflection mode using a spinning sample holder (8.0 s revolution time). The samples were measured in θ−2θ geometry. Continuous scans were recorded in the 2θ range from 4.00° to 70.00°, with a total counting time of 198.645 seconds per step. The total scan duration was approximately 70 min. Data acquisition was performed using Data Collector v4.3 and Empyrean Instrument Control Software v7.7. A synthetic stoichiometric hydroxyapatite standard (Sigma-Aldrich) was used for calibration of peak positions and instrumental broadening.

Based on the received diffractograms, full width at half maximum (FWHM) of reflection peak from (002) plane, mean size of hydroxyapatite (HA) nanocrystallites D along with z-direction, and lattice parameters a and c of the hexagonal cell were determined. The mean size D of the nanocrystallites was calculated using the Scherrer equation (Patterson, 1939).

$$D=\frac{K\lambda }{FWH{M}_{002}cos\theta },$$

where D is the mean size of the ordered crystalline domains, K is a constant related to the crystallite shape (0.9), FWHM₀₀₂ is the full width of the peak at half of the maximum intensity for 002 plane counting the apparatus broadening of 0.02 deg (limited by the detector resolution), λ is the mean wavelength of X-ray radiation, while θ is the peak position (Jackson et al., 1978). Lattice spacing d_hkl was determined from Bragg’s law for the Miller indices (300) and (002) according to the equation:

$$\lambda =2d_{hkl}sin{\theta }_{hkl}$$

While lattice parameters a and c for the hexagonal unit cell were calculated from the equation:

$$\frac{1}{d_{hkl}^2}=\frac{4}{3}\left(\frac{h^2+hk+k^2}{a^2}\right)+\frac{l^2}{c^2},$$

where h, k and l are the Miller indices that are the reciprocal intercepts of the plane on the unit cell axes (Fujisaki et al., 2006). Additionally, the unit cell volume V was calculated according to the equation (Clark and Iball, 1957):

$$V=\frac{\sqrt{3}}{2}·a^2·c$$

for a hexagonal-shaped cell.

Bragg peaks and crystallographic planes were determined using Mercury CSD 3.10.1 software (CCDC, Cambridge, UK) from the HA references (No. 2300273, Crystallography Open Database, and No. 96-901-0053, High Score Plus package software). The peak position and FWHM were calculated from the Gaussian function’s fits to every peak using the Grams/AI 8.0 (Thermo Scientific, Waltham, MA, USA) and OriginLab 2022 (OriginLab, MA, USA).

Statistical analysis

The statistical analysis was performed using the UNIVARIATE and the GLM procedure of SAS software (Statistical Analysis System, 9.4, 2013). The normality of the distribution was verified using the Kolmogorov Smirnov test. To verify the significance of differences in bone morphology, mechanical properties and mineral traits, a two-factor analysis of variance (factors: age of the animal time point, 2 levels, and Gln level, 4 levels) with the interaction of both factors was conducted. Linear and quadratic contrasts for the Gln level effect were evaluated over the entire experimental period to determine the overall trend in response to Gln supplementation. Tukey's post-hoc test was used with a P-value ≤ 0.05.

Result Table 3, Table 4, Table 5, Table 6 present least-squares means for the main effects of week of lay and dietary glutamine level, together with P-values for the main effects, their interaction, and the orthogonal linear and quadratic contrasts tested within the Gln level effect. Table 6 presents least-squares means for traits showing a significant age × Gln level interaction (P < 0.05), with detailed values for each treatment combination.

Table 3.

Body mass, bone morphology, the Seedor ratio, the relative bone weight, and geometric parameters of tibia from Japanese quail after 6 and 12 weeks of Gln supplementation¹.

TRAIT1	Week of lay			Gln Supplementation level					Effect of			Contrast2
	6	12	SEM	0 %	0.5 %	1.0 %	1.5 %	SEM	Age	Gln level	Age × Gln level	linear	quadratic
Body weight (g)	173b	185a	2.333	180	175	177	183	3.29	<0.001	0.358	0.820	0.581	0.122
Bone weight (g)	0.694	0.682	0.013	0.715	0.680	0.672	0.684	0.018	0.528	0.394	0.612	0.230	0.203
Bone length (mm)	49.2	50.3	0.839	49.1	49.5	48.4	51.9	1.19	0.384	0.175	0.512	0.164	0.185
Seedor index (mg/mm)	14.1	13.7	0.275	14.5	13.7	13.9	13.4	0.389	0.309	0.223	0.278	0.073	0.677
RBW (%)	0.402a	0.369b	0.008	0.398	0.388	0.381	0.376	0.011	0.004	0.567	0.494	0.188	0.835
Hout (mm)	2.52	2.51	0.028	2.55	2.49	2.47	2.56	0.039	0.707	0.293	0.455	0.921	0.061
Hin (mm)	1.46	1.33	0.049	1.5	1.41	1.35	1.47	0.070	0.056	0.569	0.440	0.317	0.694
Vout (mm)	2.52	2.53	0.026	2.56	2.52	2.47	2.54	0.037	0.709	0.389	0.769	0.488	0.159
Vin (mm)	1.58	1.52	0.044	1.59	1.58	1.47	1.56	0.061	0.314	0.528	0.930	0.476	0.397
MRWT (–)	0.681	0.836	0.16	0.806	1.083	0.839	0.704	0.234	0.135	0.703	0.482	0.603	0.385
CI, (%)	39.6	43.7	1.46	42.6	40.4	42.9	40.6	2.07	0.055	0.743	0.475	0.709	0.961
CSA (mm2)	3.16	3.36	0.084	3.42	3.18	3.18	3.29	0.118	0.088	0.446	0.146	0.477	0.166
CSMI (mm4)	1.69	1.76	0.061	1.82	1.69	1.59	1.79	0.085	0.429	0.245	0.476	0.598	0.066

¹Data are presented as least squares means and standard error of the mean (SEM); n = 8 birds per treatment per sampling time point.

^{a, b} Means in the same row and for the same main effect (age or Gln level) with different superscripts are significantly different (P < 0.05, Tukey’s HSD test).

²Linear or quadratic effects were evaluated by orthogonal polynomial contrasts to test the effects of Gln levels on selected traits.

RBW, the relative bone weight; H_out, transversal outer diameter; H_in, transversal inner diameter; V_out, anteroposterior outer diameter; V_in, anteroposterior inner diameter; MRWT, mean relative wall thickness; CI, cortical index; CSA, cross-sectional area; CSMI, cross-sectional moment of inertia.

Table 4.

Mechanical and material properties of tibia from Japanese quail after 6 and 12 weeks of Gln supplementation¹.

TRAIT1	Week of lay			Gln Supplementation level					Effect of			Contrast2
	6	12	SEM	0 %	0.5 %	1.0 %	1.5 %	SEM	Age	Gln level	Age × Gln level	linear	quadratic
Fyield (N)	55.8	52.6	1.48	57.5	52.5	53.7	53.1	2.092	0.128	0.322	0.271	0.208	0.298
Welastic (mJ)	13.4	12.5	0.477	13.7	11.9	13.4	12.7	0.674	0.213	0.269	0.290	0.611	0.464
Fmax (N)	64.1	62.8	1.11	64.4	62.7	60.9	65.9	1.57	0.417	0.135	0.014	0.721	0.051
Wmax (mJ)	20.8	20.0	0.896	22.1	20.5	19.9	19.2	1.27	0.541	0.432	0.378	0.107	0.754
Stiffness (N/mm)	114	110	3.60	120	114	109	106	5.09	0.380	0.273	0.223	0.056	0.746
Young`s modulus (GPa)	47.1	43.9	1.67	45.4	47.3	47.2	42.1	2.37	0.173	0.375	0.807	0.339	0.141
Elastic stress (MPa)	337	310	9.93	326	323	336	310	14.1	0.056	0.634	0.409	0.597	0.432
Ultimate stress (MPa)	390	371	10.9	369	386	383	383	15.4	0.219	0.856	0.602	0.544	0.590

¹Data are presented as least squares means and standard error of the mean (SEM); n = 8 birds per treatment per sampling time point.

²Linear or quadratic effects were evaluated by orthogonal polynomial contrasts to test the effects of Gln levels on selected traits.

F_yield, yield load; W_elastic, elastic work; F_max, fracture load; W_max, work to fracture.

Table 5.

Bone mineral components and the structure of HA crystals of tibia from Japanese quail after 6 and 12 weeks of Gln supplementation¹.

TRAIT1	Week of lay			Gln Supplementation level					Effect of			Contrast2
	6	12	SEM	0 %	0.5 %	1.0 %	1.5 %	SEM	Age	Gln level	Age × Gln level	linear	quadratic
Ca (g/kg bone ash)	385b	422a	2.63	384a	403b	414b	413b	3.72	<0.001	<0.001	<0.001	0.001	0.122
P (g/kg bone ash)	191	194	1.44	195	196	191	189	2.03	0.083	0.081	0.080	0.026	0.432
Ca:P	2.02b	2.18a	0.021	1.98c	2.07bc	2.17ab	2.19a	0.029	<0.001	<0.001	0.002	<0.001	0.379
a (nm)	0.941	0.949	0.003	0.933	0.943	0.945	0.942	0.002	0.393	0.239	0.341	0.101	0.044
c (nm)	0.686	0.686	0.009	0.686	0.686	0.685	0.687	0.012	0.982	0.999	0.998	0.989	0.923
VHA (nm3)	0.526b	0.531a	0.001	0.513b	0.531a	0.536a	0.534a	0.002	0.021	<0.001	0.004	<0.001	<0.001
D (nm)	13.1	12.9	0.032	13.0b	13.1b	12.8c	13.2a	0.046	0.063	<0.001	<0.001	0.179	0.002
Ash (%)	52.3b	53.4a	0.389	52.8	52.8	53.4	52.3	0.551	0.049	0.572	0.378	0.715	0.337

¹Data are presented as least squares means and standard error of the mean (SEM); n = 8 birds per treatment per sampling time point.

^a,b,c Means in the same row and for the same main effect (age or Gln level) with different superscripts are significantly different (P < 0.05, Tukey’s HSD test).

²Linear or quadratic effects were evaluated by orthogonal polynomial contrasts to test the effects of Gln levels on selected traits.

a, the length of the a-axis in the hexagonal lattice; c, the length of the c-axis in the hexagonal lattice; V_HA, unit-cell volume V_HA of the hydroxyapatite crystals; D, mean size of nanocrystallites along the z-direction.

Table 6.

Features for which there was an interaction of factors.

Week of lay	Supplementation level %	TRAIT1
		Fmax (N)	Ca (g/kg bone ash)	Ca:P	VHA (nm3)	D (nm)
6	0	62.6ab	358d	1.88c	0.517cd	13.2ab
	0.5	61.5ab	376d	1.91bc	0.528bc	13.0bc
	1.0	61.5ab	401c	2.16a	0.531ab	12.9bc
	1.5	70.9a	406bc	2.16a	0.529b	13.00bc
12	0	66.3ab	410abc	2.07ab	0.509d	12.8cd
	0.5	63.9ab	431a	2.23a	0.534ab	13.0bc
	1.0	60.3b	427ab	2.18a	0.541a	12.6d
	1.5	60.9b	421abc	2.23a	0.538ab	13.4a
SEM		2.22	5.26	0.041	0.002	0.065

¹Data are presented as least squares means and standard error of the mean (SEM); n = 8 birds per treatment per sampling time point.

^{a, b, c, d} Means in the same row and for the same main effect (age or Gln level) with different superscripts are significantly different (P < 0.05, Tukey’s HSD test).

F_max, fracture load; V_HA, unit-cell volume V_HA of the hydroxyapatite crystals; D, mean size of nanocrystallites D along the z-direction.

Results

Body mass, bone morphology and geometry

Body mass and bone morphometric parameters are presented in Table 3. Body weight was significantly affected by age (P < 0.001), whereas dietary Gln had no effect. Neither the main effect of dietary Gln (0–1.5 %) (P = 0.358) nor the age × Gln level interaction (P = 0.820) was significant.

Relative bone weight decreased with age (P = 0.004) and was not affected by dietary Gln (P = 0.567). No other geometric parameters differed among diets.

Material and mechanical properties

All bone mechanical and material properties are presented in Table 4. A significant age × Gln interaction was detected for F_max (P = 0.014), whereas the main effects of age (P = 0.417) and Gln level (P = 0.135) were not significant. No other material or mechanical variable differed among diets.

Bone mineral composition and the structure of HA crystals

Bone mineral composition and basal parameters of HA crystals are presented in Table 5. Bone ash content was higher at 12 wk of lay than at 6 wk (P = 0.049). Dietary Gln had no effect on ash content. Bone calcium content and the Ca:P ratio were significantly affected by both age and Gln level (P < 0.001 for all), whereas phosphorus content showed only minor variation and was not influenced by age. The mean nanocrystallite size D and unit-cell volume VHA were significantly affected by dietary Gln Gln level (P < 0.001), while the c lattice parameter remained unchanged.

Detailed age × Gln level interactions for bone calcium content, Ca:P ratio, unit-cell volume (VHA), nanocrystallite size (D), and fracture load are summarized in Table 6. Calcium content was consistently higher in supplemented groups than in controls at both sampling points, with more pronounced differences at 6 wk of lay. At 12 wk of lay, all Gln-supplemented groups exceeded control values, with the highest calcium levels observed at 0.5 % Gln. Similar interaction patterns were observed for the Ca:P ratio and VHA, indicating age-dependent responses to dietary Gln inclusion.

Discussion

Interpretation of skeletal responses to nutritional interventions in laying quail is constrained by the limited availability of species-specific data on bone metabolism and mechanical properties during lay. Consequently, discussion of the present findings is framed within a broader avian physiological context.

The present study demonstrated that dietary Gln supplementation increased tibial Ca content, the Ca to P ratio, and selected crystallographic parameters of hydroxyapatite in laying quail. These effects followed a dose-dependent pattern across dietary Gln inclusion levels. However, Gln supplementation had minimal effects on bone geometry or mechanical performance. The lack of correspondence between enhanced mineral content and unchanged bone strength suggests that a portion of the additional mineral may have been preferentially allocated to medullary bone. This tissue primarily serves a metabolic function as a rapidly mobilizable calcium reservoir during lay (Dacke et al., 1993) and contributes little to load-bearing capacity, thereby failing to improve resistance to bending loads (Fleming et al., 1998).

Several mechanisms have been proposed to explain the influence of Gln and its metabolic derivatives on skeletal metabolism. Gln can support osteoblast activity, collagen formation and bone microstructure (Tomaszewska et al., 2020). It may indirectly influence Ca/P balance by supporting intestinal absorption and systemic mineral availability (El-Deken et al., 2024; Muszyński et al., 2023), and broader metabolic benefits of Gln supplementation have been reported (El-Deken et al., 2024). In addition, Gln serves as a metabolic fuel and potential signaling molecule for osteoblasts (Yu et al., 2019). However, the present results do not allow discrimination between direct and indirect mechanism of actions, and the observed mineral-related changes should therefore be interpreted as multifactorial.

No effect of dietary Gln on relative bone weight (RBW) was observed. Analysis of RBW indicated that age-related variation followed a predictable biological pattern. Although tibial absolute mass increased with age, its proportion relative to body mass declined, reflecting allometric scaling in maturing birds. Similar age-dependent reductions in relative bone growth have been reported in laying hens and broilers (Kongpechr et al., 2019). The pattern observed in the present study thus reflects normal physiological development rather than a diet-induced effect.

The increased tibial calcium content observed in Gln-supplemented quail is consistent with studies reporting effects of Gln or glutamate on bone mineralization in laying hens (Ibrahim et al., 2024; Selle et al., 2024). Gln has been shown to modulate calcium-related traits in poultry (Muszyński et al., 2023), and supplementation with glutamic acid has been associated with increased tibial Ca and P content and altered mineral metabolism markers (El-Deken et al., 2024). These responses were accompanied by improvements in eggshell quality. The most pronounced effects were reported at inclusion levels of 0.6–0.8 %, whereas (1.0 %) did not yield additional benefits, suggesting a level-dependent plateau. Although Gln and glutamic acid are distinct compounds, they share key metabolic pathways relevant to osteoblast function, rendering these findings physiologically comparable. Nevertheless, the specific mechanisms underlying these responses require further investigation. A schematic overview of the proposed mechanisms linking dietary Gln supplementation with changes in bone mineralization is provided in Supplementary Figure S1.

Alterations in crystallite size and unit cell dimensions were statistically significant but small in magnitude. Such changes may reflect subtle differences in mineral organization or maturation. The values observed in this study were comparable to those reported in laying hens, in which mean crystallite diameters typically range from 10 to 20 nm depending on bone type (Rodriguez-Navarro et al., 2023). The absence of corresponding improvements in whole-bone mechanical properties suggests that these crystallographic modifications likely occurred in mineral fractions with limited structural contribution, such as medullary bone.

The distinction between mineral content and mechanical function is particularly relevant in laying birds. Medullary bone expands during lay under estrogenic stimulation (Whitehead, 2004; Prondvai, 2017), increasing total mineral mass without substantially enhancing mechanical resistance (Fleming et al., 1998). This phenomenon may explain why quails at 18 weeks exhibited higher mineral content and Ca:P ratio (Muszyński et al., 2023) without marked improvement in tibial strength. The mineral gains observed in the present study therefore appear to reflect physiological adaptation to egg production rather than structural reinforcement load-bearing bone.

The interaction between age and dietary Gln level for fracture load suggests that mechanical responses may depend on the stage of lay, although the overall effects were modest. Younger birds exhibited more pronounced increases in tibial Ca content following supplementation, whereas older birds did not demonstrate a clear mechanical advantage. This pattern may reflect age-related differences in calcium turnover and deposition dynamics during early versus later stages of lay, although mechanistic interpretation remains limited in the absence of histological data. Previous studies have reported that moderate Gln supplementation supports bone metabolism, whereas further increases in dietary inclusion do not result to proportional benefits (Gholipour et al., 2019). The present findings are consistent with this pattern.

The level-response relationship observed in this study indicated that dietary Gln levels of 0.5 to 1.0 % produced most of the mineral-related effects, whereas 1.5 % did not confer additional benefits. During early lay, inclusion of 1.0 to 1.5 % was associated with the highest Ca accumulation, whereas at later stages 0.5 % appeared sufficient. Phosphorus content remained unaffected, indicating that in the Ca:P ratio were driven primarily by changes in Ca content. Moderate Gln supplementation also promoted longitudinal growth of hydroxyapatite crystallites during early lay and maintained this effect later, whereas higher inclusion levels did not result in further crystallographic changes.

Age significantly influenced bone characteristics. Older quail exhibited higher mineral content, a higher Ca:P ratio, and narrower marrow cavities, reflecting medullary bone accumulation typical in laying birds (Whitehead, 2004; Muszyński et al., 2023; Prondvai, 2017). Although medullary bone can provide limited internal support to the cortex (Fleming et al., 1998), its contribution to mechanical performance remains modest. Consequently, the bone changes observed in older quail primarily reflect mineral storage rather than structural reinforcement.

Across both age groups, dietary Gln supplementation did not result in major improvements in bending strength. Thus, the observed mineral-related changes did not translate into substantial differences in tibial mechanical properties. This findings support the interpretation that Gln primarily influenced mineral metabolism and Ca allocation rather than structural properties of cortical bone.

The practical implications of the results should be interpreted with caution. Increased skeletal Ca reserves may support Ca availability for eggshell formation, although eggshell traits were not assessed in the present study. Evidence from hens indicates that supplementation with Gln or α-ketoglutarate can improve eggshell Ca content and thickness (Muszyński et al., 2023), but direct extrapolation to quail is not possible without species-specific measurements. From a skeletal health perspective, enhanced mineral content may support metabolic Ca reserves, though this does not necessarily reduce fracture risk in dual-purpose quail. By analogy, Gln-fed quail might produce eggs with improved shells quality, which could reduce egg breakage and enhance hatchability in breeding flocks.

For quail producers and breeders, the results indicate that nutritional interventions can influence traits linked to both egg production and physical robustness. Gln supplementation is relatively straightforward to implement, as it can be incorporated into premixed feeds. Importantly, Gln was well tolerated at the inclusion levels tested, with no adverse effects on growth or general health observed at up to 1.5 % of the diet. This suggests a reasonable safety margin, although substantially higher inclusion levels (above 1.5 %) are unlikely to be justified and could lead to inefficient protein use or imbalance in amino acid supply. From an economic perspective, the cost of Gln supplementation may be offset by potential improvements in egg quality and possibly by enhanced bird longevity, as healthier birds may maintain productivity longer or exhibit fewer carcass downgrades related to bone damage. From a scientific standpoint, our findings contribute to a broader understanding of how amino acid nutrition influences mineral metabolism and bone biology in birds, highlighting the close interrelationship between skeletal health and reproduction in laying poultry.

One limitation of the study is the lack of histological, hormonal and gene expression data, which restricts mechanistic interpretation. Histological analyses could help distinguish between cortical and medullary bone contributions, while hormonal measurements (e.g. estrogen-related changes) would clarify age-dependent regulation of mineral deposition and bone remodeling in laying quail. Another limitation of the present study is that bone traits were assessed at only two time points (6 and 12 wk of lay), which does not fully capture the temporal dynamics of skeletal changes throughout the laying period.

In conclusion, dietary supplementation with Gln at 0.5–1.5 % increased tibial calcium content and several mineralization-related indicators in laying quail. These effects were level- and age-dependent and primarily reflected changes in mineral metabolism rather than improvements in bone geometry or mechanical strength. The lack of parallel enhancement in biomechanical properties suggests that the additional mineral was likely incorporated into metabolically active bone compartments, such as medullary bone, which contribute little to load-bearing capacity–. From a broader perspective, Gln supplementation may support skeletal calcium reserves during the laying period without substantially improving structural bone strength.

CRediT authorship contribution statement

Ewa Tomaszewska: Writing – review & editing, Writing – original draft, Supervision, Resources, Formal analysis, Data curation, Conceptualization. Kornel Kasperek: Writing – review & editing, Writing – original draft, Resources, Methodology, Formal analysis, Data curation, Conceptualization. Kamil Drabik: Resources, Investigation, Funding acquisition, Data curation, Conceptualization. Iwona Puzio: Writing – original draft, Resources, Investigation, Data curation. Izabela Świetlicka: Writing – original draft, Validation, Methodology, Formal analysis, Data curation. Justyna Batkowska: Resources, Data curation. Michał Wójcik: Writing – original draft, Visualization, Project administration. Daniel Kamiński: Methodology, Formal analysis.

Disclosures

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Declarations of interest: None.

Appendix. Supplementary materials

Supplementary Figure 1. Schematic overview of glutamine (Gln) metabolism in the small intestine and its systemic fate. Dietary Gln is taken up by enterocytes, where it serves as a substrate for protein synthesis and is metabolized to glutamate and other intermediates of the tricarboxylic acid cycle. Glutamine-derived metabolites contribute to the synthesis of ornithine, citrulline, and proline, which are released into the bloodstream. Systemically, Gln participates in nitrogen metabolism in the liver and kidneys and provides substrates for proline and hydroxyproline synthesis, supporting collagen maturation, bone matrix formation, and medullary bone development as a calcium reservoir for eggshell formation.