Multifaceted effects of Salvia hispanica oil and Lactobacillus farciminis on quail physiology

Abstract

Background

This study evaluated the effects of Lactobacillus farciminis and Salvia hispanica oil, alone or in combination, on growth performance, antioxidant status, cecal short-chain fatty acid (SCFA) profiles, and bone biomechanical properties in Japanese quails. These natural antibiotic alternatives were selected because L. farciminis supports the intestinal barrier, while chia oil—rich in omega-3s and phenolic compounds—modulates antioxidant defense and mineral bioavailability.

Methods

A total of 216 one-day-old Coturnix coturnix japonica chicks were randomly assigned to four groups: control, probiotic (Lactobacillus farciminis, 1 × 10⁹ CFU/kg), chia oil (Salvia hispanica oil, 0.2 g/kg), and combined treatment Birds were maintained under identical rearing conditions and fed a maize- and soybean-based basal diet with or without supplementation. The trial lasted 35 days; at the end of this period, blood samples were collected from the wing vein, and following slaughter, the hind leg bones (femur and tibia) were collected along with the carcass contents for laboratory analysis.

Results

No significant effects were observed on growth performance parameters (BW, BWG, FI, FCR) or bone biomechanical properties of femur and tibia (length, width, UL, YL, DUL, DYL, stiffness) (p > 0.05). Similarly, MDA, total SCFAs, major SCFA fractions (acetate, propionate, valerate, isovalerate), and serum biochemical parameters (ceruloplasmin, albumin, total protein, globulin) were not significantly affected (p > 0.05). Antioxidant status showed selective modulation, with Lactobacillus farciminis increasing GSH levels (p < 0.001), while superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) activities were influenced by chia oil and interaction effects. In SCFA profiles, specific fractions were differentially affected. Notably, a significant probiotic × chia oil interaction was observed for SOD activity and butyrate levels.

Conclusion

The combined supplementation of Lactobacillus farciminis and Salvia hispanica oil influenced certain antioxidant and fermentation-related parameters, particularly SOD activity and butyrate concentration, although no corresponding improvements were observed in growth performance or bone characteristics.

Supplementary Information

The online version contains supplementary material available at https://doi.org/10.1186/s12917-026-05731-1.

Introduction

The ban on the use of antibiotics in animal production to promote growth, due to the development of antimicrobial resistance and the risk of residues, has increased interest in alternative feed additives that can sustainably support animal health and production performance. Among these alternatives, probiotics are considered a significant option due to their ability to support gut health and demonstrate positive effects on performance [1]. Lactic acid bacteria, found among probiotics, are the subject of intensive research due to their ability to adhere to the intestinal epithelium and their positive effects on intestinal barrier function. One of the standout species in this context, Lactobacillus farciminis, has attracted attention due to its potential to support gut integrity and contribute to host health [2]. It has been reported that probiotic microorganisms used as feed additives can not only improve the general health status of animals but also positively influence performance parameters [3]. Phytobiotics, defined as plant-derived feed additives, play a significant role in enhancing health and productivity in farm animals [4]. These compounds help regulate the immune system, assist in balancing the gut microbiota, and can suppress the proliferation of pathogenic microorganisms. As a result, digestive system functions are supported, improvements in product quality are observed, and positive changes may occur in certain characteristics related to meat and egg quality in particular [5].

In this study, chia oil (Salvia hispanica L.), selected as a phytobiotic source, is one of the functional plant-based products that has attracted attention in recent years due to its rich nutritional content. Chia seeds are rich in omega-3 fatty acids, polyunsaturated fatty acids (PUFAs), and dietary fibre, whilst also containing gluten-free proteins, vitamins, minerals, and various phenolic compounds [6, 7]. Historically used for medicinal purposes, the seeds of this plant are particularly high in fat and fibre [8]. It has been reported that chia seeds contain approximately 18–24% protein, as well as 8.65–9.99% saturated fatty acids and 7.33–10.95% monounsaturated fatty acids [9, 10]. Skeletal disorders observed particularly in fast-growing genotypes in poultry production lead to both economic losses and adverse effects on animal welfare [11]. It is known that diet plays a decisive role in bone development and strength, and it has been reported that polyunsaturated fatty acids (PUFAs) are particularly effective in regulating bone metabolism. Omega-3 fatty acids may support osteoblast activity whilst reducing the production of inflammatory cytokines that increase bone resorption [12]. Furthermore, it has been noted that monounsaturated fatty acids (MUFA) also play a role in bone formation, bone resorption and prostaglandin metabolism [13, 14].

Japanese quails (Coturnix japonica) are widely preferred in experimental research due to their rapid growth, short generation interval, early sexual maturity and low maintenance costs [15]. Thanks to these characteristics, they serve as a suitable model for investigating various physiological parameters such as growth performance, metabolic processes, gut health and bone biomechanics.

Restrictions on the use of antibiotic growth promoters have accelerated research into natural alternatives that can simultaneously support gut health, the antioxidant defence system and skeletal development. It has been reported that Lactobacillus farciminis regulates the gut environment by increasing the production of short-chain fatty acids (SCFAs), thereby potentially enhancing the solubility of minerals [2]. It is suggested that the increase in SCFA production lowers intestinal pH, thereby facilitating mineral absorption, and that this may contribute to bone metabolism [16].

Chia oil, being a rich source of alpha-linolenic acid (ALA), calcium and phenolic antioxidants, is significant for bone health and the control of oxidative stress [10]. Phenolic compounds found in chia, such as caffeic acid, quercetin and chlorogenic acid, exhibit strong antioxidant properties. Furthermore, as chia seeds contain significant levels of calcium, they may contribute to bone mineralisation [17]. It has also been reported that chia oil, which is rich in ALA (Alpha-Linolenic Acid; ALA), may support bone integrity and mineral density [18].

Studies conducted on Japanese quails have shown that chia oil can improve live weight gain and feed conversion ratio (FCR), as well as exert positive effects on lipid metabolism and the antioxidant system [19]. Similarly, it has been reported that the use of chia oil in laying quails increases the omega-3/omega-6 fatty acid ratio in egg yolk [20].

Therefore, the aim of this study is to determine the effects of Lactobacillus farciminis and Salvia hispanica oil supplements, either alone or in combination, on growth performance, antioxidant status, caecal short-chain fatty acid profiles and bone biomechanical properties in Japanese quails, and to elucidate any potential synergistic interactions. In the study, it is anticipated that the combined application will positively modulate the gut microbiota to increase SCFA production, support the activities of antioxidant enzymes—reduced glutathione (GSH), superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (Glutathione Peroxidase; GPx), improve mineral absorption, and consequently provide more pronounced improvements in bone strength and growth performance compared to single applications.

Materıals and methods

Animals, experimental design and feed

A total of 216 one-day-old chicks (Coturnix coturnix japonica) were used in the study; these chicks were randomly divided into 4 main groups (54 birds per group), and each group was further divided into 6 subgroups/replicates (9 birds per replicate). All subgroups were fed the same diet under the same rearing conditions as their respective groups. The study groups were: control (C), Lactobacillus farciminis (P) (Probiotic); 1 × 10⁹ CFU/kg), Salvia hispanica oil (CO (Chia Oil); 0.2 g/kg) and Probiotic+Chia oil (PCO; 1 × 10⁹ CFU Lactobacillus farciminis + 0.2 g/kg Salvia hispanica oil). During the experiment, while the control group was given a basic ration consisting of maize and soya beans, the experimental groups were given probiotics and chia oil at the specified doses in addition to this basic ration (Table 1). The trial period was 35 days. Ration was prepared in accordance with NRC [21] standards, and the AOAC [22] method was used for all nutrient analyses. A cage with dimensions of 22 × 95 × 45 cm; height, length, and width was used for each of the replicates. The stocking density was maintained at approximately 21 birds/m² to ensure optimal welfare and growth conditions. In addition, manual feeders and automatic nipple drinkers were used in all cages. Water and feed were given to the birds ad libitum. The temperature of the cage, which was set to 32–35 °C on the first day of the study, was gradually reduced and kept at 22 °C for the last two weeks. A continuous lighting program of 24 h (24 L:0D) was applied during the first week, followed by a 16 L:8D cycle for the remainder of the study The animals used in the study were obtained from the Kafkas University Poultry Unit, where local ethics committee approval was received. The feeds are prepared daily to ensure the effectiveness of the probiotics and phytobiotics used in each group. The probiotic (Lactobacillus farciminis) used in the study was supplied (Biacton + R) by Tarimsan Chemical Inc. Istanbul, Turkey. Biacton^®+, a commercial probiotic, was utilized in this study. As per the manufacturer’s specifications, this strain is specifically formulated to remain stable during feed preparation. Furthermore, to preserve microbial viability, feeds were prepared fresh and kept under cool, dry storage conditions. The phytobiotic source chia oil used in the study was supplied by the commercial company Botalife in Isparta, Türkiye. Information about chia oil and probiotic is shown in Table 2. All experimental procedures were performed in accordance with the international guidelines for the care and use of laboratory animals.

Table 1.

Composition of basal diets used in experiment (%)

Feed materials	%
Corn	50.00
Soybean meal	28.00
Full fat soybean	19.00
Limestone	1.30
Dicalciumphosphate	1.00
DL- Methionine	0.10
L-Lysine Hydrochloride	0.10
Vitamin premix	0.15
Mneral premix	0.15
Salt	0.20
Total	100.00
The calculated value
Crude protein, %	23.82
ME (kcal/kg)	3066.00
Ca, %	0.87
Total P, %	0.62
Analysis Values
ME (kcal/kg)	3047.00
Crude protein, %	23.77
Ca, %	0.87
Total P, %	0.60

Table 2.

Chia oil fatty acid composition and probiotic content information used in this study

Probiotic
Product Content
Lactobacillus farciminis	5 × 109 *CFU/g
*Chia oil, %
Palmitic acid	2.0–5.0
Palmitoleic acid	0.5
Oleic acid	13.0
Linolenic acid	1.0
Linoleic acid	11.0
Stearic acid	3.0

*CFU Colony forming units

*Botalife

Growth performance

The body weight (BW) of all birds in each subgroup was measured individually on a weekly basis (at 0, 7, 14, 21, 28, and 35 days of the experiment). Body weight gain (BWG) for each subgroup was determined by calculating the difference between the consecutive weekly weight values. Feed intake (FI) was also monitored weekly throughout the 35-day experimental period. Based on these weekly measurements, the feed conversion ratio (FCR) was calculated to evaluate the efficiency of nutrient utilization. To evaluate growth performance, individual body weights (BW) were measured weekly from day 0 to day 35. We determined body weight gain (BWG) by calculating the difference between successive weekly records. Concurrently, feed intake (FI) was monitored at weekly intervals. These measurements allowed us to calculate the feed conversion ratio (FCR) as an indicator of nutrient utilization efficiency.

Antioxidant status

At the end of the experiment, blood samples were collected from the wing veins of a total of 24 birds, one from each subgroup, into anticoagulant (EDTA) tubes. After separating whole blood from the blood samples, the remaining samples were centrifuged at 3000 rpm for 15 min to obtain plasma and stored at −20 °C until analysis. Antioxidant enzymes superoxide dismutase (SOD), glutathione peroxidase (GPx) and catalase (CAT) activities were determined using commercial kits (Cayman Chemical Company, USA) and an ELISA device (Epoch, Biotek, USA). Whole blood glutathione (GSH) analysis was determined colorimetrically (Epoch, Biotek, USA) [23]. Malondialdehyde (MDA) value and ceruloplasmin values in plasma were determined by specific methods, respectively [24, 25]. A commercial test kit (Biolabo, Maizy, France) was used to determine albumin and total protein values. The globulin value was calculated by subtracting albumin from total protein [26].

Cecal short-chain fatty acid concentrations

One animal from each subgroup was randomly selected and euthanized by cervical dislocation, and the cecum was dissected. At the end of the study, the cecum contents were stored at −20 °C. Then, these samples were thawed at 4 °C. The thawed samples were diluted 10-fold by mixing with double distilled water in sterile screw-capped tubes. The caecum was centrifuged at 4000 rpm for 15 min at 4 °C to homogenize the digestion. The supernatant was placed in a 750 µl Eppendorf tube and 150 µl of iced 25% metaphosphoric acid solution was added and mixed. Then, the tubes were kept in a styrofoam filled with dry ice at low temperature for 30 min. This cold application was done to ensure the precipitation of proteins. Then, the tubes were centrifuged again at 10,000 rpm at + 4 °C for 10 min. The analysis of the supernatant was performed by gas chromatography (Shimadzu GC-2010, Shimadzu Co., Kyoto, Japan). The GC column was a 30 m×0.53 mm inner diameter column (Teknokroma TRB-FFAP, Teknokroma, Barcelona, Spain). A flame ionization detector was used to determine the SCFA concentrations in the cecum content. The temperatures were fixed at 250 °C in the injector port and flame ionization detector (FID). The temperature was kept constant during the temperature program and the first 4 min after injection and was increased at a rate of 4 °C/min until it reached 160 °C after injection and finally kept constant at this temperature for 4 min. The carrier gas was helium. The injection volume used was a fixed 1 µL and duplicate analyses were performed [27].

Bone biomechanics

At the end of the experiment, a total of 24 animals (one from each subgroup) were slaughtered, and the left femur and left tibia from each animal were collected for biomechanical analyses. The left femur and left tibia were cleaned of all tissues and their lengths and widths were measured with a digital caliper. The bone samples were stored at −20 °C for later analysis. Prior to testing, the samples were conditioned in 0.09% saline solution to prevent drying. The femurs and tibias were subjected to three-point bending tests with an Instron 5944 test frame (Instron, Norwood, MA, USA). The loading rate was 5 mm/min. The span lengths were 25 mm for tibias and 20 mm for femurs. To ensure uniform load distribution within the biological structures, a 2 N preload was applied to all specimens. To evaluate bone behavior under bending loads, a three-point bending test was executed in the anterior-posterior plane. The load was applied precisely at the midpoint of the bone shaft using a loading rate of 5 mm/min.To evaluate the effects of Lactobacillus farciminis and Salvia hispanica oil supplementation on bone metabolism, internal mechanical data at the tissue level are required Based on the literature, calculating parameters such as the elasticity modulus and ultimate strength in mechanical tests for materials used in conditions affecting bone metabolism will yield more accurate results in the evaluation process. Researchers can use formulae based on the sources ([28] Load-displacement data were determined for each sample as shown Fig. 1. While the stiffness values were calculated from the slope of the linear region of the load-displacement curves, the ultimate load (UL) and the displacement at ultimate load (DUL) were also calculated from the load-displacement curves. The load at which the permanent deformation of the system begins is the yield load (YL). The displacement at which the permanent deformation begins is the displacement at yield load (DYL) [29]. During the tests, load and displacement data were collected and used to create the Load-Displacement curve shown in Fig. 2.

Fig. 1.

Test setup

Fig. 2.

Load-displacement curve

Statistics analysis

Statistical analysis of the data was performed using IBM SPSS Statistics for Windows, Version 27.0 (IBM Corp., Armonk, NY, USA). Prior to conducting the parametric analyses, model assumptions were rigorously evaluated using Shapiro-Wilk and Kolmogorov-Smirnov tests, skewness and kurtosis coefficients, residual histograms, and normal Q-Q plots. Although moderate deviations from normality and homogeneity of variance assumptions were observed in certain variables, the skewness and kurtosis values for most parameters remained within acceptable limits (−2 to + 2) [30, 31], and residual analyses indicated no severe violations of normality. Homogeneity of variance was evaluated using the Levene test alongside the median-based Brown-Forsythe approach. Given that the experimental design was strictly balanced with equal g Dietary supplementation with Lactobacillus farciminis and chia oil selectively modulated the antioxidant parameters depending on the specific variable evaluated. No statistically significant main or interaction effects were observed for malondialdehyde (MDA) levels (p > 0.05). Group sample sizes (n = 6 per group), the two-way analysis of variance (ANOVA) was considered robust against these moderate assumption violations. For growth performance criteria (body weight, body weight gain, feed intake, and feed conversion ratio), the replicate cage (subgroup mean) was defined as the experimental unit. For blood antioxidant status, cecal short-chain fatty acid profiles, and bone biomechanical parameters, the individually sampled bird (one randomly selected bird per replicate cage) served as the experimental unit. Where significant differences were identified, post-hoc pairwise comparisons were performed using the Bonferroni method, and the interaction effects between main factors (probiotic × chia oil) were assessed. The level of statistical significance was set at p < 0.05.

Results

Two-way ANOVA results indicated that neither probiotic nor chia oil supplementation, alone or in combination, had a statistically significant effect on growth performance parameters (p > 0.05). At the beginning of the experiment, no significant differences were observed among the groups in terms of initial body weight (p > 0.05). Similarly, final body weight, body weight gain, feed intake, and feed conversion ratio (FCR) were not significantly affected by the main effect of probiotic supplementation (p = 0.173, p = 0.160, p = 0.621, and p = 0.270, respectively). Likewise, the main effect of chia oil supplementation was not significant for any of the growth performance parameters (p > 0.05). Furthermore, no significant interaction effect (probiotic × chia oil) was detected for any parameter (p > 0.05). The effect of probiotics and chia oil on the growth performance of quails is presented in Table 3.

Table 3.

Influence of probiotic and chia oil on growth performance

Performance Parameters	Groups				Significance
	Control	P	CO	PCO	Probiotic	Effect Size	Chia Oil	Effect Size	Probiotic* Chia Oil	Effect Size
	x̄- S x̄	x̄- S x̄	x̄- S x̄	x̄- S x̄	p	η²	p	η²	p	η²
Initial Live Weight, g	13.22 ± 0.48	13.55 ± 0.38	13.41 ± 0.41	13.26 ± 0.38	0.825	0.002	0.908	0.001	0.565	0.017
Final Live Weight, g	221.90 ± 3.88	224.32 ± 3.69	217.60 ± 3.03	224.47 ± 2.66	0.173	0.091	0.534	0.020	0.507	0.022
Live Weight Gain, g	208.68 ± 3.64	210.77 ± 3.54	204.19 ± 3.00	211.21 ± 2.29	0.160	0.096	0.521	0.021	0.439	0.030
Feed Intake, g	660.77 ± 32.90	647.86 ± 18.76	659.36 ± 33.48	643.73 ± 32.62	0.621	0.012	0.921	0.001	0.960	0.001
Feed Conversion Ratio	3.16 ± 0.13	3.07 ± 0.06	3.23 ± 0.15	3.04 ± 0.13	0.270	0.060	0.875	0.001	0.703	0.007

Statistically significant *p < 0.05, **p < 0.01, ***p < 0.001, effect size: η², The mean (x) and standard error (Sx) values of 6 replicates in each group

Dietary supplementation with probiotics and chia oil selectively modulated the antioxidant parameters depending on the specific variable evaluated. No statistically significant main or interaction effects were observed for malondialdehyde (MDA) levels (p > 0.05). For glutathione (GSH) levels, chia oil supplementation alone did not have a significant effect (p > 0.05), whereas probiotic supplementation showed a statistically significant moderate effect (p < 0.001; η² = 0.331). The interaction effect was not statistically significant (p > 0.05). Superoxide dismutase (SOD) activity was significantly affected by chia oil supplementation (p < 0.01) as well as by the probiotic × chia oil interaction (p < 0.001). The interaction effect demonstrated a high effect size (η² = 0.302). However, probiotic supplementation alone did not significantly influence SOD activity (p > 0.05). Similarly, catalase (CAT) activity was significantly influenced by chia oil supplementation (p < 0.01) and by the interaction between probiotic and chia oil (p < 0.01), both showing moderate effect sizes. Probiotic supplementation alone had no significant effect (p > 0.05). Glutathione peroxidase (GPx) activity was significantly affected by both probiotic and chia oil supplementation, as well as their interaction (p < 0.001). Chia oil exhibited a very high effect size (η² = 0.792), indicating a dominant role in influencing this parameter. The interaction effect was also high (η² = 0.490). No statistically significant main or interaction effects were observed for ceruloplasmin, albumin, total protein, or globulin levels (p > 0.05). The effect of experimental diets on antioxidant status is given in Table 4.

Table 4.

Influence of probiotic and chia oil on antioxidant status

Blood Parameters	Groups				Significance
	Control	P	CO	PCO	Probiotic	Effect Size	Chia Oil	Effect Size	Probiotic* Chia Oil	Effect Size
	x̄- S x̄	x̄- S x̄	x̄- S x̄	x̄- S x̄	p	η²	p	η²	p	η²
MDA (µmol/L)	2.79 ± 0.1	2.73 ± 0.09	2.57 ± 0.06	2.65 ± 0.12	0.122	0.065	0.904	0.001	0.492	0.013
GSH (mg/dL)	20.25 ± 2.68c	27.35 ± 2.22ab	34.45 ± 1.79a	34.27 ± 3.49a	0.0001***	0.331	0.266	0.034	0.123	0.065
SOD (U/mL)	27.56 ± 1.79b	39.37 ± 1.33a	32.55 ± 1.81b	31.8 ± 1.36b	0.424	0.018	0.001**	0.251	0.0001***	0.302
CAT (nmol/min/mL)	2.96 ± 0.09b	3.31 ± 0.05a	3.27 ± 0.05a	3.24 ± 0.02a	0.058	0.096	0.012*	0.161	0.004**	0.211
GPx (nmol/min/mL)	20.62 ± 1.28c	44.36 ± 1.94b	61.11 ± 1.94a	57.78 ± 2.37a	0.0001***	0.792	0.0001***	0.353	0.0001***	0.490
Ceruloplasmin (mg/dL)	13.18 ± 1.07	12.90 ± 0.96	12.81 ± 1.11	12.85 ± 1.06	0.841	0.001	0.905	0.001	0.881	0.001
Albumin (g/dL)	1.63 ± 0.04	1.60 ± 0.02	1.61 ± 0.05	1.62 ± 0.07	0.950	0.001	0.850	0.001	0.675	0.005
Total protein (g/dL)	3.81 ± 0.06	3.78 ± 0.03	3.75 ± 0.03	3.78 ± 0.04	0.397	0.020	0.970	0.001	0.424	0.018
Globulin (g/dL)	2.18 ± 0.06	2.17 ± 0.04	2.14 ± 0.05	2.16 ± 0.08	0.607	0.007	0.902	0.001	0.838	0.001

Mean (x) and standard error (Sx) values of 6 replicates in each group. statistically significant *p < 0.05, **p < 0.01, ***p < 0.001, effect size: η² *a, b,c; Differences between mean values indicated with different letters in the same row are statistically significant (p < 0.05)*

The results indicated that the main effects of probiotic and chia oil supplementation, as well as their interaction, were not statistically significant for acetic, propionic, valeric, isovaleric acids, and total short-chain fatty acids (SCFAs) (p > 0.05). Isobutyric acid levels were significantly affected by probiotic supplementation (p < 0.05; η² = 0.226), whereas the effects of chia oil and the interaction were not significant (p > 0.05). Butyric acid levels were significantly influenced by both probiotic supplementation (p < 0.05; η² = 0.203) and the probiotic × chia oil interaction (p < 0.05; η² = 0.274). Additionally, caproic acid and isocaproic acid levels were significantly affected by probiotic supplementation (p < 0.05), while chia oil and interaction effects were not significant for these parameters (p > 0.05). Branched-chain fatty acid (BCFA) levels were significantly affected by chia oil supplementation (p < 0.05; η² = 0.204), whereas probiotic supplementation and the interaction effect were not statistically significant (p > 0.05).The effects of experimental diets on some short-chain fatty acids in the cecum (µmol/g) are given in Table 5.

Table 5.

Influence of probiotic and chia oil on cecal some short-chain fatty acid concentrations (µmol/g)

SCFA Parameters	Groups				Significance
	Control	P	CO	PCO	Probiotic	Effect Size	Chia Oil	Effect Size	Probiotic* Chia Oil	Effect Size
	x̄- S x̄	x̄- S x̄	x̄- S x̄	x̄- S x̄	p	η²	p	η2	p	η²
Acetic acid	50.63 ± 2.36	51.16 ± 3	50.43 ± 2.49	54.40 ± 0.96	0.345	0.045	0.522	0.021	0.469	0.027
Propionic acid	8.87 ± 3.21	3.26 ± 0.77	7.06 ± 2.24	8.27 ± 2.84	0.379	0.039	0.521	0.021	0.180	0.088
Isobutyric acid	0.42 ± 0.07a	0.26 ± 0.02b	0.43 ± 0.08a	0.32 ± 0.05ab	0.025*	0.226	0.569	0.016	0.707	0.007
Butyric acid	3.11 ± 0.55b	7.16 ± 0.96a	6.57 ± 0.84a	6.18 ± 0.83ab	0.035*	0.203	0.142	0.105	0.013*	0.274
Valeric acid	0.60 ± 0.11	0.57 ± 0.07	0.90 ± 0.15	0.64 ± 0.09	0.209	0.078	0.115	0.119	0.296	0.054
Isovaleric acid	1.95 ± 0.0	1.82 ± 0.15	1.99 ± 0.01	2.14 ± 0.16	0.965	0.001	0.125	0.114	0.222	0.074
Caproic acid	0.17 ± 0.01a	0.15 ± 0.02b	0.18 ± 0.01a	0.15 ± 0.01b	0.010*	0.286	0.743	0.006	0.743	0.006
Isocaproic acid	1.50 ± 0.22b	1.95 ± 0.0a	1.81 ± 0.17ab	2.13 ± 0.17a	0.025*	0.228	0.144	0.103	0.694	0.008
BCFA	2.98 ± 0.13	2.65 ± 0.17	3.33 ± 0.17	3.09 ± 0.21	0.120	0.116	0.035*	0.204	0.806	0.003
Total SCFA	64.99 ± 3.84	63.65 ± 3.35	66.49 ± 2.5	71.30 ± 2.91	0.592	0.015	0.167	0.093	0.347	0.044

Mean (x) and standard error (Sx) values of 6 replicates in each group. statistically significant *p < 0.05, **p < 0.01, ***p < 0.001, effect size: η² *a, b,c; Differences between mean values indicated with different letters in the same row are statistically significant (p < 0.05)*. BCFA (branched-chain fatty acids): isobutyric acid + isovaleric acid + isocaproic acid. Total SCFA: acetic acid + propionic acid + butyric acid + isobutyric acid + isovaleric acid + valeric acid + isocaproic acid

The two-way ANOVA results presented in Table 6. showed that probiotic and chia oil supplementation had no statistically significant effects on the biomechanical properties of either the femur or tibia (p > 0.05). For both bones, none of the evaluated parameters—including length, width, ultimate load (UL), displacement at ultimate load (DUL), yield load (YL), displacement at yield load (DYL), and stiffness—were significantly affected by the main effects of probiotic or chia oil supplementation, nor by their interaction (p > 0.05). Examination of effect sizes indicated that most parameters exhibited low effect sizes (η² < 0.06). Although some parameters approached small-to-moderate effect levels, these effects were not statistically significant.

Table 6.

Influence of probiotic and chia oil on femur and tibia biomechanical properties

		Groups				Significance
		Control	P	CO	PCO	Probiotic	Effect Size	Chia Oil	Effect Size	Probiotic* Chia Oil	Effect Size
		x̄±  Sx	x̄±  Sx	x̄± Sx	x̄± Sx	p	η²	p	η²	p	η²
	Length, mm	42.38 ± 0.32	43.68 ± 0.74	42.86 ± 0.58	42.83 ± 0.29	0.232	0.071	0.209	0.078	0.724	0.006
	Width, mm	3.33 ± 0.13	3.30 ± 0.10	3.27 ± 0.10	3.53 ± 0.13	0.218	0.075	0.457	0.028	0.329	0.048
	UL, N	36.92 ± 2.04	41.38 ± 2.39	38.34 ± 2.21	40.70 ± 2.89	0.671	0.009	0.881	0.001	0.176	0.090
	DUL, mm	0.61 ± 0.09	0.58 ± 0.04	0.57 ± 0.03	0.50 ± 0.06	0.814	0.003	0.321	0.049	0.367	0.041
Femur	YL, N	33.87 ± 2.06	39.97 ± 2.69	36.42 ± 2.23	37.36 ± 2.89	0.313	0.051	0.992	0.001	0.173	0.091
	DYL, mm	0.52 ± 0.01	0.53 ± 0.03	0.50 ± 0.02	0.38 ± 0.04	0.278	0.059	0.146	0.103	0.358	0.042
	Stiffness, N/mm	74.79 ± 7.68	77.51 ± 3.59	77.94 ± 5.49	100.09 ± 5.49	0.197	0.082	0.103	0.127	0.092	0.135
	Length, mm	53.09 ± 0.49	54.58 ± 0.91	53.76 ± 1.03	54.18 ± 0.57	0.239	0.069	0.863	0.002	0.505	0.022
	Width, mm	3.04 ± 0.12	2.83 ± 0.05	2.87 ± 0.08	2.85 ± 0.07	0.173	0.091	0.387	0.038	0.240	0.068
Tibia	UL, N	43.02 ± 3.90	36.29 ± 0.36	36.48 ± 1.56	39.05 ± 1.57	0.366	0.041	0.411	0.034	0.052	0.176
	DUL, mm	0.71 ± 0.02	0.67 ± 0.04	0.73 ± 0.03	0.76 ± 0.04	0.848	0.002	0.108	0.124	0.209	0.078
	YL, N	40.38 ± 4.41	35.79 ± 0.38	34.87 ± 1.28	38.26 ± 1.66	0.809	0.003	0.541	0.019	0.119	0.117
	DYL, mm	0.61 ± 0.04	0.62 ± 0.04	0.65 ± 0.05	0.71 ± 0.04	0.368	0.041	0.119	0.117	0.565	0.017
	Stiffness, N/mm	69.07 ± 6.39	59.45 ± 3.57	58.86 ± 3.67	57.17 ± 1.42	0.189	0.085	0.149	0.101	0.351	0.044

Statistically not significant (p > 0.05), effect size: η², The mean (x) and standard error (Sx) values of 6 replicates in each group

UL Ultimate Load, DUL Displacement at Ultimate Load, YL Yield Load, DYL Displacement at Yield Load

Discussion

The utilisation of probiotics as feed additives is widely regarded as advantageous due to their numerous physiological benefits, including the inhibition of pathogens, the enhancement of nutrient absorption, and the overall improvement of animal health and production performance [32]. In poultry, probiotic supplementation has been associated with enhancements in various performance indicators, including laying performance, egg quality, body weight gain, and feed conversion ratio (FCR) [3]. In a similar manner, phytobiotics have been documented to demonstrate growth-promoting and immunomodulatory properties ([33] With enhancements in FCR, feed intake (FI), and average daily gain (ADG) observed in birds receiving supplementation [34].However, the present findings demonstrate that probiotic and chia oil supplementation did not influence growth performance either individually or synergistically. In the present study, both single and combined supplementations of the probiotic and chia oil failed to yield measurable improvements in growth performance criteria. The initial homogeneity of the experimental groups confirms that the lack of variance in final body weight, weight gain, and feed efficiency was not biased by baseline weight differences. Furthermore, the non-significant interaction effect suggests that these two feed additives do not exert an additive or synergistic influence on phenotypic growth traits under these conditions.

These findings diverge from certain previous investigations. For instance, Alagawany et al., [19] reported enhanced weight gains and FCR with chia oil, while Durna et al., [35] observed variable body weight responses to Lactobacillus farciminis in Japanese quails. Such contradictions in literature are common and typically stem from fluctuations in dietary inclusion rates, administration periods, baseline physiological health, or environmental variations among trials.

In the present study, both single and combined supplementations of the probiotic and chia oil failed to yield measurable improvements in growth performance criteria. The initial homogeneity of the experimental groups confirms that the lack of variance in final body weight, weight gain, and feed efficiency was not biased by baseline weight differences. Furthermore, the non-significant interaction effect suggests that these two feed additives do not exert an additive or synergistic influence on phenotypic growth traits under these conditions. These findings diverge from certain previous investigations. For instance, Alagawany et al., [19] reported enhanced weight gains and FCR with chia oil, while Durna et al., [35] observed variable body weight responses to Lactobacillus farciminis in Japanese quails. Such contradictions in literature are common and typically stem from fluctuations in dietary inclusion rates, administration periods, baseline physiological health, or environmental variations among trials Furthermore, the non-significant interaction between probiotic and chia oil suggests that their combined use did not result in additive or synergistic benefits. The observed discrepancies between our findings and some previous literature may be attributed to differences in dosage, supplementation duration, animal species, and physiological status, all of which are recognized to influence the efficacy of dietary additives. The absence of any statistically significant differences in initial live weight indicates that the experimental groups are homogeneous, thereby ensuring the reliability of subsequent comparisons. The absence of observed effects on performance parameters suggests that, under the present experimental conditions, neither additive was sufficient to induce measurable improvements. Furthermore, the non-significant interaction effect suggests that no synergistic or additive response occurred between the two supplements. The observed discrepancies in the present study may be attributed to differences in dosage, supplementation duration, animal species, and physiological status, all of which have been demonstrated to influence the efficacy of dietary additives. Indeed, previous studies have reported variable responses to Lactobacillus farciminis in Japanese quail, affecting live weight (LW) and FCR but not always live weight gain (LWG) or FI [35]. In a similar vein, chia oil supplementation has been documented to enhance LW, LWG, and FCR without affecting FI in quail [19]. Moreover, chia seeds have been demonstrated to promote growth performance and feed efficiency [36]. In the context of poultry, the effects of probiotic supplementation on LW, FI, and FCR have been shown to be inconsistent [37]. From a mechanistic perspective, these inconsistencies can be explained by the complex and dynamic nature of the gut ecosystem. The mechanisms through which probiotics exert their effects are predominantly attributable to three main factors: competitive exclusion, the production of antimicrobial metabolites, and the enhancement of intestinal barrier integrity. However, the efficacy of probiotics is contingent upon the initial microbial balance. In animals with a stable gut microbiota, the colonisation of exogenous probiotics may be limited, resulting in minimal changes in performance-related traits. Conversely, phytobiotics primarily exert their effects through antioxidant and anti-inflammatory pathways, which are mediated by bioactive compounds such as phenolics, flavonoids, and terpenoids. These compounds have been demonstrated to enhance nutrient absorption by reducing oxidative stress and modulating immune responses [38]. In instances where inclusion levels are minimal or supplementation periods are brief, the potential for such effects to translate into quantifiable performance responses may be inadequate. Furthermore, the observed variability among studies may be attributable to differences in probiotic strains and phytobiotic composition. It is important to note that probiotic effects are strain-specific, and phytobiotic activity is influenced by factors such as plant source, extraction method, and storage conditions. Animal age, physiological status, and environmental stressors further contribute to variability by modulating gut microbiota composition and host response. Finally, the duration of supplementation plays a critical role, as short-term exposure may be insufficient to induce stable microbial or metabolic adaptations. In this context, the absence of performance-related effects in the present study may be attributed to an already balanced gut microbiota, limited dose and duration of supplementation, or optimal rearing conditions that reduced the potential for observable improvements. This lack of performance enhancement aligns with the core principles observed in previous research by Khan et al., [37], suggesting that when poultry are reared under optimal, non-stressed environmental conditions, the baseline gut microbiota remains highly stable. Consequently, the competitive exclusion mechanism of exogenous probiotics like L. farciminis or the growth-promoting efficiency of phytobiotics becomes less pronounced compared to trials executed under active physiological or environmental stress.

Oxidative stress occurs when free radicals are present in excess in biological systems. Free radicals are produced from regulated and unregulated sources (e.g. mitochondrial respiration, enzymatic activities, electron leakage). They are also formed in response to environmental and nutritional stress factors. They disrupt redox homeostasis by disrupting antioxidant defenses in the body, thus leading to oxidative stress. Dietary antioxidants can be used to correct redox imbalances. Important dietary antioxidants include vitamins, trace elements, some fatty acids, and phytonutrients such as beta-carotene, polyphenols, flavonoids, etc. [39]. With the knowledge that dietary antioxidants protect against oxidative stress, our study investigated the protection of probiotics and phytobiotics against oxidative stress. Probiotics have their own antioxidant enzymatic systems [40]. Bifidobacterium animalis has been shown to scavenge hydroxyl radicals and superoxide anion in vitro by improving antioxidant activity [41]. Thus, this mechanism of action may necessarily show antioxidant effects. Plants have antioxidant effects, especially due to their redox properties, with the phenolic compounds they contain. Therefore, plant phenolic compounds act as reducing agents, hydrogen donors, singlet oxygen inhibitors and metal chelators [38]. The observed discrepancies in findings can be attributed to the differential and target-specific responses of Lactobacillus farciminis and Salvia hispanica oil within redox-related biological pathways. The hypothesis that Lactobacillus farciminis exerts its effects through microbial metabolites such as short-chain fatty acids and bioactive peptides is postulated, given the evidence that such metabolites influence antioxidant enzyme regulation. In particular, the consistent increase in superoxide dismutase activity reported in various studies suggests a selective stimulatory effect on this enzyme, which is primarily responsible for the initial neutralisation of superoxide radicals. Of particular significance was the observation of a substantial interaction between Lactobacillus farciminis and Salvia hispanica oil on superoxide dismutase activity, indicating a synergistic effect. This finding suggests that combined supplementation enhances enzymatic superoxide detoxification more effectively than either additive alone. Conversely, the more variable responses observed for catalase and glutathione peroxidase may be ascribed to their more intricate regulatory control and reliance on intracellular redox conditions. Salvia hispanica oil, which is rich in phenolic compounds, may further contribute to these effects through redox-active mechanisms and modulation of gene expression pathways, although its impact appears to be more dependent on bioavailability and metabolic conversion. The observed variability across studies is likely driven by differences in dose, duration, host physiology, and microbiota composition, highlighting that Lactobacillus farciminis and Salvia hispanica oil exert selective but potentially complementary effects, with a clear synergistic interaction at the level of superoxide dismutase activity. By correlating our selective antioxidant responses with prior art, the substantial interaction effect observed on SOD and CAT activities validates the synergistic hypothesis between L. farciminis metabolites and chia oil phenolics. While Alagawany et al., [19] highlighted that chia oil predominantly triggers SOD synthesis due to its high concentration of flavonoids, our findings further demonstrate that the co-supplementation with a probiotic strain complements this first line of enzymatic defense. In one study, it was observed that different doses of chia oil applied to poultry increased the activity of Superoxide Dismutase (SOD), but did not change the values of Glutathione (GSH) and Glutathione Peroxidase (GPx) [19]. Similarly, in another study where different doses of the same probiotic strain were applied as in our study, an increase was observed in GSH, SOD and GPx, while no change was recorded in catalase (CAT) [35]. The overlap of these two findings suggests that the phenolic compounds and probiotic metabolites in chia oil particularly trigger SOD synthesis, but the stimulatory effect on the glutathione system remains weaker. On the other hand, a significant increase in GPx activity was reported in a study using mixed plant extract (phytobiotic) [42]. Flavonoid and tannin derivatives in the extract may have increased the activity of this enzyme by acting as an inducer for GPx. In contrast, in a study conducted with the same phytobiotic formulation in geese, it was reported that although there was an increase in GSH in the early period, GSH, SOD, GPx and CAT levels did not exceed threshold values at the end of the trial [43]. This situation shows that although the animals responded to the stimulating effect in the short term, adaptation mechanisms may have stepped in and brought the antioxidant enzyme levels back to balance in the long term. In a study, the use of microencapsulated probiotics and prebiotics in broiler diets increased total antioxidant capacity (TOAC) in serum [44]. In another poultry study where a different probiotic source was preferred, a significant effect was only achieved on SOD activity [37]. This result suggests that probiotic metabolites specifically support the synthesis of SOD, which scavenges superoxide radicals, but do not provide an induction towards glutathione and catalase. On the other hand, in a study conducted on lambs with different doses of the same probiotic strain as in our study, SOD, CAT and GPx activities increased, while GSH was not affected [35].

Probiotics, like antibiotics, produce antimicrobial substances with a similar level of effectiveness (in combination with glucose and modification of intestinal pH) as organic acids, bacteriocins, short-chain fatty acids (SCFA) or hydrogen peroxide [45]. The composition of the gut microbiota and its metabolites are essential components of overall health in living organisms. Short-chain fatty acids (SCFAs) are among the primary metabolites produced by the gut microbiota [46]. SCFAs themselves or additives that cause their production may be considered promising alternatives to antibiotics in poultry. In light of this information; dietary additives may also have an antimicrobial effect through the SCFAs they create in the intestinal flora. Also, short-chain fatty acids are a result of bacterial fermentation in the cecum. They stimulate cell growth-differentiation in the intestine, improving intestinal integrity and suppressing the proliferation of pathogenic microorganisms by lowering the pH of the digestive tract [47]. Acetic, propionic and butyric acids are formed as a result of saccharolytic fermentation and have health-promoting effects. SCFAs act as mediators in the communication between the gut microbiome and the immune system [48]. Butyric acid (C4:0) affects metabolic processes that have been confirmed. Butyric acid supplementation has been used to prevent obesity, hyperinsulinemia, and hypertriglyceridemia. It also activates brown adipose tissue (BAT) via vagal nerve signaling [49]. Isobutyric acid is a branched short-chain fatty acid that is the product of proteolytic (decomposition) fermentation in the large intestine (with the participation of proteolytic microbiota from the Proteobacteria group) [50].

There is a view that short-chain fatty acids (SCFAs) and medium-chain fatty acids (MCFAs), particularly compounds such as caproic acid, may exert a synergistic effect on bacteria. In this mechanism, it is suggested that MCFAs increase permeability by damaging the cell wall of microorganisms, thereby allowing SCFAs to enter the bacterial cytoplasm more easily and exert their antibacterial effects [51]. Current findings indicate that the effects of probiotic and chia oil supplements on SCFA profiles are not homogeneous across all fatty acids but exhibit a selective pattern of action. The absence of significant changes in the levels of the primary SCFAs, acetic and propionic acids, suggests that the interventions did not substantially alter the overall fermentation pattern. In contrast, it was determined that probiotic administration had significant effects on the levels of isobutyric acid, butyric acid, caproic acid and isocaproic acid. This suggests that specific microbial metabolic pathways were selectively affected. The increase in butyric acid levels is particularly noteworthy, as butyric acid has previously been shown to play a key role in maintaining gut health and epithelial integrity [52]. This finding supports the functional importance of probiotic supplementation. The interactive effect on butyric acid suggests that the combination of Lactobacillus farciminis and chia oil may enhance certain microbial fermentation processes and potentially create a synergistic effect. Conversely, the pronounced effect of chia oil on branched-chain fatty acids (BCFAs) may be related to changes in protein fermentation or microbial composition. The absence of an interaction effect observed for many parameters in the study indicates that the combined use of probiotics and chia oil does not consistently produce an additive or synergistic effect across all SCFA components. Overall, the findings suggest that these two supplements do not alter total SCFA production, but rather selectively influence specific fatty acid fractions, pointing to potential changes in gut microbial activity and metabolic functions. In a study where 1% thyme powder was added to the diet of broiler chickens as a phytobiotic, the production of short-chain fatty acids was 61% higher in the experimental groups compared to the control [53]. As a phytobiotic, Safflower is able to regulate SCFAs, especially acetic, butyric and total acids, in the intestinal content of carp [54]. Addition of essential oil mixture (peppermint oil, juniper oil, rosemary oil and thyme oil) as phytobiotic to the drinking water of breeder quails at doses of 0.1 ml/L and 0.3 ml/L affected acetic acid, butyric acid, isocaproic acid and SCFA values linearly with the dose [44]. Green tea extract and black tea extract stimulated caeca SCFA production in rat diets [55]. Probiotics of the same strain as our study, added to the drinking water of breeding quails at various doses (0.5 g/10 L and 1 g/10 L), affect isocaproic acid but, did not affect the values of acetic acid, propionic acid, isobutyric acid, butyric acid, isovaleric acid, valeric acid, caproic acid, total SCFA and BCFA of the cecum content [56]. In a study with the same strain as the probiotic in the study, the use of probiotics in quails (at doses of 0.1 g/kg and 0.3 g/kg) affected acetic acid, isocaproic acid and SCFA values linearly in proportion to the dose [35]. In the context of poultry, short-chain fatty acids (SCFAs) represent pivotal microbial metabolites, which are produced through the fermentation of dietary fibres within the gastrointestinal tract. The influence of these metabolites on the host’s physiology has been demonstrated to be of pivotal significance. The effects of these hormones are mediated through three main mechanisms: the activation of G protein–coupled receptors (GPCRs), which modulates cellular signalling pathways involved in immune response and energy metabolism; the inhibition of histone deacetylase (HDAC) activity, leading to increased histone acetylation and epigenetic regulation of gene expression; and the subsequent modulation of anti-inflammatory responses in intestinal and peripheral tissues. Among SCFAs, butyrate functions as a primary energy source for intestinal epithelial cells and contributes to mucosal integrity, whereas acetate and propionate play a role in immune regulation and metabolic balance. It has been demonstrated that these mechanisms enhance gut barrier function, suppress the proliferation of pathogenic microorganisms, and optimise microbiota–host interactions. Consequently, SCFAs play a critical role in maintaining gut health, improving disease resistance, and supporting sustainable production performance in poultry [57]. The observed variability among studies can be explained by the selective, pathway-specific modulation of gut microbial fermentation rather than a uniform alteration in total short-chain fatty acid (SCFA) production. Probiotics such as Lactobacillus farciminis have been demonstrated to influence microbial community structure and metabolite output, leading to targeted changes, particularly in butyric, isobutyric, caproic, and isocaproic acids. Conversely, major SCFAs, including acetate and propionate, frequently exhibit no alteration in their levels. The increase in butyric acid, in conjunction with the observed interaction between probiotic and Salvia hispanica, is a subject that merits further investigation. The selective modulation of butyric, isobutyric, and caproic acids in our study underscores a specific metabolic shift rather than a total alteration of the fermentation pattern, a phenomenon that mirrors the selective trends reported by Durna and Yıldız [56]. The significant probiotic × chia oil interaction on butyrate levels is particularly noteworthy when connected to the wider literature; it implies that the polyunsaturated fatty acids and fibrous fractions of chia oil may act as a selective substrate (prebiotic effect) that actively favors the proliferation of butyrate-producing commensal bacteria when stimulated by the stabilizing presence of L. farciminis.

In the poultry industry, leg abnormalities are an increasingly prevalent issue, stemming from an imbalance between rapid growth rates and skeletal mineralization capacity [11]. Dietary micro- and macronutrients play an important role in skeletal health, particularly through fatty acid profiles and mineral intake. Polyunsaturated and monounsaturated fatty acids modulate bone metabolism via prostaglandin synthesis and osteoblast/osteoclast activity, with the potential to reduce bone resorption and stimulate bone formation [12–14]. Due to its high alpha-linolenic acid (omega-3) and calcium content, Salvia hispanica oil has been hypothesized to enhance bone mineral density and structural strength [17, 18]. Conversely, the effect of probiotics on bone health is explained primarily through indirect mechanisms; by increasing short-chain fatty acid (SCFA) production and lowering intestinal pH, probiotics can increase the solubility of minerals in their ionized form, thereby facilitating their absorption through colonic epithelial cells [16, 58].The present findings indicate that probiotic and Salvia hispanica oil supplementation did not influence the biomechanical properties of the femur and tibia under the conditions of this study. The absence of significant effects across all measured parameters-including length, width, ultimate load, displacement at ultimate load, yield load, displacement at yield load, and stiffness-suggests that these dietary interventions were insufficient to physically alter cortical bone strength or mechanical resistance. The consistently low effect sizes further support the conclusion that the biological impact of these treatments on bone biomechanics was minimal at this developmental stage. These results mirror prior findings where the addition of chia oil as a phytobiotic did not alter the macro-mechanical properties of bones in quails [59], whereas other fiber- or oil-rich sources like flaxseed sometimes show more robust osteo-protective preservation depending on inclusion rates [60].

Furthermore, while probiotics have shown variable or insignificant effects on bone stiffness in certain young quail models [61], the indirect gut-bone axis mechanism driven by mineral solubility requires a longer induction period or a higher developmental stress threshold to manifest as physical changes in cortical bone density. This confirms that while the metabolic framework for enhanced gut barrier function and mineral availability was supported through increased butyrate levels and SOD activity, the rapid 35-day skeletal growth cycle of Japanese quails outpaces the structural translation of these dietary benefits into measurable biomechanical strength. Therefore, differing results in the literature remain a natural reflection of the highly multifactorial nature of bone metabolism under varying dietary, species-specific, and environmental conditions.

These findings suggest that the health of poultry bones is influenced not only by mineral intake, but also by a network of nutrient-microbiota-metabolic interactions. Skeletal problems, which are particularly prevalent in fast-growing strains, stem from an imbalance between growth rate and bone mineralisation capacity. From a mechanistic perspective, dietary fatty acids modulate bone metabolism via prostaglandin synthesis and osteoblast/osteoclast activity. Specifically, monounsaturated and omega-3 fatty acids have the potential to reduce bone resorption and stimulate bone formation by modulating the inflammatory response. In this context, chia seeds have the potential to have a multifaceted effect on bone health due to their high calcium content and omega-3 fatty acid content. The effect of probiotics on bone health, however, is explained primarily through indirect mechanisms. The regulation of the gut microbiota is known to increase the production of short-chain fatty acids (SCFAs), which in turn lowers intestinal pH. This, in turn, increases the solubility of minerals in their ionised form and facilitates the absorption of minerals, with calcium being a prime example. In addition, SCFAs enhance the growth of epithelial cells and the function of the barrier they form, which in turn improves the absorption of nutrients. It is thought that a combination of probiotics and phytobiotics (e.g. chia seeds) could produce complementary and potentially synergistic effects: one via the microbiota and the other directly through nutrient content. However, the manifestation of this effect may vary depending on factors such as dose, bioavailability, the animal’s growth rate, and current gut health. Therefore, differing results in the literature can be regarded as a natural reflection of the multifactorial nature of bone metabolism. In connection with the structural insights provided by [59, 61], our biomechanical results demonstrate that the indirect gut-bone axis mechanism-driven by increased mineral solubility via SCFA production-requires either a longer induction period or a higher developmental stress threshold to manifest as quantifiable physical changes in cortical bone density. This confirms that while the metabolic framework for enhanced bone mineralization was established through increased butyrate levels, the rapid skeletal growth rate of Japanese quails within a 35-day cycle clinically outpaces the structural translation of these dietary benefits.

Conclusion

In conclusion, under the conditions of this study, supplementation with Lactobacillus farciminis and Salvia hispanica oil, alone or in combination, did not influence growth performance or the biomechanical properties of the femur and tibia, and most SCFAs remained unaffected, indicating limited impact on overall metabolic and structural outcomes. However, a more selective and biologically meaningful response was observed at the level of intestinal fermentation and redox-related parameters. In particular, significant modulation of butyric acid alongside a probiotic × chia oil interaction suggests a targeted shift in microbial metabolic activity toward butyrate-producing pathways, which is considered beneficial for intestinal epithelial integrity and nutrient utilization. Similarly, the observed synergistic increase in superoxide dismutase activity indicates a complementary interaction between Lactobacillus farciminis-derived metabolites and phenolic compounds of Salvia hispanica oil, enhancing first-line enzymatic defense against reactive oxygen species more effectively than either additive alone. Although these effects were not translated into measurable improvements in growth or bone parameters, the selective synergy observed at the level of butyrate production and superoxide dismutase activity suggests a potential functional benefit of combined supplementation on gut-associated metabolic and cellular defense mechanisms, which may become more pronounced under conditions of physiological stress or altered microbiota stability.

Authors’ contributions

OD, GY, MMC and KO executed the experiment; KO completed the manuscript; GY, OD, OM, DK and FKEE were in charge of laboratory analyses. KO was responsible for supervision and writing of the manuscript.

Funding

This study has not been financially supported by any person or institution.

Data availability

The data supporting this study’s findings are available from the corresponding author upon reasonable request.

Declarations

Ethics approval

This study was approved by the Kafkas University Animal Experiments Local Ethics Committee (KAUHADYEK/2022-061).

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.