https://orcid.org/0000-0002-6789-1288
Figures
Abstract
This study aimed to examine the effect of dietary supplementation with sex hormones, garlic, and fennel extracts in layer breeders on the molecular sex ratio of their progeny. One hundred layer breeders, aged sixty-five weeks, were assigned to five treatments with five replicates of four hens each in a completely randomized design (CRD) for five weeks. The experimental treatments consisted of: (1) a control diet (corn and soybean meal-based), (2) control diet + testosterone (1 mg/kg), (3) control diet + progesterone (1 mg/kg), (4) control diet + fennel extract (400 mg/kg), and (5) control diet + garlic extract (400 mg/kg). In the third and fifth weeks of the experiment, blood samples were collected from the wing vein of layer breeders to measure sex hormone levels. At the end of the fifth week, eggs were gathered over two consecutive days and incubated at 37.5°C. The results suggest that fennel extract increased serum testosterone levels compared to the control throughout the study period (P = 0.057). Garlic and fennel extracts and progesterone increased the female sex ratio, while testosterone treatment increased the male sex ratio compared to the control (P = 0.056), although these differences were not statistically significant (P > 0.05). The experimental treatments significantly influenced the percentage of embryos produced (P < 0.05). No significant effect was observed on blood glucose levels (P = 0.076), though numerical differences were noted. Treatment 2 (testosterone) resulted in the lowest female sex ratio and blood glucose levels, whereas treatments 3 (progesterone), 4 (fennel extract), and 5 (garlic extract) yielded the highest female sex ratios and blood glucose levels. These findings suggest that the treatments’ effects may be linked to their impact on blood glucose levels. The results indicate that dietary interventions affect the offspring sex ratio.
Citation: Bardel Z, Saki AA, Mirzaie-Asl A, Zamani P (2026) Effect of sex hormones, garlic and fennel extracts in layers’ breeders diet on inherited offspring sex. PLoS One 21(7): e0338813. https://doi.org/10.1371/journal.pone.0338813
Editor: Lamiaa Mostafa Radwan, Ain Shams University Faculty of Agriculture, EGYPT
Received: April 7, 2024; Accepted: November 27, 2025; Published: July 9, 2026
Copyright: © 2026 Bardel et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All relevant data are within the manuscript and its Supporting Information files.
Funding: The author(s) received no specific funding for this work.
Competing interests: The authors have declared that no competing interests exist.
Introduction
Efficiency could be significantly enhanced by controlling offspring sex in the poultry industry. In the layer industry, approximately half of all chickens are culled post-hatching because they are male, a practice deemed wasteful yet economically necessary due to the lack of market for male layer chickens, which neither lay eggs nor exhibit the rapid growth and large breast muscles of broiler breeds [1,2]. Consequently, male chickens are considered a byproduct of egg production and are euthanized immediately after hatching. Manipulating hen reproduction to produce only female offspring could increase productivity, reduce waste, improve profitability, and address animal welfare concerns [2].
In birds, males are the homogametic sex (ZZ) and females are the heterogametic sex (ZW), making the female bird the determinant of offspring sex. The mechanism by which female birds influence offspring sex remains unclear, despite extensive research on the evolutionary and ecological implications [3,4]. An unidentified factor complicates the intricate process of sex determination in birds. Moreover, it remains challenging to identify the variables that influence sex and sex ratio [5–7].
Substantial evidence suggests that female birds can manipulate offspring sex ratios prior to fertilization. Across various bird species, females adjust sex ratios in response to social, environmental, and parental factors [8]. Navara [9] identified food quality, season, stress, female condition, laying order, latitude, and mate quality as key influences on offspring sex ratios. Recent studies suggest that maternal steroid hormones regulate sex ratio determination [2,10]. Sex hormones have been shown to alter sex ratios in birds, and their impact on gender in poultry has been investigated [11].
Research has demonstrated a relationship between offspring sex genetics and plasma hormone levels in hens during sex determination. Pinson [12] reported that maternal steroids can affect sex chromosome segregation. Several studies have identified testosterone as a regulator of sex ratios in birds, though the timing and mechanism of its influence remain unclear [2]. In local Iraqi breeder hens, corticosterone level variations directly influence primary and secondary sex ratios oppositely, impacting offspring sex and inversely affecting hatching rates, as stress reduces the hatching percentage [6]. Corticosterone, progesterone, and testosterone could alter sex ratios at elevated or reduced levels before ovulation [6,12–14]. Supplementation with prednisolone (5, 10, and 20 mg/kg feed) in broiler breeders (Arbor-Acres Plus) shifted initial sex ratios toward males, with the most significant effect at 20 mg/kg [7]. Studies have reported that elevated corticosterone concentrations correlate with increased progesterone levels in blood, contributing to sex hormone imbalances [15]. The timing and quantity of hormones can affect offspring sex ratios [11].
Phytoestrogens are biologically active plant-derived compounds structurally similar to 17-beta-estradiol, capable of binding to estrogen receptors and exerting estrogenic or antiestrogenic effects [16]. Lignans, a class of phytoestrogens, are present in garlic [17]. Coumarin in fennel exhibits aromatase inhibitory, estrogenic, reductase inhibitory, and antiandrogenic activities, while flavonoids and anethole in fennel also display estrogenic properties [18].
Studies have shown that medicinal herbs can influence sex hormones, including estrogen, progesterone, and testosterone. Diets supplemented with fennel have been reported to affect sex hormone levels in animals [19], with fennel extract reducing estrogen and increasing progesterone levels in female rats [20]. Genistein implants in local Iraqi chickens increased blood estrogen and the female offspring sex ratio [21]. In-ovo injection of garlic extract altered sex ratios in broiler chickens and exerted positive effects [22,23] while 200 mg of dry garlic extract in broiler diets increased plasma estrogen levels [23]. However, in-ovo garlic injection had no effect on offspring sex ratios in broilers [24]. Fennel extract treatment influenced serum progesterone in female rats and serum estrogen and testosterone in male rats [25]. These effects likely involve interactions among multiple hormones and physiological systems. Stress and elevated corticosterone levels are known to suppress reproduction [26].
Several mechanisms have been proposed to explain how female birds manipulate offspring sex ratios through hormones. These include: 1) Selective follicle atresia [27], occurring in follicles prior to meiotic segregation [28]; 2) Asynchronous follicular development [29], where maternal hormones influence follicle development, though the mechanism for sex-specific differentiation remains unclear [2]; and 3) Non-random segregation of sex chromosomes, considered the most likely mechanism for altering sex ratios [2,9,30]. It has been reported that hormones probably affect chromosome separation through the release of intracellular calcium, which is involved in altering the structure of tubulin, actin, and the cytoskeleton [31,32]. Additionally, steroid hormones (estrogen, progesterone, and androgens) regulate telomerase activity and chromosome length via cell cycle regulators, receptors [33], and gene pathways linked to meiotic segregation [34], thereby influencing sex ratios.
Numerous studies have explored the impact of hormone injection on offspring sex ratios in birds, including [6,13,14,27]. Fewer studies have demonstrated that dietary components can influence sex ratios [7,35]. Modifying diets to alter sex ratios is more practical than hormone injections. Given that the effect of hormones on offspring sex ratios remains unclear and few studies have explored the impact of sex hormones and medicinal plants in diets on bird sex ratios. Therefore, this study investigates the effects of sex hormones and garlic and fennel extracts in the diet of layer breeders on offspring sex ratios.
Materials and methods
This study was approved by the Research Ethics Committee of Bu-Ali Sina University, Hamedan, Iran, under approval ID IR.BASU.REC.1402.056.
Experimental animals and diet
This study utilized 100 birds from W80 layer breeders strain, aged 65 weeks, assigned to five treatments with five replicates per treatment, each containing four hens. Two roosters were assigned to each treatment at the start of the experiment. A natural insemination method was used. Roosters were rotated among replicates every two days, with the exchanges occurring simultaneously. The average body weight was similar across replicates, and a 15: 9 hour light: dark cycle was maintained.
Initially, steroid hormone doses (0.25 and 0.75 mg/kg of feed) were evaluated in a preliminary trial to confirm no adverse effects on performance or blood hormone levels in layer breeder hens. Subsequently, a higher dose (1 mg) was selected and tested for its effect on offspring sex ratios.
The experimental treatments consisted of: 1) a control diet (corn and soybean meal-based); 2) control diet + 1 mg/kg testosterone (testosterone undecanoate); 3) control diet + 1 mg/kg progesterone (medroxyprogesterone acetate); 4) control diet + 400 mg/kg fennel extract; 5) control diet + 400 mg/kg garlic extract.
To ensure homogeneity, garlic and fennel extracts were pre-mixed with a small portion of feed before being blended into the entire ration. Testosterone and progesterone were dissolved in oil before incorporation into the feed.
The garlic and fennel extracts used in this study were procured from the Academic Center for Education, Culture, and Research, Karaj, Iran. Testosterone was produced by Organon Company, France, and progesterone was produced by Iran Hormone Company, Tehran, Iran.
The amino acid profiles of corn, soybean meal, and barley were analyzed using Near-Infrared Reflectance Spectroscopy (NIRS). Standardized ileum digestible (SID) amino acid contents were calculated using coefficients provided by Degosa Evonik, Tehran, Iran. Experimental diets were then formulated based on the amino acid profiles, following the W80 layer breeder catalog recommendations (Table 1).
Performance
At the end of each week, residual feed in each cage was weighed, fresh feed was supplied to the layer breeders, and feed intake was calculated on a hen-day. Eggs were collected and weighed daily. Performance traits, including feed intake, egg mass, production percentage, and average egg weight, were determined using the following formulas [36]:
The following abbreviations are used: BD (bird-day), NH (number of hens), DR (number of days the rooster was presented in the pen), DFI (daily feed intake), and WFI (weekly feed intake of the pen).
The following abbreviations are defined: HD denotes hen-day, NH represents the number of hens, PP indicates production percentage, NE refers number of eggs, EW signifies average egg weight, TWE denotes total weekly egg weight, EM represents egg mass, and FCR indicates feed conversion ratio.
Blood parameter
During the third and fifth weeks of the experimental period, the same bird from each cage was selected to monitor changes in hormone levels (estrogen, progesterone, and testosterone). Blood samples (2 mL) were collected from the wing vein, and the serum was separated and stored at −20˚C until analysis. Sex hormone concentrations were quantified using ELISA kits, following the manufacturer’s instructions.
After five weeks, two hens from each replicate were selected, and blood samples were collected from their wing veins into heparinized tubes. The samples were centrifuged at 2500 rpm for 15 minutes to separate the plasma. Blood parameters (glucose, triglyceride, aspartate aminotransferase, alkaline phosphatase, calcium, total protein, magnesium, high-density lipoprotein, low-density lipoprotein, cholesterol) were then analyzed using a chemistry analyzer with Pars Azmun kits (DiaSys Diagnostic Systems GmbH Company).
Fertility assessment
Throughout the 5-week experiment, two eggs per replicate were selected weekly. Each egg was broken on a flat surface, and fertilization was assessed as described in Fig 1. Unfertilized eggs exhibited a white spot on the yolk, whereas fertilized eggs displayed a white spot encircled by a white ring.
(A) Unfertilized egg, displaying a white spot on the yolk. (B) Fertilized egg, characterized by a white spot on the yolk encircled by a white ring.
Embryonic development
Following the five-week experiment, 110 hatchable eggs were collected over two days and incubated at 37.5 ˚C with 60% relative humidity. On day 18, the eggs were removed from the incubator, and the embryos were carefully extracted, cleaned of residual material, and placed in labeled containers. The embryos were then stored at −20°C until DNA extraction.
DNA extraction and molecular sexing
Embryos were retrieved from a −20˚C freezer, and 20 mg of tissue (pectoralis muscle) was immediately excised using a scalpel. The tissue was ground in liquid nitrogen using a mortar and pestle. DNA was extracted using the Sina Pure DNA Kit (SinaClon Company, Karaj, Iran), and its concentration was determined via NanoDrop. For polymerase chain reaction (PCR) amplification of the CHD1 gene, 1 μl of nucleic acid (adjusted based on concentration) was used with the specific SF/SR primer pair (SF:5’AGTGCATTGCAGAAGCAATATT-3’, SR:5’ GCCTCCTGTTTATTATAGAATTCAT-3’) [37]. Following a 2-minute denaturation at 94°C, CHD1 gene fragments were amplified using a thermal touchdown protocol: 2 cycles of 94°C for 30 s, 60°C for 30 s, and 68°C for 60 s; 2 cycles of 94°C for 30 s, 57°C for 30 s, and 68°C for 60 s; 2 cycles of 94°C for 30 s,53°C for −30 s, and 68°C for 60 s; and 30 cycles of 94°C for 30 s, 50°C for 30 s, and 68°C for 60 s, followed by a final elongation step at 68°C for 5 minutes [30]. The CHD1 gene fragments, 350 base pairs (CHD1-W) and 500 base pairs (CHD1-Z), were amplified from the W- and Z-chromosomes, respectively, corresponding to female (ZW) and male (ZZ) chicken chromosomes [37]. Subsequently, 6 μl of PCR products were electrophoresed on a 1% agarose gel, and bands were visualized under UV light using a gel documentation system (Fig 2).
Lane 1: DNA ladder. Lanes 2, 3, 5, and 6: Male embryo DNA with a 500 bp band. Lanes 4, 7, 8, 9, 10, 11, 12, 13, 14, and 15: Female embryo DNA with 500 bp and 350 bp bands.
Statistical analysis
This experiment was conducted in a completely randomized design (CRD) with five treatments, five replicates, and four hens per replicate. Data on sex determination, incubation, and fertilization were compared using a Two-way chi-square test, while other data collected during the study were analyzed using the GLM procedure in SAS software.
Performance data, including feed intake, feed conversion ratio, egg mass, production percentage, and egg weight, along with plasma hormone levels (estrogen, progesterone, and testosterone) collected throughout the experiment, were analyzed as repeated measures in a completely randomized design (CRD) using the following statistical model:
Where Yijk represents the dependent variable for treatment i, week j, and replicate k; µ is the overall mean; Ti is the effect of treatment i; Wj is the effect of week j; Ti × Wj is the interaction effect between treatment i and week j; Eaik is the mean error; Ebijk is the residual effect, with i, j and k = 1, 2, 3, 4, 5.
Blood parameters, including glucose, triglyceride, AST, alkaline phosphatase, calcium, total protein, magnesium, HDL, LDL, and cholesterol, were analyzed in a completely randomized design using the following model:
Where Yij is the dependent variable for treatment i, replicate j; µ is the overall mean; Ti is the effect of treatment i; and eij is the mean error.
The normality of residual distributions for production traits and blood parameters was assessed using Kolmogorov-Smirnov and Shapiro-Wilk tests. As residuals for all traits followed a normal distribution, no data transformation was required. Treatment means were compared using Duncan’s multiple range test at a significance level of P < 0.05.
Results
Offspring sex
The experimental treatments approached statistical significance in altering sex ratio (P = 0.056).Testosterone treatment increased the proportion of male offspring, whereas progesterone, fennel extract, and garlic extract increased the female sex ratio in treatments 3, 4, and 5, respectively (Table 2).
Performance
The treatments had no significant effect on performance traits throughout the experimental period (P > 0.05). In contrast, egg mass, egg weight, and feed conversion ratio were significantly affected by week (P < 0.05) (Table 3).
Fertility and incubation
The experimental treatments had no significant effect on egg fertility (P > 0.05). However, treatment 2 (testosterone) exhibited the highest fertilization percentage compared to the control, whereas treatments 3 (progesterone) and 4 (fennel extract) showed the lowest fertilization percentages (Table 4).
Experimental treatments significantly influenced embryo percentage (P < 0.05). Treatments 2 (testosterone), 5 (garlic extract), and 3 (progesterone) yielded the highest embryo percentages compared to the control, in descending order. Conversely, treatment 4 (fennel extract) exhibited the lowest embryo percentage compared to the control (Table 4).
Blood parameters
Experimental treatments significantly affected estrogen levels during the study period (P < 0.05), with reduced estrogen observed in the second period compared to the first week. Conversely, treatments 2 (testosterone) and 5 (garlic extract) resulted in the lowest testosterone levels, while treatment 4 (fennel extract) yielded the highest testosterone levels (P = 0.057), though no significant difference was found compared to the control (P > 0.05) (Table 5).
Table 6 presents the effects of experimental treatments on blood parameters. Progesterone, fennel, and garlic extracts increased glucose levels, whereas testosterone decreased them compared to the control; however, the highest glucose level, observed with garlic extract, lacked statistical significance (P = 0.076). Calcium levels were significantly elevated by garlic extract and significantly reduced by testosterone, progesterone, and fennel extract compared to the control (P < 0.05). Total protein levels increased with garlic extract and testosterone but decreased with progesterone and fennel extract (P < 0.05). Magnesium levels rose with garlic extract and testosterone and declined with progesterone and fennel extract, but these changes showed no statistical significance (P = 0.08).
Discussion
Although numerous studies have examined laying hen nutrition [20,38,39], its impact on the genetic sex ratio of chickens remains largely underexplored. Investigating the effects of steroid hormones and medicinal plants could offer novel insights into this relationship. Steroid hormones critically regulate genetic and physiological traits in birds [40], while medicinal plants, with their nutritional and therapeutic properties, may influence egg-laying quality [41,42] and, consequently, chicken sex ratios. Thus, analyzing these factors could enhance our understanding of how nutrition and hormones affect the genetic sex ratio of chickens.
Numerous studies, as reviewed by Navara [9], have demonstrated the effects of steroid hormones on the sex ratio of birds. Corticosterone, progesterone, and testosterone at both high and low levels, can influence sex ratios when administered 4–5 hours before ovulation [2,6,13,14,43]. Notably, testosterone injection (1.5 mg per bird) 5 hours before ovulation has been shown to increase the male sex ratio [2]. In Comb White Leghorn laying hens, hormones may affect the sex chromosome contributed by the heterogametic female to the oocyte. However, as testosterone treatments in earlier studies were often administered chronically throughout follicular development or well before the chromosomal segregation, determining their precise impact on offspring sex remains challenging [12]. Wrobel et al. [11] identified corticosterone and testosterone receptors in hen ovarian follicles. Furthermore, evidence suggests that testosterone may enhance meiotic segregation, particularly in the germinal disk region.
The experimental treatments significantly influenced the sex ratio of offspring. Testosterone supplementation increased male offspring to 69.57%, while progesterone, fennel, and garlic extracts elevated female offspring to 68.75%, 72.73%, and 64.71%, respectively. Pinson [12] reported that testosterone injections increased male offspring by 70%, suggesting that testosterone administration during sex chromosome segregation enhances male offspring production. In this study, dietary testosterone similarly elevated the male sex ratio relative to females. Conversely, injecting 2 mg of progesterone several hours before ovulation significantly reduced the male offspring proportion by 25%, compared to 0.25 mg progesterone (61% male) or control oil (63% male) [13]. Jamshidi et al. [24] found that 2–4% tomato powder in broiler breeder diets had no effect on offspring sex. In our study, dietary garlic extract increased the female sex ratio in layer breeders, with progesterone and fennel supplementation similarly elevating female offspring proportions.
Performance
In this study, performance characteristics, including feed intake, egg mass, production percentage, and average egg weight remained unaffected by the experimental treatments throughout the study period. Mohammadi and Ansari-Pirsaraei [44] reported that testosterone enhances performance and egg characteristics in laying hens toward the end of the laying period. Additionally, growth hormone and testosterone may influence follicular diameters and egg production performance, though these findings contrast with the current study’s results across the entire period. In their study, testosterone was administered via injection at a dose (0.5 mg/kg body weight) different from that used here and was combined with growth hormone.
Progesterone injections were associated with ovarian atrophy and reduced egg production in hens [39]. However, this study observed no such adverse effects from progesterone. This discrepancy may be attributed to the dosage used, which was 2.5 mg daily for 21 days of age in the prior study. Elevated blood progesterone levels have been reported to halt egg-laying in turkeys, even at low doses [45], yet no such effect was observed in this study. The divergence from previous findings may be attributed to the higher progesterone doses (exceeding 0.17, 0.33, 0.5, and 1.5 mg/kg of body weight) administered for 21 days in earlier research, where ovulation ceased within two days of 0.33 mg daily injections. In this study, daily feed intake ranged from 111.13 to 113.65 grams, with daily hormone treatments of 0.111 to 0.114 mg/day.
Phytoestrogen supplementation improved performance and egg quality in quails [46]. Fennel extract, containing estrogen-like compounds, may promote egg production [20]. Research has demonstrated that a diet supplemented with 50 mg/kg of fennel extract improved performance, immune response, and hatchability in post-molted broiler breeder hens, though further studies are required to clarify the underlying mechanisms. Conversely, fennel essential oil at doses of 200, 400, and 600 mg showed no significant effects on laying hen performance [47], nor did a diet with 1% fennel oil [48]. In this study, experimental treatments had no impact on performance. These discrepancies may stem from variations in bird type, fennel extraction method, bird age, and extract dosage.
A meta-analysis demonstrated that garlic significantly affects laying hen performance [42]. Subgroup analysis revealed that garlic extract significantly influenced egg weight, while garlic powder and oil produced similar effects on egg weight. Factors such as country, breed, hen age, garlic preparation form, inclusion levels, number of hens, and treatment duration were identified as predictors of study outcomes. However, a basal diet containing 2.0 g/kg fermented garlic powder showed no significant effect on egg production or egg weight in layer breeder hens. Consequently, the authors suggested that fermented garlic powder at this level could serve as a feed additive for layer breeder diets [38].
Incubation
The highest embryo percentages were observed in treatment 2 (testosterone), 5 (garlic extract), and 3 (progesterone), respectively, compared to the control group. In contrast, treatment 4 (fennel extract) exhibited the lowest embryo percentage.
Kazemifard et al. [20] reported a linear decrease in hatchability of fertile eggs (from 90% to 86%, P = 0.0052) with increasing dietary fennel extract levels. In this study, dietary fennel extract reduced the embryo percentage (from 68.18% to 50%) and egg fertility (from 90.48% to 87.50%), though this reduction showed no statistical significance.
Antol in fennel may exert an estrogenic effect, potentially increasing estrogen levels in males and reducing testosterone levels, which could decrease sperm production in the seminiferous tubules [49]. Although the direct effects of medicinal plants and sex hormones on the percentage of non-embryonated eggs remain underexplored, several factors may contribute. These include advanced laying hen age, the influence of medicinal plants on sex hormones and the reproductive systems [49], and the modulation of plant compounds or hormones in physiological processes [50,51] related to fertilization and embryo formation. Furthermore, the potential impact of medicinal plants on rooster sperm quality warrants further investigation [52]. In this study, the observed decrease in embryo percentage may be attributed to fennel’s effects on the male reproductive system. While no significant difference in egg fertility was observed, this may reflect fennel extract’s adverse impact on egg viability.
Lim et al. [38] reported that dietary garlic supplementation in layer breeder diets increased egg production and hatchability (from 85% to 88%), consistent with this study’s findings (from 68.18% to 73.91%). Garlic supplementation also enhanced sperm vitality and survival in roosters and improved hen egg fertilization rates [38,53]. Musavi et al. [54] demonstrated that garlic boosts sperm production in roosters, regulates testosterone metabolism, and protects sperm from oxidative damage [52]. They hypothesized that garlic improves semen quality parameters, including sperm vitality, and increases male breeder survival rates. These findings indicate that garlic’s positive effects on the male reproductive system may increase embryo production compared to the control group.
Dietary progesterone supplementation increased embryo percentages compared to the control group. Adams [55] highlighted that hatchability decreased with increasing estrogen levels. Estrogen may influence hatchability by affecting calcium metabolism during egg development in the oviduct [50] or through its impact on fat metabolism [20,51]. Both calcium and estrogen are essential for luteinizing hormone (LH) and progesterone production and secretion [56].
Blood parameters
Recent studies have reported that calcium and magnesium may influence the sex ratio [57]. Therefore, the present study investigated the impact of blood calcium and magnesium levels on sex determination. Our findings revealed that garlic supplementation significantly increased calcium levels, whereas testosterone, progesterone, and fennel extract significantly decreased calcium levels compared to the control group. Although magnesium levels were numerically affected by the experimental treatments, these changes did not reach statistical significance. It has been reported that fennel root powder increases calcium and phosphorus levels in broilers [58] however, in this study using fennel extract in laying hens, calcium levels decreased, and phosphorus remained unchanged. This discrepancy may be attributed to several factors, including the form of fennel used (extract vs. root powder), the dosage administered, and the species or physiological status of the birds (broilers vs. laying hens). Each of these variables can influence the bioavailability and metabolic impact of phytogenic compounds, thereby affecting mineral absorption and regulation differently across experimental conditions.
It has been reported that estrogen may influence calcium metabolism during ovum formation and hormone secretion [51]. In addition to estrogen, our findings suggest that testosterone and progesterone also play a role in modulating calcium levels, as both hormones were associated with a significant decrease in blood calcium concentrations in laying hens. These hormonal effects may reflect complex regulatory mechanisms involved in reproductive physiology and mineral homeostasis. In this study, no significant association was found between blood calcium and magnesium levels and sex determination in layer breeders hens Testosterone injections administered 5 hours before ovulation increased blood testosterone levels, increasing the male sex ratio [12]. Throughout the study, treatments significantly influenced testosterone levels, with the lowest levels in treatments 2 (testosterone), 5 (garlic extract), and 3 (progesterone), and the highest in treatment 4 (fennel extract); however, these differences showed no statistical significance compared to the control group. Estrogen and progesterone levels remained unaffected by the treatments. Previous research indicates that blood testosterone levels in broiler breeders peak 1 hour post-testosterone injection, returning to baseline within 4 hours [59]. Progesterone injections depressed plasma estradiol concentrations in both ad libitum and restricted-fed hens, with plasma testosterone levels significantly lower (P < 0.001) in injected hens compared to non-injected ones [39]. Yang et al. [60] highlighted that aromatase inhibitors affect broiler chicken gender, with estrogen and testosterone levels serving as indicators of sexual differentiation.
Dietary fennel supplementation has been shown to reduce cholesterol levels in birds [48]. Testosterone injections in aged laying hens may increase plasma levels of growth hormone, LDL, HDL, cholesterol, and estradiol [61]. Additionally, garlic supplementation effectively lowers plasma triglycerides, cholesterol, and glucose levels in animals and humans, and reduces egg cholesterol in laying hens [62–67]. Similarly, dietary garlic is associated with reduced blood levels of triglycerides, LDL, and total cholesterol in birds [53,68–70]. Variations across studies may arise from differences in the form of the substance used (e.g., whole seeds, powders, or extracts), the administered dosage, and the species or physiological status of the birds.
The highest plasma total protein levels were observed in laying hens fed diets with 1% garlic powder and 1.5% thyme powder [71]. In this study, total protein levels increased with garlic and testosterone supplementation but decreased with progesterone and fennel extract. No significant effects were observed on HDL, LDL, triglycerides, or cholesterol levels due to the treatments. While some studies report that garlic influences egg production rates, others find no such response, potentially due to variations in preparation methods, product storage, bioactive component concentrations, administered proportions, and study duration [72].
Male sex determination may be influenced by elevated glucose levels in mammals [9,73,74]. It has been reported that an increase in glucose in the maternal bloodstream can impair the blastocysts development in female embryos. This is attributed to the higher mobility of Y sperm, which benefits from increased glucose in cervical mucus during ovulation, facilitating Y-sperm survival and uptake [75].
In mice, circulating glucose levels were reported to influence the sex ratio. when glucose concentrations were experimentally reduced using dexamethasone, fewer male offspring were produced [74]. It has been reported that testosterone significantly affects glucose metabolism, exerting a protective effect in males, but it can have adverse effects in females [76]. Less is known about the relationship between blood glucose levels and sex ratios in birds [9]. In this study, treatments affected plasma glucose levels. Testosterone yielded the lowest glucose levels and the highest male sex ratio, suggesting that reduced glucose concentrations may increase male offspring proportions in birds. Conversely, garlic, fennel extracts, and progesterone resulted in the highest glucose levels compared to the control group, correlating with increased female offspring.
Conclusion
In the layer poultry industry, culling half of male chickens highlights the need for optimized production. Producing only female chickens could significantly enhance industry productivity. This study investigates dietary manipulation as amethod to alter sex ratios, demonstrating greater practicality and efficiency compared to injection-based approaches. Contrary to expectations of elevated hormone levels, the results revealed a significantly different outcome than predicted.
This study found that testosterone supplementation increased the male sex ratio, whereas progesterone, fennel, and garlic extracts elevated the female sex ratio. These changes were not attributed to alterations in sex hormone levels. Blood glucose levels decreased in the testosterone group but increased in the fennel, garlic, and progesterone groups compared to the control, with the increase approaching statistical significance. The treatments’ effects on sex ratio may be linked to their effect on blood glucose levels. Thus, dietary interventions may modulate sex ratios through unknown mechanisms, possibly related to medicinal plants. Given that the treatments significantly influenced embryo production percentages, further research is needed to clarify the underlying mechanisms.
This study suggests that dietary interventions may enable manipulation of offspring sex ratios. However, further research across diverse avian species is required to assess the feasibility and wider implications of this approach.
Supporting information
S1 File. Raw images: Raw gel data of different treatments were shown in this file.
https://doi.org/10.1371/journal.pone.0338813.s001
(PDF)
S2 File. Raw data: The raw data of the other parameter (sex ration, performance, Fertility and incubation, plasma hormone and blood parameters) were shown in this file.
https://doi.org/10.1371/journal.pone.0338813.s002
(RAR)
References
- 1. Gerken M, Jaenecke D, Kreuzer M, Martin D. Growth, behaviour and carcass characteristics of egg-type cockerels compared to male broilers. World’s Poultry Science J. 2003;59(1):46–9.
- 2. Wrobel ER. Responsiveness of the chicken germinal disc region to testosterone and corticosterone. University of Georgia; 2018.
- 3. West SA, Shuker DM, Sheldon BC. Sex-ratio adjustment when relatives interact: a test of constraints on adaptation. Evolution. 2005;59(6):1211–28. pmid:16050098
- 4. Szász E, Kiss D, Rosivall B. Sex ratio adjustment in birds. Ornis Hungarica. 2012;20(1):26–36.
- 5. Navara KJ, Hill GE, Mendonca MT. Yolk testosterone stimulates growth and immunity in house finch chicks. Physiol Biochem Zool. 2006;79(3):550–5. pmid:16691520
- 6. Al-Maksousi SK, Al-Hayani WK, Hussein FM. Effect of the level of corticosteron hormone in the blood of local iraqi chicken mothers on the sex ratio of the produced offsprings. Plant Archives. 2019;19(2):1411–5.
- 7. El-Sagheer Mohamed M, Sayed MAM, Abu-Elmagd RM. An attempt to alter the primary sex ratio in the offspring of Arbor Acres Plus broiler breeders by prednisolone administration in feed. AJAS. 2022;0(0):0–0.
- 8. Rosenfeld CS, Roberts RM. Maternal diet and other factors affecting offspring sex ratio: a review. Biol Reprod. 2004;71(4):1063–70. pmid:15229140
- 9. Navara KJ. Hormones rule the roost: hormonal influences on sex ratio adjustment in birds and mammals. Fascinating life sciences. Springer International Publishing; 2018. 123–54.
- 10. Navara KJ. The role of steroid hormones in the adjustment of primary sex ratio in birds: compiling the pieces of the puzzle. Integr Comp Biol. 2013;53(6):923–37. pmid:23900275
- 11. Wrobel ER, Molina E, Khan NY, Akingbemi BT, Mendonca MT, Navara KJ. Androgen and mineralocorticoid receptors are present on the germinal disc region in laying hens: potential mediators of sex ratio adjustment in birds?. General Comp Endocrinol. 2020;287:113353.
- 12. Pinson SE. The effects of steroid hormone treatment during meiotic segregation on primary sex ratios in the domestic chicken. University of Georgia; 2011.
- 13. Correa SM, Adkins-Regan E, Johnson PA. High progesterone during avian meiosis biases sex ratios toward females. Biol Lett. 2005;1(2):215–8. pmid:17148170
- 14. Gam AE, Mendonça MT, Navara KJ. Acute corticosterone treatment prior to ovulation biases offspring sex ratios towards males in zebra finches Taeniopygia guttata. J Avian Biol. 2011;42(3):253–8.
- 15. Etches RJ, Croze F. Plasma concentrations of LH, progesterone, and corticosterone during ACTH- and corticosterone-induced ovulation in the hen (Gallus domesticus). Gen Comp Endocrinol. 1983;50(3):359–65. pmid:6309606
- 16.
Warren MP. The effects of phytoestrogen supplementation in postmenopausal women. Thousand Oaks, CA: SAGE Publications Sage CA; 2002.
- 17. Musk SR, Clapham P, Johnson IT. Cytotoxicity and genotoxicity of diallyl sulfide and diallyl disulfide towards Chinese hamster ovary cells. Food Chem Toxicol. 1997;35(3–4):379–85. pmid:9207900
- 18. Helal EGE, AL Jalaud NA-A, El-Aleem MA, Ahmed SS. Effect of phytoestrogen (Fennel) on some sex hormones and other physiological parameters in male albino rats. Egyptian J Hospital Med. 2019;74(7):1616–20.
- 19. Abbas A, Ikram R, Hasan F, Sarfaraz S, Bukhari QUA. Fennel fortified diet: New perspective with regard to fertility and sex hormones. Pak J Pharm Sci. 2020;33(6):2595–600. pmid:33867335
- 20. Kazemifard M, Kermanshahi H, Rezaei M, Golian A. Effect of different levels of fennel extract and vitamin D3 on performance, hatchability and immunity in post molted broiler breeders. Iranian J Appl Animal Sci. 2013;3(4):733–45.
- 21. Al-Ghurairi RAS, Al – Hayani WKhA, Maaeni YMA. The influence of genistein implantation on offspring sex ratios and their relation to estrogen levels in the blood of iraqi chickens. Iraqi J Agric Sci. 2023;54(4):1016–25.
- 22. Fazli N, Hassanabadi A, Mottaghitalab M, Hajati H. Manipulation of broiler chickens sex differentiation by in ovo injection of aromatase inhibitors, and garlic and tomato extracts. Poult Sci. 2015;94(11):2778–83. pmid:26362979
- 23. Bardel Z. The effect of in ovo injection of letrozole, Urtica dioica root and garlic extracts on sex differentiation and growth performance of broiler chickens. Bu-Ali Sina University; 2018.
- 24. Jamshidi L, Khatibjoo A, Mottaghitalab M, Shirzadi H. Effect of garlic or tomato powder feeding and its comparison with in-ovo injection of garlic or tomato hydroalcoholic extracts on performance, blood parameters, sex differentiation and hatchability of broiler breeders. J Animal Sci. 2022;53(4):233–44.
- 25. Hajiuon B. Effects of garlic (Allium sativum L.) hydroalcoholic extract on estrogen, progesterone and testosterone levels in rats exposed to cell phone radiation. Zahedan J Res Med Sci. 2014;16(12):20–5.
- 26. Breuner CW. Stress and reproduction in birds. Hormones and reproduction of vertebrates. Elsevier; 2011. 129–51.
- 27. Pike TW, Petrie M. Potential mechanisms of avian sex manipulation. Biol Rev Camb Philos Soc. 2003;78(4):553–74. pmid:14700391
- 28. Wrobel ER. Responsiveness of the chicken germinal disc region to testosterone and corticosterone. University of Georgia; 2018.
- 29. Rutkowska J, Cichoń M. Maternal testosterone affects the primary sex ratio and offspring survival in zebra finches. Animal Behav. 2006;71(6):1283–8.
- 30. Aslam MA. Offspring sex ratio bias and sex related characteristics of eggs in chicken. Wageningen University and Research; 2014.
- 31. Manavathi B, Kumar R. Steering estrogen signals from the plasma membrane to the nucleus: two sides of the coin. J Cell Physiol. 2006;207(3):594–604. pmid:16270355
- 32. Mooseker MS, Graves TA, Wharton KA, Falco N, Howe CL. Regulation of microvillus structure: calcium-dependent solation and cross-linking of actin filaments in the microvilli of intestinal epithelial cells. J Cell Biol. 1980;87(3 Pt 1):809–22. pmid:6893989
- 33. Bayne S, Liu J-P. Hormones and growth factors regulate telomerase activity in ageing and cancer. Mol Cell Endocrinol. 2005;240(1–2):11–22. pmid:16005142
- 34. Wrobel ER, Bentz AB, Lorenz WW, Gardner ST, Mendonca MT, Navara KJ. Corticosterone and testosterone treatment influence expression of gene pathways linked to meiotic segregation in preovulatory follicles of the domestic hen. PLoS One. 2020;15(5):e0232120.
- 35. Aslam MA, Groothuis TGG, Smits MA, Woelders H. Effect of corticosterone and hen body mass on primary sex ratio in laying hen (Gallus gallus), using unincubated eggs. Biol Reprod. 2014;90(4):76. pmid:24554734
- 36. Dilawar MA, Mun HS, Rathnayake D, Yang EJ, Seo YS, Park HS, et al. Egg quality parameters, production performance and immunity of laying hens supplemented with plant extracts. Animals (Basel). 2021;11(4):975. pmid:33807460
- 37. Wang Y, Jin G, Ma M, Xiang X. Sex differences in serum steroid hormone levels during embryonic development in hen eggs. Poult Sci. 2019;98(11):6053–62. pmid:31065723
- 38. Ik Lim C, Kwon Kim H, Nyeong Heo K, Sun You A, Jun Choo H. Effects of dietary supplementation of fermented garlic powder on productive performance, hatchability, and biochemical blood parameters of layer breeders. JAHP. 2022;11(1).
- 39. Mojtaba Z, Reza T, Shirin H. Effects of progesterone injection on performance, plasma hormones and ovarian morphology of ad libitum and restricted fed broiler breeder hens. Afr J Biotechnol. 2009;8(22):6481–9.
- 40. Drobniak SM, Sudyka J, Cichoń M, Arct A, Gustafsson L, Lutyk D, et al. Differential effects of steroid hormones on levels of broad-sense heritability in a wild bird: possible mechanism of environment × genetic variance interaction?. Heredity (Edinb). 2022;128(1):63–76. pmid:34921237
- 41. Khan RU, Fatima A, Naz S, Ragni M, Tarricone S, Tufarelli V. Perspective, opportunities and challenges in using fennel (Foeniculum vulgare) in poultry health and production as an eco-friendly alternative to antibiotics: a review. Antibiotics (Basel). 2022;11(2):278. pmid:35203880
- 42. Ogbuewu IP, Okoro VM, Mbajiorgu CA. Meta-analysis of the responses of laying hens to garlic (Allium sativum) supplementation. Animal Feed Sci Tech. 2021;275:114866.
- 43. Pinson SE, Parr CM, Wilson JL, Navara KJ. Acute corticosterone administration during meiotic segregation stimulates females to produce more male offspring. Physiol Biochem Zool. 2011;84(3):292–8. pmid:21527820
- 44. Mohammadi H, Ansari-Pirsaraei Z. Follicle diameters, egg weight, and egg production performance in old laying hens injected with growth hormone and testosterone. J Agricul Sci Tech. 2018;18:949–59.
- 45. Bacon WL, Liu H-K. Progesterone injection and egg production in turkey hens. Biol Reprod. 2004;71(3):878–86. pmid:15140793
- 46. Akdemir F, Sahin K. Genistein supplementation to the quail: effects on egg production and egg yolk genistein, daidzein, and lipid peroxidation levels. Poult Sci. 2009;88(10):2125–31. pmid:19762866
- 47. Taki A, Salari S, Bojarpour M, Sari M, Taghizadeh M. The effect of foeniculum vulgare essence on production performance, egg quality and reproductive parameters of laying hens. Iranian J Animal Science Res. 2014;6(2):140–9.
- 48. Samantaray L, Nayak Y. The influences of black pepper, turmeric and fennel essential oils supplementation in feed on egg quality characteristics of layers. JAHP. 2022;10(4).
- 49. Mirsayed F, Shiravi F, Heydari A, Nasrabadi M. The effect of intraperitoneal injection of alcoholic extract Foeniculum vulgare seed on gonadotropic and testosterone hormones in male wistar rats. J Animal Biol. 2008;1(1):49–56.
- 50. Common RH, Rutledge WA, Bolton W. The influence of gonadal hormones on serum riboflavin and certain other properties of blood and tissues in the domestic fowl. J Endocrinol. 1947;5(3):121–30. pmid:20259242
- 51. Bird S. The influence of ingested estrogens on feed intake, metabolic rate and lipemia in male fowl. Endocrinology. 1946;39:149–54. pmid:20996890
- 52. Okoro V, Nwokeocha A, Ijezie C, Mbajiorgu C, Mbajiorgu E. Effect of varying dietary supplemental inclusion levels of onion and garlic on semen quality characteristics of Hubbard white breeder broiler cocks aged 35–41 weeks old. Indian J Animal Res. 2016;50(6):922–9.
- 53. Hossain M, Begum M, Kim I. Effect of Leuconostoc mesenteroides KCCM35046 fermented aged garlic extract on egg production, egg quality, odour gas emissions, targeted E. coli colony, haematological characteristics and fatty acids composition of egg yolk in laying hens. J Appl Animal Res. 2016;44(1):458–65.
- 54. Musavi H, Tabnak M, Alaei Sheini F, Hasanzadeh Bezvan M, Amidi F, Abbasi M. Effect of garlic (Allium sativum) on male fertility: a systematic review. J Herbmed Pharmacol. 2018;7(4):306–12.
- 55. Adams JL, McGibbon WH, Casida LE. The fertility and hatchability of estrogen-fed pullets and carry-over effects on their progeny. Poultry Science. 1951;30(5):707–12.
- 56. Onagbesan OM, Peddie MJ. Calcium-dependent stimulation of estrogen secretion by FSH from theca cells of the domestic hen (Gallus domesticus). Gen Comp Endocrinol. 1989;75(2):177–86. pmid:2509282
- 57. Arangasamy A, Selvaraju S, Parthipan S, Somashekar L, Rajendran D, Ravindra JP. Role of calcium and magnesium administration on sex ratio skewing, follicular fluid protein profiles and steroid hormone level and oocyte transcripts expression pattern in Wistar rat. Indian J of Anim Sci. 2015;85(11).
- 58. Lotfollahian H, Rasi HM, Najmabadi A. Investigating the effect of fennel root powder on productive performance, immune system and meat quality of broiler chickens. J Anim Prod. 2023;25(4):445–59.
- 59. Navara KJ, Pinson SE, Chary P, Taube PC. Higher rates of internal ovulations occur in broiler breeder hens treated with testosterone. Poult Sci. 2015;94(6):1346–52. pmid:25834248
- 60. Yang X, Zheng J, Na R, Li J, Xu G, Qu L, et al. Degree of sex differentiation of genetic female chicken treated with different doses of an aromatase inhibitor. Sex Dev. 2008;2(6):309–15. pmid:19276633
- 61. Mohammadi H, Ansari-Pirsaraei Z. Changes in some blood parameters and production performance of old laying hens due to growth hormone and testosterone injection. J Anim Physiol Anim Nutr (Berl). 2014;98(3):483–90. pmid:23808354
- 62. Chi MS, Koh ET, Stewart TJ. Effects of garlic on lipid metabolism in rats fed cholesterol or lard. J Nutr. 1982;112(2):241–8. pmid:7057262
- 63. Konjufca VH, Pesti GM, Bakalli RI. Modulation of cholesterol levels in broiler meat by dietary garlic and copper. Poult Sci. 1997;76(9):1264–71. pmid:9276889
- 64. Lim KS, You SJ, An BK, Kang CW. Effects of dietary garlic powder and copper on cholesterol content and quality characteristics of chicken eggs. Asian Australas J Anim Sci. 2006;19(4):582–6.
- 65. Azeke MA, Ekpo KE. Egg yolk cholesterol lowering effects of garlic and tea. J Med Plants Res. 2009;3(12):1113–7.
- 66. Canogullari S, Karaman M, Erdogan Z, Baylan M, Kucukgul A, Duzguner V. Effect of garlic powder on egg yolk and serum cholesterol and performance of laying hens. Bulletin Vet Inst Pulawy. 2009;53(3):515–9.
- 67. Adebiyi F, Ologhobo A, Adejumo I. Modulation of cholesterol in laying chickens fed sun-dried garlic powder. J Exp Agricul Int. 2017;19(2):1–7.
- 68. Yalçın S, Onbaşılar EE, Reisli Z, Yalçın S. Effect of garlic powder on the performance, egg traits and blood parameters of laying hens. J Sci Food Agric. 2006;86(9):1336–9.
- 69. Aderemi F, Alabi O, Ayoola O. Evaluating pepper (Capsicum annuum) and garlic (Allium sativum) on performance egg trait and serum parameters of old layers. J Biol Agricul Healthcare. 2013;3(7):90–6.
- 70. Aswal P, Kumar A, Singh PK. Garlic (Allium sativum) and Amla (Emblica officinalis) powder addition in the diet affects production performance of white leghorn laying hens. Ind J Anim Nutr. 2017;34(1):80.
- 71. Heydari M, Eila N, Afshar M. The effect of mixture of thyme and garlic powder on laying hen performance and some blood parameters. J Animal Sci Res. 2017;23:57–67.
- 72. Adjei-Mensah B, Hamidu JA, Konfe H, Onagbesan O, Ameyapoh YA, Tona K. Evaluating the effects of dietary allicin-rich extract on the production performance, egg quality, caecal bacterial population and lipid profile of laying hens. Euro Poul Sci. 2023;87:1–16.
- 73. Helle S, Laaksonen T, Adamsson A, Paranko J, Huitu O. Female field voles with high testosterone and glucose levels produce male-biased litters. Animal Behav. 2008;75(3):1031–9.
- 74. Cameron EZ, Lemons PR, Bateman PW, Bennett NC. Experimental alteration of litter sex ratios in a mammal. Proc Biol Sci. 2008;275(1632):323–7. pmid:18048284
- 75. Yazdani Aliabadi M, Javadnouri M. Natural methods affecting fetal sex selection and related ethical considerations: a review. Iran J Obstetrics Gynecol Infertil. 2016;19(5):11–27.
- 76. Leutner M, Matzhold C, Bellach L, Wohlschläger-Krenn E, Winker R, Nistler S, et al. Increase in testosterone levels is related to a lower risk of conversion of prediabetes to manifest diabetes in prediabetic males. Wiener klinische Wochenschrift. 2021:1–6.