Growth performance, biochemical outcomes, and testicular histological features in male Japanese quails supplemented with milk thistle seeds

Abstract

Background

Due to its rich content of active phytochemicals, milk thistle is regarded as a promising nutritional supplement for quails, particularly in regions with limited financial resources. Thus, our study aimed to evaluate the possible beneficial impact of aqueous extract of milk thistle seeds (MTS) at two graded concentrations (10 and 20 ml/L of drinking water) on male Japanese quails during their reproductive period.

Results

The phytochemical analysis revealed the presence of 29 active compounds, including nine flavonoids and ten phenolic compounds. The supplemented groups showed significant improvements in body weight gain. MTS1 group exhibited a notable decrease in daily feed consumption, while MTS2 group showed a significant increase in daily water consumption. There was a dose-dependent increase in cecum length. The total count of intestinal bacteria decreased in a dose-dependent manner. Incorporating aqueous extract of MTS at concentration of 10 ml /L resulted in a significant increase in total protein and packed cell volume. Similar increases in globulin and decreases in the albumin/globulin ratio and aspartate aminotransferase (AST) were observed with both doses of supplementation. A significant decrease in total cholesterol and AST was observed in the high-dose group. Significantly higher plasma testosterone and triiodothyronine levels were observed only in the high-dose group, while plasma thyroxine levels were similarly increased in both supplemented groups. Intervention with MTS resulted in dose-dependent increases in cloacal gland index and cloacal foam production. Both supplemented groups showed significant increases in the diameter of seminiferous tubules and the number of Sertoli cells.

Conclusion

Marked growth-promoting, antibacterial, and reproductive-enhancing effects were observed when incorporating aqueous extract of MTS into the quails’ drinking water, particularly at a dosage of 20 ml/L.

Background

The increasing need to enhance livestock productivity by improving animal growth rate, feed efficiency, stress resistance, and overall well-being using natural products has been a focal point of research (Achilonu et al., 2018). The Japanese quail is recognized for its rapid growth, prolific egg-laying, small size, space-efficient nature, robust reproductive abilities, short lifecycle, disease resilience, early maturation, outstanding egg-laying capabilities, and swift hatching period, setting it apart from other poultry breeds. These attributes make it a preferred species for scientific studies in laboratories and a valuable protein source in economic terms (Batool et al., 2023; Lima et al., 2023). However, the significant prevalence of male aggression resulting from an imbalanced male-to-female ratio presents a substantial challenge in attaining reproductive efficiency and maintaining the well-being of animals (Waly et al., 2023). The presence of multiple males can induce stress in both sexes, likely due to aggressive behavior from the roosters toward the hens during forced copulation (Moyle et al., 2010). The ratio of males to females during mating is believed to adversely influence bird productivity, fertility, and hatchability (Haghighi et al., 2016). Injuries stemming from male aggression and feather pecking during mating can have adverse effects on quail production (Abuoghaba et al., 2022). Elevated encounters with males resulting from skewed male-to-female ratios are anticipated to raise the reproductive expenses for females linked to multiple matings or resistance, thereby reducing the incremental advantages of subsequent matings for females (Jeffery et al., 2018). Hence, implementing dietary approaches that improve reproductive abilities could present a viable solution to reduce the necessary number of male quails required to sustain optimal fertility levels. Our laboratory emphasized the potential advantages of including natural products like Zingiber officinale roots and Eruca sativa seeds in the diet of Japanese quails to enhance reproductive traits. These findings suggest a way to mitigate male aggression and reduce the expenses associated with maintaining a large number of males (Abdelfattah et al., 2023; Waly et al., 2023).

From another perspective, implementing methods to boost productivity in industrial poultry farming inevitably increases the pressure on the birds’ physiological systems. Increased feeding intensity and compromised welfare diminish birds’ resilience, leading to higher morbidity rates, safety concerns, and reduced productivity (Bagno et al., 2021). To mitigate the adverse effects of technological pressures in the poultry industry, various dietary supplements are employed. Incorporating functional feed additives into poultry diets enhances overall physiological regeneration, thus mitigating the adverse impacts of intensive farming practices and ensuring the production of competitive, high-quality products (Kiczorowska et al., 2017).

Milk thistle (Silybum marianum) is an annual or biennial plant belonging to the Asteraceae family. Its seeds are rich in flavonolignans, as well as polymeric and oxidized polyphenolic compounds (Khazaei et al., 2022). Phenolic compounds have been shown to decrease the population of pathogenic microbes in the gut, thereby preventing nutrient loss. This contributes to enhanced intestinal health, improved digestion, and nutrient absorption, and ultimately leads to better production performance (Das et al., 2020). They also improve pituitary–gonadal axis and germ cells development (Amevor et al., 2022). Flavonolignans have been observed to stabilize cell membranes and promote liver regeneration (Federico et al., 2017). The addition of milk thistle extract to the broiler chicken diet led to increased counts of red and white blood cells, higher hemoglobin levels, and elevated γ-globulin levels suggesting the stimulation of hematopoiesis processes, enhancement of immune system functions, and overall body growth (Bagno et al., 2021). Incorporating milk thistle seeds (MTS) into broiler chicken feed resulted in achieving the highest body weight with the lowest feed conversion per unit of body weight gain, in addition to a decrease in the crude fat content in leg muscles (Janocha et al., 2021). MTS supplementation protected against decreased testosterone production and histopathological damage in the testes of quails fed a high-energy diet (Çeribaşı et al., 2020). In rabbits, the consumption of MTS resulted in higher sperm concentration, viability, motility, testosterone levels, and fertility (Attia et al., 2017). The inclusion of milk thistle meal in the diets of laying hens with varying levels of metabolizable energy led to a decrease in ileal E. coli count and an improvement in lipid profile (Hashemi Jabali et al., 2018). In light of the limited research conducted on investigating the effects of MTS on nutritional and physiological aspects of male Japanese quails, this study was undertaken to address this knowledge gap, explore the potential inclusion of MTS in quail drinking water, and assess its potential for improving growth, enhancing antibacterial defenses, and supporting reproductive health in quails.

Methods

Preparation of milk thistle extract

Recently harvested green MTS were gathered from the Faculty of Agriculture farm located at Al-Azhar University, Assiut, Egypt. A voucher specimen was deposited at the Assiut University Herbarium in Egypt. Twenty grams of MTS were ground using an electric blender and transferred to a nonmetallic container. One liter of hot water was then poured over the powdered seeds. The container was left at room temperature overnight, and the resulting infusion was added to the drinking water of the treated groups at concentrations of 10 and 20 ml/L. The preparation of the MTS aqueous extract followed the method described by Rehman et al. (2011).

Gas chromatography–mass spectrometry (GC–MS) analysis

It was performed at the Functional and Therapeutic Foods Laboratory, Faculty of Agriculture, Alexandria University, Egypt, using a trace GC-TSQ mass spectrometer (Thermo Scientific, Austin, TX, USA). Identification of plant-derived constituents was achieved by comparing their mass spectra with the mass spectral databases WILEY 09 and NIST.

High-performance liquid chromatography (HPLC)

The analysis of phenolic and flavonoid compounds was performed using an HPLC system (Agilent Series 1100) from Agilent, USA. This system includes an auto-sampling injector, solvent degasser, two LC pumps (Series 1100) with ChemStation software, and a UV/Vis detector set at 250 nm for phenolic acids and 360 nm for flavonoids. The analysis employed a C18 column (125 mm × 4.60 mm, 5 µm particle size). For phenolic acids, separation was achieved using a gradient mobile phase consisting of methanol as solvent A and acetic acid in water (1:25) as solvent B. The gradient program started with 100% B for the initial 3 min, followed by 50% A for 5 min, increased to 80% A for 2 min, and returned to 50% A for another 5 min at a detection wavelength of 250 nm. For flavonoids, separation utilized a mobile phase composed of acetonitrile (A) and 0.2% (v/v) aqueous formic acid (B) with an isocratic elution program (70:30). The solvent flow rate was maintained at 1 ml/min, and separation occurred at 25 °C. Injection volumes were set at 25 μL.

Experimental groups

Ninety male Japanese quails, aged 8 weeks, were used in this study and sourced from the Poultry Research Farm at the Faculty of Agriculture, Assiut University, Egypt. All birds were wing banded and housed in battery cages, following species-specific requirements. They were evenly divided into three groups, each containing three replicates of 10 quails: a control group with no additives, group 1 (MTS1) treated with aqueous extract of MTS at concentration of 10 ml/L of drinking water, and group 2 (MTS2) treated with aqueous extract of MTS at concentration of 20 mL of drinking water. Feed and water were provided ad libitum, and the composition of the feed mixtures is detailed in Table 1. Throughout the trial, the quails remained healthy, and no signs of disease were observed. Each quail was considered as an individual experimental unit. To maintain blinding throughout the study, the animal care technician in charge of the quails was kept unaware of the group allocations. The main objective of this study was to evaluate alterations in hepatic functional enzymes and reproductive outcomes following the interventions.

Table 1 Composition and calculated nutrient content of male Japanese quail diet

Evaluation of carcass traits

At the conclusion of the 18-week experiment, six birds were randomly selected from each treatment group and subjected to a 6-h fasting period before slaughter. A total of 18 quails were then slaughtered by cutting the jugular vein, and carcass weights were recorded after bleeding, defeathering, eviscerating, and removing the head and feet to obtain dressed carcasses. Carcass trait measurements, including carcass weight percentage, gizzard weight percentage, heart weight percentage, liver weight percentage, spleen weight percentage, testes weight percentage, and bursa, were taken after dissecting out the organs.

Analysis of biochemical parameters

At 18 weeks of age, during slaughter, six blood samples per group were collected and transferred to anticoagulant tubes. The blood samples were then centrifuged at 3000 rpm for 15 min, and the plasma samples were stored at -40 °C until analysis. Total protein (TP), albumin, and globulin concentrations were calculated by subtracting albumin from total protein. Glucose, cholesterol, low-density lipoprotein-cholesterol (LDL-C), high-density lipoprotein-cholesterol (HDL-C), triglyceride, aspartate aminotransferase (AST), and alanine aminotransferase (ALT) were determined in blood plasma using commercial kits from Bio Diagnostics Company, Egypt. Plasma hormone levels of triiodothyronine (T3) and thyroxine (T4), and testosterone were assayed by ELISA. Blood hemoglobin (HB) was estimated using commercial kits, while hematocrit value was determined by centrifuging blood in heparinized micro hematocrit tubes for 5 min at 15,000 rpm.

Assessment of cloacal gland attributes and foam output

Thirty male quails (10 single males per group) were measured for cloacal gland area using a digital caliper and foam production. The cloacal gland volume (CVOL) was calculated using the formula CVOL = (4/3) × 3.1416 × a × b2, where a = 0.5 × long axis and b = 0.5 × short axis, following Chaturvedi et al. (1993). Cloacal gland foam production (CFP) was subjectively scaled from 1 (no foam) to 5 (maximum foam), and foam was collected by manual squeezing and weighed after collection on aluminum paper, as described by Mohan et al. (2002) and Satterlee et al. (2002)

Quantifying the population of specific bacterial strains in intestinal contents

At 18 weeks of age, during slaughter, cecum contents were collected from 18 birds (six birds per treatment). Two birds per replicate cage were sampled separately in sterile tubes and used for microbial assay. Sterilized PBS (99 mL) was added (1:100) to 1 g of fresh material, and subsequent dilutions were prepared. 100 µl from the dilutions (10^–5 to 10^–7) was inoculated in nutrient agar plate and then incubated at 37 °C for 24 h for the enumeration of the total bacterial count. Also, 100 µl from the dilutions (10^–3 to 10^–5) was inoculated in eosin methylene blue and Salmonella–Shigella agar plates, then incubated at 37 °C for 24 h for the enumeration of the E. coli count and the Salmonella count, respectively.

Histological examination

The testes were promptly fixed in 10% neutral buffered formalin (pH 7.2). The paraffin-embedded method was employed for preparing testes sections. Subsequently, they were dehydrated in ethanol solutions (ranging from 70 to 100%) to eliminate residual water, cleared in xylene, and embedded in wax. Sections of 5 μm thickness were obtained from paraffin blocks using a rotary microtome, followed by deparaffinization in xylene. Standard hematoxylin and eosin staining protocol (Bancroft & Gamble, 2008) was applied for general histological examination. Morphometric measurements were conducted on the diameter of seminiferous tubules and the length of the germinal epithelium, measured in micrometers from the basal membrane to the luminal edge. The number of Sertoli cells per seminiferous tubule was also enumerated. These parameters were assessed in 20 randomly selected round or nearly round seminiferous tubules from each slide.

Statistical analysis

The data were expressed as mean ± standard error of mean (SEM) and analyzed using one-way analysis of variance (ANOVA) in SAS 9.2 software. Prior to analysis, the data underwent normality testing using the Shapiro–Wilk test, and the homogeneity of variances was verified to ensure adherence to the assumptions of normal distribution and equal variances. Treatment means were compared using Duncan’s multiple range test, with statistical significance defined as P < 0.05.

Results

Analysis of the bioactive compounds present in the aqueous extract of MTS using GCMS and HPLC

The GC–MS analysis of MTS revealed the presence of 29 bioactive phytochemical compounds (Table 2), with the most abundant ones being 9-octadecenoic acid (21.68%), á-sitosterol (15.39%), and pentanenitrile, 5-(methylthio) (10.62%). Additionally, the analysis identified nine flavonoid components and ten phenolic compounds in the extract (Table 3). Notable flavonoids included 7-OH flavone (15.32 μg/gm extract), luteolin (11.66 μg/gm extract), and catechin (9.85 μg/gm extract), while prominent phenolic compounds comprised benzoic acid (12.69 μg/gm extract), gallic acid (12.31 μg/gm extract), and chlorogenic acid (11.25 μg/gm extract).

Table 2 Phytochemical compounds of aqueous extract of milk thistle seeds according to GC–MS analysis

Table 3 Flavonoids and phenolic compounds in the aqueous extract of milk thistle seeds according to HPLC analysis

Effect of aqueous extract of MTS on growth performance and carcass characteristics of male Japanese quails

At the beginning of the experiment, there were no significant differences in body weight (BW) among the various groups. However, by the end of the study, the final BW and BW difference significantly improved in the supplemented groups compared to the control group. In comparison with the control group, the average daily feed consumption significantly decreased in MTS1 group, while there was no significant change in MTS2 group. Additionally, average daily water consumption significantly increased in MTS2 group, with no significant change in MTS1 group compared to the control group. Moreover, there was an equipotent increase in the water/feed consumption ratio observed in the treated groups compared to the control group. On the other hand, carcass traits, including live body weight and carcass attributes, did not show significant differences compared to the control group (Table 4).

Table 4 Effect of adding aqueous extract of milk thistle seeds to drinking water on growth performance and carcass characteristics of male Japanese quails

Effect of aqueous extract of MTS on plasma biochemical and hematological parameters of male Japanese quails

Incorporating aqueous extract of MTS at concentration of 10 ml /L of drinking water resulted in a significant increase in TP and packed cell volume (PCV) compared to the control group. Similar increases in globulin and decreases in the albumin/globulin ratio and AST were observed with both doses of supplementation. However, there were no significant differences observed among all groups regarding albumin, TG, HDL-C, LDL-C, and Hb. A significant decrease in TC and AST was observed in the high-dose group without a significant change in the low-dose group compared to the control. Significantly higher plasma T3 levels were observed only in the high-dose group, while plasma T4 levels were similarly increased in both supplemented groups (Table 5).

Table 5 Effect of adding aqueous extract of milk thistle seeds to drinking water on plasma biochemical and hematological parameters and thyroid hormones of male Japanese quails

Effect of aqueous extract of MTS on testosterone level, testicular morphological characters, and cloacal gland traits of male Japanese quails

Significantly higher plasma testosterone was observed only in the high-dose group. Intervention with MTS resulted in dose-dependent increases in cloacal gland index (CGI) and CVOL. CFP showed a similar increase in both supplemented groups, while foam weight, weight of right and left testes, width of right and left tsetse, and testes percentage remained unchanged across all groups (Table 6).

Table 6 Effect of adding aqueous extract of milk thistle seeds to drinking water on testosterone hormone level, testicular morphological characters, and cloacal gland traits of male Japanese quails

Effect of aqueous extract of MTS on intestinal morphology and cecal bacterial count in male Japanese quails

Only a dose-dependent increase was observed in cecum length compared to the control group. The total count of intestinal bacteria, Salmonella, and E. coli decreased in a dose-dependent manner (Table 7).

Table 7 Effect of adding aqueous extract of milk thistle seeds to drinking water on intestinal morphology and cecal microbiota in male Japanese quails

Effect of aqueous extract of MTS on the testicular histological features in male Japanese quails

Hematoxylin and eosin-stained sections of the testis from the control group exhibited a normal structure (Fig. 1a and d). The seminiferous tubules were densely packed and enclosed by a regular basement membrane. Interstitial tissue containing connective tissue and Leydig cells was present between the tubules. Within the seminiferous tubules, two types of cells, namely the germinal epithelium and Sertoli cells, were typically observed. These cells resided between the tubule lumen and the basement membrane, with the germinal epithelium comprising the spermatogenic lineage. Spermatozoa were observed attached to the Sertoli cells by their heads and occupied the lumen of the seminiferous tubules. In MTS1 group (Fig. 1b and e), there was a nonsignificant increase in the diameter of seminiferous tubules and in the length of the germinal epithelium compared to the control group. However, there was a significant increase in the number of Sertoli cells, as depicted in Fig. 1g, h, and i. In MTS2 group (Fig. 1c and f), both the diameter of seminiferous tubules and the number of Sertoli cells showed a significant increase compared to the control group (Fig. 1g and i). Although the cells of the germinal epithelium appeared noticeably crowded in MTS2 group, the increase in the length of this germinal epithelium was nonsignificant compared to the control group (Fig. 1h).

Fig. 1

Photomicrographs of testis sections stained by H&E a–f, bars = 100 μm a–c, and 50 μm d–f. a and d in Control group, b and e in MST1 group, c and f in MST2 group; showing closely packed seminiferous tubules (St) with regular basement membranes. Normal interstitial tissue (▲) contains Leydig cells. Different stages of spermatogenesis in the germinal epithelium. Prominent elongated Sertoli cells (yellow arrow) with intact spermatozoa (red arrow). g diameter of seminiferous tubules (µm), h length of germinal epithelium (µm), and i number of Sertoli cells per seminiferous tubule in all experimental groups. Values in the column with unlike superscript letters are significantly different

Discussion

Flavonoids exhibit growth-promoting effects by increasing the expression of growth hormone and hepatic growth hormone receptors, subsequently leading to an increase in insulin-like growth factor-1 concentration, thereby promoting animal growth (Kamboh & Zhu, 2013). The enhanced net BW gain can be attributed to the effective utilization of nutrients, primarily facilitated by the antimicrobial, antifungal, and antioxidant properties of the bioactive components present (Hashemi Jabali et al., 2018). The significant increase in intestinal villus height observed with the addition of MTS in the diet (Hashemi Jabali et al., 2018) could contribute to the improved nutrient absorption and overall performance of birds.

The reduction in feed intake observed in the present study in birds receiving drinking water containing 20 ml/L MTS is consistent with the results reported in broiler chickens (Schiavone et al., 2007) and laying hens (Hashemi Jabali et al., 2018), mostly owing to the phytochemicals in MTS. Apigenin, a flavonoid belonging to the flavone subgroup, exhibited suppressive effects on food intake by activating neuropeptide appetite suppressors in hypothalamic neuronal cells (Mosqueda-Solis et al., 2017). Polyphenols boost the gene encoding for gut anorexigenic hormone peptides and hypothalamic anorectic neuropeptides, leading to heightened feelings of fullness (Wang et al., 2021). Linoleic acid inhibits the appetite through decreasing the expression of neuropeptides Y and agouti-related protein and increasing the levels of leptin (Cao et al., 2007).

Elevated levels of globulins suggest improved immunopotency, as immunoglobulins are a subtype of globulin proteins (Zhan et al., 2017). Consistent with our findings, both raw and heated MTS have been shown to enhance humoral immunity and increase immunoglobulin G titer in broiler chickens (Toubkanlou et al., 2018). The abundance of amino acids in MTS (Apostol et al., 2017) enhances the availability of precursors, thereby boosting protein synthesis in the liver and intensifying somatic growth (Szabó et al., 2005). Both the elevation of globulin levels and the reduction in albumin/globulin ratio are associated with enhanced immune system function (Bovera et al., 2016), potentially benefiting the overall health of birds.

The hypocholesterolemic effect of MTS is attributed to its phytochemicals. Plant-derived phytosterols, such as campesterol and sitosterol, decrease transintestinal cholesterol excretion, compete with dietary and biliary cholesterol for micellar solubilization in the intestinal lumen, reduce cholesterol transport to the liver, and lower the bile acid hydrophobic/hydrophilic ratio (Cedó et al., 2019). Additionally, polyunsaturated fatty acids (PUFAs) (Orabi et al., 2020), flavonoids (Mahdavi et al., 2020), and polyphenolic components (Islam et al., 2015) inhibit the HMG-CoA reductase enzyme, which is responsible for a key step in cholesterol manufacture. The hypocholesterolemic effect observed with MTS in this study might be a reflection of increased availability of cholesterol predecessor for the steroidogenic pathway in Leydig cells, leading to enhanced testosterone manufacture and consequently increased cholesterol elimination (Kilby et al., 2021).

The hepatoprotective effect of MTS is evidenced by a decrease in plasma hepatic metabolizing enzymes, akin to what was observed in male Japanese quails intoxicated with cadmium (Saleemi et al., 2019) and broiler chickens (Toubkanlou et al., 2018). As plasma membrane damage and protein leakage is probably the most common reason for elevated blood liver metabolizing enzymes, their reductions signify cell membrane stabilization (McGill, 2016). This outcome stems from suppression of lipid peroxidation by phytochemical constituents of MTS (Viktorova et al., 2019). Flavonoids contribute to the stabilization of membrane integrity through direct means, such as enhancing resistance to reactive oxidants (Veiko et al., 2020) and activating the plasma membrane redox system (Rizvi & Pandey, 2010). Flavonoids also indirectly shield the membranes from attacks by free radicals by interacting with their biological constituents (Hapner et al., 2010). ALT and AST enzymes facilitate the transfer of an amino group from specific amino acids, namely L-alanine and L-aspartate, to α-ketoglutarate. This process results in the production of L-glutamate along with either pyruvate or oxaloacetate (McGill, 2016). The alterations noticed in the levels of AST and ALT in the plasma of Japanese quails receiving milk thistle extract may suggest modifications in transamination processes and adjustments in their metabolic status (Bagno et al., 2021).

The increase in PCV observed in Japanese quails following MTS administration indicates its hematological boosting effect, consistent with previous findings in broiler chickens (Bagno et al., 2021). Flavonoids, such as apigenin and luteolin, have been shown to direct hematopoietic stem cell differentiation toward the erythroid lineage and increase the expression of hemoglobin genes and erythroid transcription factors (Samet et al., 2015). Chlorogenic acid, a polyphenol, enhances the resistance of erythrocytes to hemolysis, osmotic fragility, and oxidative stress (Cheng et al., 2021). Another polyphenolic compound, ferulic acid, promotes the growth of hematopoietic progenitor cell colonies and the production of erythropoietin (Ma et al., 2011). Additionally, feeding polyunsaturated fatty acids such as linoleic acid stimulates the bone marrow microenvironment (Limbkar et al., 2017).

Plasma T4 levels showed a significant increase with both doses of MTS, while plasma T3 levels increased only with the high dose. To our knowledge, there is currently no data available on the effects of MTS on thyroid hormone levels. However, certain phytochemicals present in MTS have demonstrated effectiveness in modulating thyroid secretory activity. Naringenin enhances the function of the pituitary–thyroid axis, as indicated by elevated serum levels of TSH and thyroglobulin. Additionally, it increases the presence of T4-bound thyroglobulin within the colloid thyroid follicles of elderly male rats (Miler et al., 2017). Rutin enhances the expression of thyroid sodium/iodide symporter and thyroperoxidase, as well as the bioactivity or responsiveness of thyroid stimulating hormone (Gonçalves et al., 2013). The rise in thyroid hormones linked to PUFA consumption may occur through the upregulation of enzymes responsible for their production or the downregulation of enzymes involved in their breakdown (Lachowicz et al., 2008). A concomitant rise in testosterone levels was found alongside thyroid hormone levels in the MTS groups. Thyroid hormones control gonadotropin production within the endocrine reproductive axis. Additionally, they influence androgen output by directly and indirectly modulating the expression and function of steroidogenic pathway (Flood et al., 2013). The decrease in cholesterol levels observed in the MTS supplemented groups may be attributed to the sustained elevation of T3 hormone, which enhances the breakdown of fatty acids by increasing the expression and activity of Cpt1α (Jackson-Hayes et al., 2003). This enzyme plays a crucial role in transporting and oxidizing fatty acids in the mitochondria.

The rise in testosterone output observed in MTS2 group aligns with findings from studies involving the dietary supplementation of MTS oil in male broiler Japanese quails fed with a high-energy diet (Çeribaşı et al., 2020) and MTS powder in rabbit bucks (Attia et al., 2017). PUFAs boost the reproductive performance of young roosters by enhancing the hormonal activity of the hypothalamic–hypophyseal–gonadal axis and increasing the transcription levels of their receptors (Feng et al., 2015). Flavonoids enhance the transcript abundance of the steroidogenic acute regulatory protein, crucial for facilitating the cholesterol influx into the mitochondria. This process results in heightened testosterone production by Leydig cells in the testes (Martin & Touaibia, 2020). Similarly, ferulic acid, a phenolic compound found in MTS, boosted the activation of the cAMP-dependent Star promoter, as well as the expression and levels of the corresponding gene and protein (Basque et al., 2023). Caffeic acid, another phenolic agent, increases testicular steroidogenic key enzymes Δ5-3β- and 17β-hydroxysteroid dehydrogenase (Akomolafe et al., 2018). The enhancement in morphometric testicular characteristics following MTS supplementation is in consistent with that found in male Japanese quails fed high fat diet in conjunction with MTS (Çeribaşı et al., 2020). The enlargement of seminiferous tubules in the MTS2 group may be attributed to the elevation of testosterone levels and the influence of testicular paracrine/autocrine factors, which are pivotal in controlling the multiplication, meiosis, and differentiation phases of spermatogenesis (Çeribaşı et al., 2020). The augmented diameter of seminiferous tubules indicates hyperplasia of the germinal epithelium, likely attributed to the elevated transcript levels of proliferating cell nuclear antigen (Abu-Dief et al., 2018) or the downregulation of cell suicidal genes (Malkani et al., 2020). The notable increase in Sertoli cell count due to dietary intervention might contribute to enhanced testicular androgen production by promoting the maturation and viability of Leydig cells (O’Donnell et al., 2022).

Earlier studies have shown that the powder of ginger roots (Abdelfattah et al., 2023) and seeds of Eruca sativa L. (Waly et al., 2023) enhanced the morphological characteristics of the cloacal gland and the foam it produced, which aligns with our results. The parallel rise in CGI and CVOL alongside the increase in CFP suggests a strong causative link between cloacal gland size and foam production rate (Abdelfattah et al., 2023). The cloacal gland size showed a direct relationship with foam discharge, foam weight, fertility, and plasma testosterone concentration (Biswas et al., 2007). Elevated CFP has beneficial consequences on sperm motility, metabolism (Singh et al., 2011b), oviductal transport (Singh et al., 2011a), and mating preference (Singh et al., 2012).

The dose-dependent increase in cecum length was noted similar to increase villus height, crypt depth, and goblet cell numbers in laying roosters (Hashemi Jabali et al., 2018). The favorable effects of MTS on intestinal morphological parameters may be attributed to a decrease in free radicals within the mucosal membrane, leading to reduced lipid peroxidation (Yi et al., 2012). Flavonoids play crucial roles in the development of the intestinal epithelium by inhibiting inflammatory signaling, increasing the expression of tight-junction proteins, and suppressing pathogenic microorganisms (He et al., 2021; Hu et al., 2019). Polyphenols sustain intestinal cell survival by restricting the generation of free radicals, activating phase II detoxification enzymes, and decreasing the production of short-chain fatty acids in the cecum (Yuan et al., 2016).

Dietary inclusion of MTS succeeded in reducing intestinal total bacterial count including salmonella and E. coli align with that seen in laying hens (Hashemi Jabali et al., 2018). Phenolic phytochemicals possess lipophilic characteristics, allowing them to permeate cell membranes and mitochondria. This penetration can cause structural alterations and changes in membrane permeability, potentially resulting in the release of cellular contents (Allievi & Gualandris, 1984). The suggested antibacterial mechanisms of flavonoids include inhibition of nucleic acid synthesis, modification of cytoplasmic membrane function, inhibiting energy metabolism, reduction of cell attachment and biofilm formation, alteration of membrane permeability, and mitigation of pathogenicity (Cushnie & Lamb, 2005, 2011; Xie et al., 2015). Damage of cell membrane, downregulation of virulence factor gene expression (Yoon et al., 2018) generation of cytotoxic agents, and enhancement of the phagocytic action of leukocytes and macrophages (Das, 2018) are responsible for the bactericidal activity of PUFAs. According to our findings, MTS can be used as an antibacterial agent to minimize the risk of promoting antibiotic resistance in quail populations.

Conclusions

These findings highlight the advantages of adding MTS to the drinking water of male Japanese quails, especially at a concentration of 20 ml/L, to improve growth development, intestinal microflora, testicular histology, and hemato-biochemical parameters.