Open Veterinary Journal, (2025), Vol. 15(9): 4301-4309

Research Article

10.5455/OVJ.2025.v15.i9.37

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The effect of polyunsaturated fatty acids along with mixture of medicinal plants on production performance, blood parameters, and reproduction performance in laying hens

Yadollah Chashnidel¹, Matin Movagharnezhad^2* and Mohammad Mehdi Jafary charati³

¹Associate Professor, Animal Nutrition, Faculty of Animal and Fishery Sciences, Sari University of Agriculture and Natural Resources, Sari, Iran

²Animal Nutrition, Faculty of Animal and Fishery Sciences, Sari University of Agriculture and Natural Resources, Sari, Iran

³Department of Animal Science, Faculty of Agriculture, University of Babol, Babol, Iran

*Corresponding Author: Matin Movagharnezhad. Ph.D. at Animal Nutrition, Faculty of Animal and Fishery Ssciences, Sari University of Agriculture and Natural Resources, Sari, Mazandaran, Iran. Email: matin.movagharnezhad [at] gmail.com

Submitted: 29/04/2025 Revised: 31/07/2025 Accepted: 16/08/2025 Published: 30/09/2025

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Abstract

Background: The combined effects of conjugated linoleic acid (CLA), omega-6 fatty acids, and medicinal plants such as fennel powder and thyme have not been fully investigated in laying hens. Their potential synergistic impact on production traits, blood parameters, and reproductive performance in Lohman selected leghorn (LSL) laying hens remains unclear.

Aim: This study investigated the effects of different dietary levels of CLA and omega-6 fatty acids, combined with medicinal plant supplements (fennel and thyme powder), on productive performance and egg quality traits in Lohmann LSL laying hens. Blood lipid profiles, ovarian follicle development, and liver health were also evaluated.

Methods: Sixty 34-week-old hens (1.5 ± 0.2 kg) were divided into four treatments with five replicates (three hens per replicate) over 42 days. Treatments included: control (no additives), T1 (0.025% CLA/omega-6 + 0.025% medicinal plants), T2 (0.0375% CLA/omega-6 + 0.0375% medicinal plants), and T3 (0.05% CLA/omega-6 + 0.05% medicinal plants). Production performance, egg quality, blood lipid parameters, ovarian follicle activity, and liver fat content were assessed.

Results: Hens fed T2 treatment (0.075% each of CLA/omega-6 and medicinal plants) showed superior production traits (p < 0.05). Egg production percentage significantly differed between treatments and control (89.31% vs. 87.07%, p < 0.05). Haugh unit scores improved with higher supplementation levels (82.14 vs. 78.66 for T3 vs. control, p < 0.05). Blood cholesterol levels decreased in supplemented groups (131.16 mg/dl in T2 vs. 148.75 mg/dl in control, p < 0.05). Liver fat percentage was significantly reduced in T2 and T3 groups (34.38% and 33.79% vs. 39.43% in control, p < 0.05). Large yellow follicle weight increased in T2 treatment (43.83 vs. 37.79 g in control, p < 0.05).

Conclusion: Dietary inclusion of 0.075% (0.0375% CLA/omega-6 combined with 0.0375% medicinal plants) optimized production performance, egg quality, blood lipid profiles, and reproductive health in Lohman LSL hens. This supplementation level demonstrated the most pronounced benefits for enhancing poultry productivity and metabolic health.

Keywords: CLA, Omega-6, Medicinal plant, Blood parameter, Laying hen.

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Introduction

Polyunsaturated fatty acids (PUFAs) present in diets play essential roles in metabolic and developmental processes in poultry. Conjugated linoleic acid (CLA) and omega-6 (linoleic acid) are unsaturated fatty acids that have demonstrated positive nutritional effects in poultry feeding, particularly in laying hens (El-Hack et al., 2020; Vargas-Ramella et al., 2020).

The requirement for linoleic acid in mature chickens is complicated by their ability to store linoleate in adipose tissue. Most vegetable oils, including corn and linseed oil, are rich in linoleic acid—a component that supports egg development and enhanced laying hen productivity (Aydin and Çavuşoğlu, 2022; Sun et al., 2023). Recent studies have shown that CLA supplementation at levels below 2% of the diet improves production traits in laying hens (Pisulewski and Szymczyk, 2003; Schäfer et al., 2011), while higher levels (5 g/kg) can alter yolk color and texture (Ahn and Smith, 2001; Aydin et al., 2001).

Contemporary research indicates that CLA, when used with other fatty acid sources, significantly increases yield, yolk fatty acid concentration, and egg consistency (Alvarez et al., 2004; Giannenas et al., 2006). Studies from 2018 to 2023 have demonstrated that CLA supplementation increases yolk CLA content proportionally to dietary levels, accompanied by changes in yolk fatty acid saturation profiles (Bölükbaşı and Kaynar, 2018; Skoufos et al., 2022). Moreover, CLA has been shown to improve serum lipid profiles and increase antioxidant capacity in laying hens, while also regulating pH levels and maintaining egg quality during storage at various temperatures (Yanfu et al., 2020; Ghasemi-Sadabadi et al., 2021).

Omega-6 fatty acids, when consumed in balanced amounts, provide numerous benefits. Flaxseed supplementation as an omega-6 source can significantly enhance egg production and blood parameters (Basmacioglu et al., 2003). Recent findings suggest that omega-6 fatty acid supplementation influences egg size and improves intestinal and liver health indices (Alagawany et al., 2019). The ratio of omega-6 to omega-3 fatty acids in laying hen diets significantly affects production performance, blood biochemistry, immunity, and carcass traits (Tufarelli et al., 2017).

The use of medicinal plants in poultry nutrition has gained considerable attention in recent years. Natural additives such as thyme and fennel can improve performance outcomes in laying hens (Dorman and Deans, 2000; Ognik et al., 2019; Ghasemi-Sadabadi et al., 2021). When using various sources of unsaturated fatty acids, incorporating plant-based antioxidant compounds from fennel and thyme can be particularly beneficial due to their ability to prevent lipid peroxidation and cellular damage (Chehrei et al., 2011; Abdel-Wareth et al., 2021).

Research has demonstrated that medicinal plant mixtures show stronger beneficial biological effects than individual components. In broiler chickens, thyme promotes growth, enhances starch digestion, and increases dry matter absorption (Shahriari et al., 2008; Eitan et al., 2009; Tufarelli et al., 2017). Recent studies show that 2% herbal mixtures of thyme and other plants significantly contribute to improved egg yield, enhanced egg traits, and better biochemical indicators in laying hens (Mousavi et al., 2014; Ognik et al., 2019). Dietary inclusion of fennel in laying hens can boost production performance, enhance egg and shell quality, and improve fertility parameters (Nobakht et al., 2010; Taki et al., 2013; Sun et al., 2023).

Despite these individual benefits, the combined effects of CLA, omega-6 fatty acids, and medicinal plant mixtures on laying hen performance remain inadequately studied. This research aims to address this knowledge gap by investigating the synergistic effects of these nutritional components.

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Method and Materials

Experimental location and environmental conditions

The experiment was conducted at the poultry breeding and maintenance unit of Sari University of Agricultural Sciences and Natural Resources, Iran. The average ambient temperature throughout the trial was maintained at 21°C ± 2°C, with 16 hours of light and 8 hours of darkness provided daily.

Supplement used

The additives used in this research were produced by Simorgh Behin Darou Company (Babolsar, Mazandaran, Iran). The herbal supplements were prepared in powdered form from dried plant materials. The CLA and omega-6 supplements were used in equal proportions as specified in the treatment formulations.

Chemical composition of supplements

The supplement contained omega-6 fatty acids primarily as linoleic acid (cis-9, cis-12-octadecadienoic acid), and CLA isomers including cis-9, trans-11 CLA (80%–90%) and trans-10, cis-12 CLA (3%–5%). The medicinal plant mixture consisted of equal parts fennel powder (Foeniculum vulgare) and thyme powder (Thymus vulgaris), both containing active compounds including thymol, carvacrol, and anethol.

Animals and diet formulation

Sixty Lohman selected leghorn (LSL) laying hens, 34 weeks old (1.5 ± 0.2 kg body weight), were housed in galvanized wire cages (25 × 51 × 45 cm) with five cages per treatment and three birds per cage. The experiment lasted 42 days, with a 2-week pre-experimental adaptation period. Feed was provided at 115 g/day (according to breed recommendations for this age and production stage), and water was supplied ad libitum. The experimental treatments were: Control: Basal diet without supplementation; T1: Basal diet + 0.025% CLA/omega-6 + 0.025% medicinal plants (total 0.05%); T2: Basal diet + 0.0375% CLA/omega-6 + 0.0375% medicinal plants (total 0.075%); T3: Basal diet + 0.05% CLA/omega-6 + 0.05% medicinal plants (total 0.1%). The ingredient and chemical composition of the basal diet are shown in Tables 1 and 2. Experimental diets were formulated based on corn-soybean meal and prepared according to NRC (1994) nutrient requirements for laying hens using UFFDA software. No mortality occurred during the experiment.

Table 1. Composition and nutrient levels of the basal diet.

Table 2. Chemical composition of CLA, omega-6 and medicinal plant mixture supplement.

Production performance measurements

Birds were weighed at 34 and 39 weeks of age. Feed intake was recorded weekly on a cage basis. Eggs were collected and weighed daily. Production parameters calculated included egg production percentage (% hen/day), average egg weight, egg mass (egg production × egg weight), and feed conversion ratio (g feed/g egg).

Egg quality assessment

Egg quality parameters were evaluated at 34 and 39 weeks using 24 randomly selected eggs per treatment between 08:00 and 12:00. Parameters measured included egg weight, yolk color (Roche scale), shell thickness, Haugh unit (calculated according to Motamedi et al., 2016), and shell weight. For shell weight determination, eggshells were completely cleaned of all adherent albumen, dried at room temperature for 24 hours, and weighed. Shell weight was expressed as a percentage of total egg weight.

Blood sampling and biochemical analysis

At 39 weeks of age, blood samples were collected from the wing vein into heparinized tubes from 12 birds per treatment. Samples were centrifuged at 3,000×g for 20 minutes to obtain plasma, which was stored at −20°C until analysis. Biochemical parameters, including total cholesterol, high-density lipoprotein (HDL), low-density lipoprotein (LDL), triglycerides, total protein, glucose, and uric acid, were measured using commercial diagnostic kits (Pars Azmun, Tehran, Iran) and a semi-automatic biochemistry analyzer (Model: RT-9200, Rayto, China).

Liver morphology and fatty liver assessment

At the end of the experimental period, all birds were humanely euthanized following ethical guidelines. Livers were immediately excised and weighed to determine absolute weight. Fatty liver syndrome was assessed using a five-point macroscopic scoring system (Gilbert et al., 1984): Score 1=normal liver; Score 2=slightly enlarged with minor discoloration; Score 3=moderately enlarged and soft with yellowish-brown color; Score 4=significantly enlarged, soft and fragile with light yellow color; Score 5=severely fatty with pale appearance.

For biochemical quantification, representative liver samples (~5 g) were collected and stored at −80°C. Total liver fat content was determined using the Soxhlet extraction method (AOAC, 1984). Samples were freeze-dried, ground, and fat was extracted using petroleum ether for 6 hours. Extracted fat was quantified gravimetrically and expressed as a percentage of dry liver weight.

Reproductive organ assessment

At 39 weeks of age, four birds per treatment were randomly selected for reproductive morphology assessment. After weighing, birds were anesthetized and humanely euthanized. Reproductive organs (ovary and oviduct) were separated and weighed. Follicles were classified and counted as: large yellow follicles (LYFs, >10 mm diameter), small yellow follicles (SYFs, 5-10 mm diameter), large white follicles (LWFs, 3–5 mm diameter), and medium white follicles (MWF, 1–3 mm diameter) according to Renema (2001). Stroma weight included the remaining ovarian tissue after follicle removal.

Statistical analysis

Data were analyzed using analysis of variance under the General Linear Model procedure in SAS software (version 9.4; SAS Institute Inc., Cary, NC). When significant differences were detected (p < 0.05), means were compared using Fisher’s Least Significant Difference post-hoc test. Statistical significance was declared at p < 0.05. Data are presented as means ± SEM.

Ethical approval

This study was approved by the Animal Research Ethics Committee (JAN/016/2019) and complied with Animal Welfare Declaration guidelines (code: MU. ECRA .2022.12).

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Results

Production performance

The effects of different supplementation levels on production performance are presented in Table 3. Feed intake was numerically higher in the T2 (102.94 g/d) and T3 (103.29 g/d) groups compared to the control (99.88 g/d) and T1 (101.11 g/d), although this difference was not statistically significant (p=0.241 Egg production percentage showed significant differences among treatments (p=0.026). The T2 and T3 treatments achieved superior egg production rates (89.31% and 89.05%, respectively) compared to the control (87.07%) and T1 (87.90%). The T2 treatment demonstrated the highest production percentage with no significant difference from T3, but both differed significantly from the control and T1 treatments. Regarding egg weight, no significant differences were observed among treatments (p=0.105), although there was a linear increase with supplementation level (57.65, 58.24, 58.94, and 59.18 g for control, T1, T2, and T3, respectively). Feed conversion ratio was not significantly affected by treatments (p=0.318), with T2 showing numerically the best conversion efficiency (2.00) throughout the experimental period.

Table 3. Effect of different supplementation levels on production performance.

Egg quality

Supplementation effects on egg quality characteristics are shown in Table 4. Haugh unit scores improved significantly with increasing supplementation levels (p=0.041), ranging from 78.66 in the control to 82.14 in the T3 treatment. The T3 treatment showed the highest Haugh unit value, significantly different from control and T1, while T2 was intermediate. Yolk color (Roche scale) was significantly enhanced by supplementation (p=0.012). The highest yolk color score was observed in T3 treatment (8.57), which was not significantly different from T2 (8.21) but differed significantly from control (7.41) and T1 (7.82). Shell thickness and shell weight showed numerical improvements with increasing supplementation levels, although differences were not statistically significant (p=0.71 and p=0.126, respectively).

Table 4. Effect of different supplementation levels on egg quality characteristics.

Blood parameters

Blood biochemical parameters are presented in Table 5. Glucose levels averaged 217.72 mg/dl across treatments with no significant differences (p=0.143), although values increased numerically with supplementation levels. Total cholesterol concentrations differed significantly among treatments (p=0.037), with mean values of 148.75, 142.92, 131.16, and 135.27 mg/dl for control, T1, T2, and T3, respectively. The T2 treatment showed the lowest cholesterol level, significantly different from the control. Triglyceride levels were significantly affected by supplementation (p=0.081), decreasing from 156.42 mg/dl in the control to 143.34 mg/dl in T3. HDL concentrations increased significantly in T2 (48.85 mg/dl) and T3 (49.16 mg/dl) treatments compared to control (42.01 mg/dl) (p=0.023). LDL levels decreased significantly with supplementation (p=0.026), with T3 showing the lowest value (59.13 mg/dl) compared to control (76.62 mg/dl). Total protein and uric acid levels were not significantly affected by treatments (p=0.418 and p=0.237, respectively).

Table 5. Effect of different supplementation levels on blood parameters.

Liver morphology

Results for liver parameters are presented in Table 6. Liver weight was significantly affected by treatments (p=0.036), with control and T1 groups showing higher weights (46.18 and 42.63 g, respectively) compared to T2 (40.94 g) and T3 (38.78 g). Fatty liver scores differed significantly among treatments (p=0.014), with control showing the highest score (4.59) and T2 and T3 showing the lowest scores (3.31 and 3.29, respectively). The percentage of liver fat was significantly reduced in T2 (34.38%) and T3 (33.79%) treatments compared to control (39.43%) and T1 (38.96%) (p=0.03). These differences in liver morphology among treatments are also illustrated in Figure 1.

Table 6. Effect of different supplementation levels on liver morphology.

Fig. 1. Liver status scoring system used for macroscopic fatty liver assessment: Score 1: Normal, healthy liver with a firm texture and dark brown color. Score 2: Slightly enlarged liver with minor discoloration. Score 3: Moderately enlarged, soft liver with yellowish-brown color. Score 4: Significantly enlarged, soft and fragile liver with light yellow color. Score 5: Severely fatty, paled appearance.

Reproductive performance

Reproductive organ parameters are shown in Table 7. Ovary and oviduct weights were not significantly affected by treatments (p=0.421 and p=0.086, respectively), although oviduct weight increased numerically with supplementation levels. The number of LYFs was significantly increased in supplemented groups (p=0.027), with T3 showing the highest number (5.8) compared to control (5.2). SYF numbers were also significantly affected (p=0.003), increasing from 17.8 in control to 21.6 in T3. LYF weight was significantly influenced by treatments (p=0.037), with T2 showing the highest weight (43.83 g) compared to the control (37.79 g). Other reproductive parameters, including LWFs, largest follicle weight, and stroma weight, showed numerical improvements but were not statistically significant.

Table 7. Effect of different levels of supplements on reproductive performance.

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Discussion

Production performance

The improved production performance observed with CLA and omega-6 supplementation aligns with recent findings. El-Hack et al. (2020) demonstrated that linoleic acid supplementation below 1% enhanced production efficiency and egg weights in laying hens, while levels above this threshold provided no additional benefits. Our results support this, showing optimal effects at 0.0375% supplementation levels. Recent studies by Tufarelli et al. (2017) and Ghasemi-Sadabadi et al. (2021) reported that although fatty acid supplementation may not always significantly influence individual parameters, the overall production performance consistently improved. The synergistic effects of CLA and medicinal plants observed in our study may be attributed to enhanced nutrient utilization and improved metabolic efficiency. The beneficial effects of medicinal plants on performance may result from their phenolic compounds, which demonstrate antimicrobial and antifungal activities. This activity is primarily attributed to thymol and carvacrol present in thyme and fennel extracts (Ahn and Smith, 2001; Sivropoulou et al., 2006; Abdel-Wareth et al., 2021). These compounds may modulate DNA methylation in genes associated with yolk synthesis, such as ApoB and VLDLR, contributing to improved production efficiency.

Egg quality

The improvements in egg quality parameters, particularly Haugh unit and yolk color, are consistent with recent research. Schäfer et al. (2011) reported that egg quality characteristics improved in laying hens receiving 0.75% CLA and linoleic acid supplementation. The enhancement of shell quality may be attributed to CLA’s ability to increase calbindin-D28k expression, facilitating calcium transport and deposition in the shell matrix. Recent studies by Berenjian et al., (2018), Skoufos et al. (2022), and Sun et al. (2023) have shown that medicinal plants enhance calcium metabolism through multiple mechanisms. Fennel contains phytoestrogens that increase oviduct activity and enhance albumin protein synthesis. The plant compounds may also stimulate carbonic anhydrase activity in the shell gland, facilitating bicarbonate ion production essential for calcium carbonate formation (Bolukbasi and Erham, 2006).

Blood parameters

The significant improvements in blood lipid profiles align with contemporary research. Tiwari et al. (2017) reported that omega-3 and omega-6 fatty acids at levels above 0.5% beneficially affected HDL, LDL, and VLDL levels while reducing cholesterol and triglycerides. Our findings demonstrate similar effects at lower supplementation levels when combined with medicinal plants. The lipid-lowering effects may be attributed to several mechanisms: CLA may increase LDL receptor expression by activating SREBP-2, while omega-6 fatty acids may enhance ApoA-I synthesis through peroxisome proliferator-activated receptors-α activation. Additionally, these fatty acids can increase lecithin-cholesterol acyltransferase activity, promoting HDL maturation and reverse cholesterol transport (Vargas-Ramella et al., 2020).

Liver health

The significant reduction in liver fat content and improved liver scores represent important findings for poultry health. Ghasemi-Sadabadi et al. (2021) recently reported similar protective effects of PUFAs against oxidative stress-induced liver damage. The improvements may result from enhanced mitochondrial membrane fluidity and cellular respiration, preventing cytoplasmic lipid accumulation. CLA may reduce profibrotic gene expression (TGF-β1, type I collagen) in hepatic stellate cells, while plant phenolic compounds enhance nitric oxide production in sinusoidal endothelial cells, improving hepatic blood flow. These mechanisms collectively contribute to better liver function and reduced fatty liver syndrome incidence.

Reproductive performance

The enhanced follicular development observed with supplementation supports recent findings on fatty acid effects on reproductive function. Omega-6 fatty acids may increase prostaglandin E1 production, enhancing glycogen accumulation in granulosa cells. CLA supplementation may reduce ovarian stroma lipid accumulation while decreasing oxidative stress in yellow follicles through elevated superoxide dismutase activity. Plant phenolic compounds, particularly thymol, may inhibit 5-alpha reductase activity, preventing testosterone conversion to dihydrotestosterone and preserving follicular growth. These synergistic mechanisms demonstrate how combined supplementation optimizes reproductive parameters in laying hens.

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Conclusion

The results demonstrate that dietary supplementation with 0.075% (0.0375% CLA/omega-6 fatty acids combined with 0.0375% fennel and thyme) significantly improved production performance, egg quality, and metabolic health in Lohman LSL laying hens. This supplementation level achieved the best egg production percentage (89.31%), enhanced Haugh unit scores (80.63), improved blood lipid profiles (reduced cholesterol to 131.16 mg/dl), and decreased liver fat content (34.38%). The synergistic effects of these nutritional components provide a cost-effective approach to enhancing poultry productivity while maintaining bird health and welfare.

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Acknowledgments

The authors are thankful to Director Simourgh Behin Darou Co., Iran, for providing facilities required for this research.

Funding

This research received no specific grant from funding agencies.

Conflict of interest

The authors declare that there is no conflict of interest.

Authors’ contributions

All authors contributed to the conception and design of the study. Materials preparation, collection, and analysis were done performed by [Matin Movagharnezhad] in collaboration with [Yadollah Chashnidel] and [Mohammad Mehdi Jafari Charati]. The first draft of the manuscript was written by [Matin Movagharnezhad], and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.

Data availability

The data from this research are available upon reasonable request to the corresponding author.

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