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. 2024 Jun 22;103(9):104016. doi: 10.1016/j.psj.2024.104016

ELOVL5 and VLDLR synergistically affect n-3 PUFA deposition in eggs of different chicken breeds

Caiyun Jiang 1, Yuanhang Shi 1, Xuefeng Shi 1, Jin Yan 1, Lin Xuan 1, Longyu Zhuang 1, Junying Li 1, Guiyun Xu 1, Jiangxia Zheng 1,1
PMCID: PMC11287006  PMID: 39018654

Abstract

There was no significant difference in the composition and content of fatty acids in eggs among different breeds initially, but following the supplementation of flaxseed oil, Dwarf Layer were observed to deposit more n-3 polyunsaturated fatty acid (PUFA) in eggs. Currently, there is limited research on the mechanisms underlying the differences in egg composition among different breeds. Therefore, in this study, 150 twenty-four-wk-old hens of each breed, including the Dwarf Layer and White Leghorn, were fed either a basal diet or a diet supplemented with 2.5% flaxseed oil. After 28 d, eggs and liver samples were collected to determine fatty acid composition, and serum, liver, intestine, and follicles were collected for subsequent biochemical, intestinal morphology, and lipid metabolism-related genes expression analysis. Duodenal contents were collected for microbial analysis. The results showed that there was no significant difference in the content and deposition efficiency of total n-3 PUFA in the liver of the 2 breeds, but the content and deposition efficiency of total n-3 PUFA in the egg of Dwarf Layer were significantly higher than those of White Leghorn after feeding flaxseed oil. Flaxseed oil and breeds did not have significant effects on cholesterol (CHO), free fatty acids (NEFA), low-density lipoprotein (LDL), and estrogen (E2) levels. After feeding with flaxseed oil, the villus height and the villus-to-crypt ratio in both breeds were increased and duodenal crypt depth was decreased. The villus-to-crypt ratio (4.78 vs. 3.60) in the duodenum of Dwarf Layer was significantly higher than that in White Leghorn after feeding with flaxseed oil. Flaxseed oil can impact the gut microbiota in the duodenum and reduce the microbiota associated with fatty acid breakdown, such as Romboutsia, Subdolibranulum, Lachnochlostridium, and Clostridium. This may mean that less ALA can be decomposed and more ALA can be absorbed into the body. Additionally, after feeding flaxseed oil, the mRNA levels of elongation enzymes 5 (ELOVL5), fatty acid desaturase 1 (FADS1), and fatty acid transporter 1 (FATP1) in the liver of Dwarf Layer were significantly higher than those in White Leghorn, while the mRNA levels of peroxisome proliferator-activated receptor alpha (PPAR), carnitine palmitoyl transferase 1 (CPT1), Acyl CoA oxidase 1 (ACOX1), and Acyl-CoA synthetase (ACSL) were significantly lower than those in White Leghorn. The mRNA level of FABP1 in the duodenum of Dwarf Layer was significantly higher than that of White Leghorn, while the mRNA level of FATP1 was significantly lower than that of White Leghorn. The protein levels of ELOVL5 in the liver of Dwarf Layer and very low-density lipoprotein receptor (VLDLR) in the follicles were significantly higher than those of White Leghorn. In summary, after feeding flaxseed oil, the higher ratio of villus height to crypt depth in Dwarf Layer allows more α-linolenic acid (ALA) to be absorbed into the body. The higher mRNA expression of FADS1, ELOVL5, and FATP1, as well as the higher protein expression of ELOVL5 in the liver of Dwarf Layer enhance the conversion of ALA into DHA. The higher protein expression of VLDLR in follicles of Dwarf Layer allows more n-3 PUFA to deposit in the follicles. These combined factors contribute to the Dwarf Layer's ability to deposit higher levels of n-3 PUFA in eggs, as well as improving the deposition efficiency of n-3 PUFA.

Key words: polyunsaturated fatty acid, lipid metabolism-related gene, chicken, breed

INTRODUCTION

Eggs enriched in n-3 polyunsaturated fatty acid (PUFA), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA), have enhanced nutritional value in human health (Kralik and Kralik, 2017). Due to the health-related benefits of functional eggs, the demand for animal protein sources with functional nutritional characteristics is significantly increasing. With the improvement of people's living standards and the popularization of dietary nutrition knowledge, consumers are increasingly concerned about the relationship between dietary ingredients and health (Chen et al., 2023). By changing the diet of layers, the content of specific nutrients in eggs can be increased, thereby producing nutritional enhanced eggs (Zaheer, 2015). In recent years, the functional nutrition of eggs has attracted the attention of many researchers (Kralik and Kralik, 2017; Alagawany et al., 2019; Grcevic et al., 2019; Liu et al., 2020; Lu et al., 2020). In our previous research, we found significant differences in n-3 PUFA content in eggs from different breeds after feeding flaxseed oil, including Dwarf Layer, White Leghorn, Silky fowl, Beijing-you, and Shouguang (Jiang et al., 2023). The contents of α-linolenic acid (ALA) (212.45 mg/yolk), DHA (100.32 mg/ yolk), and total n-3 PUFA (320.59 mg/yolk) in eggs of Dwarf Layer are the highest among the 5 breeds (Jiang et al., 2023). In a study on broiler chickens, it was also found that there were significant differences in the contents of saturated fatty acid (SFA), monounsaturated fatty acid (MUFA) and n-3 PUFA in the muscles of different chickens breeds (Cömert et al., 2016). Fats that are metabolized in chicken are derived from 3 main sources: dietary fat, depot fat, and fat from de novo fatty acid synthesis (from feed carbohydrates) (Alvarenga et al.,2011). Intestinal nutrient uptake is highly complex and largely depends on the interaction between digestive secretions, intestinal absorption surfaces, and intestinal epithelial permeability (Caspary, 1992; Choct, 2009; Metzler-Zebeli et al., 2017). Intestinal surface absorption conditions are one of the main regulatory factors for regulating the absorption efficiency of dietary fatty acids and other nutrients. Morphologically, the length of intestinal villus and the depth of crypts indicate the absorption capacity of the intestine (Caspary, 1992). Long villi in the small intestine lead to increased nutrient absorption, while shortening of villi and enlargement of crypts can lead to poor nutrient absorption, increased gastrointestinal secretion, diarrhea, decreased disease resistance, and overall performance decline (Xu et al., 2003). Therefore, a well-conditioned intestinal surface enhances the ability to absorb nutrients.

Intestinal microbiotas help the host digest and produce short-chain fatty acids, amino acids, hormones and other active substances, which further affects the host's local tissues and systemic metabolism (Valdes et al., 2018; Chadaideh and Carmody, 2021). The gut microbiota plays an important role in lipid metabolism and absorption. Some evidence suggests that microorganisms are important for lipid absorption. For example, compared to conventional mice, germ-free mice fed with a high fat diet showed an increase in fecal lipid levels (Rabot et al., 2010). In addition, under fasting and feeding conditions, routine germ-free zebrafish showed increased long-term and short-chain fatty acid uptake by intestinal epithelial cells (Semova et al., 2012). The small intestine microbiota participates in its digestion and absorption by regulating the intestinal epithelium, which is crucial for the host to adapt to changes in dietary lipids (Martinez-Guryn et al., 2018). Due to the lack of teeth and jaw muscles, birds mainly use their stomachs to crush and digest feed and absorb nutrients from the small intestine (including the duodenum, jejunum, and ileum) (Xing et al., 2017). Due to the function of the duodenum is to digest and absorb nutrients, the microorganisms in this area may affect the health of the intestine and the utilization of nutrients, thereby affecting the production performance of chickens. More than 95% of fat is digested in the duodenum, the released products are mainly absorbed by the distal duodenum (Noy and Sklan, 1995). Although the actual functions of most microbial communities are not yet clear, some previous observations in chickens have shown a significant correlation between duodenal microbiota and fat deposition (Yan et al., 2017; Wen et al., 2019). Many internal microorganisms found in the duodenum may have an impact on the health and production performance of chickens (Wen et al., 2019). Environmental factors, including age, gender, geographical, and medication use, collectively shape the host gut microbiota (Jackson et al., 2018; Rothschild et al., 2018). Among them, diet plays a crucial role in the composition of host gut microbiota (Fujisaka et al., 2018; Bolte et al., 2021). In human adults, changes in gut microbiota were observed after supplementation with omega-3 polyunsaturated fatty acids (Costantini et al., 2017). More and more evidences indicate a correlation between n-3 PUFA and gut microbiota. n-3 PUFA can affect the composition of the gut microbiota, while the gut microbiota can reciprocally impact the metabolism and absorption of these fatty acids. However, knowledge about the relationship between omega-3 polyunsaturated fatty acids and gut microbiota is limited. n-3 PUFA has significant value in the study of duodenal microbiota, which as an important site for fatty acid metabolism and absorption.

Due to the lack of Δ 12 desaturase and Δ 15 desaturases, chickens are unable to synthesize n-3 PUFA through de novo synthesis (Cherian, 2015; Reza Akbari Moghaddam Kakhki and Kiarie, 2019). Therefore, the main source of n-3 PUFA is obtained from external sources in poultry feed. On the one hand, it absorbs and deposits long-chain polyunsaturated fatty acids such as DHA directly from the feed. On the other hand, ALA is obtained from external feeds and gradually converted into long-chain polyunsaturated fatty acids such as EPA and DHA. The conversion of ALA to EPA and DHA primarily takes place in the liver (Bourre, 2005), involving desaturation and elongation processes (Wang et al., 2006). The conversion steps are shown in Figure 1. Δ 6 desaturases and Δ 5 desaturases enzymes introduce double bonds into the precursor of n-3 PUFA, leading to the formation of long-chain PUFA. These enzymes are encoded by the fatty acid desaturase 1 (FADS1) and desaturase 2 (FADS2) genes, respectively (Nakamura and Nara, 2004). The elongation of extremely long chain fatty acids is responsible for elongation enzymes 1 (ELOVL1), elongation enzymes 2 (ELOVL2), elongation enzymes 5 (ELOVL5) (Jump et al., 2005). Acyl CoA oxidase 1 (ACOX1) and liver carnitine palmitoyl transferase 1 (CPT1) are involved in the oxidation of fatty acids (Kersten, 2014). Fatty acid transporter 1 (FATP1) is an evolutionarily conserved protein located on the plasma membrane, facilitating the transport of fatty acids (Huang et al., 2021). Fatty acid-binding proteins (FABPs) are abundant intracellular proteins that bind long-chain fatty acids, facilitating intracellular dietary lipid transport and directing them to various cell destinations (Rodriguez et al., 2017).

Figure 1.

Figure 1

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The conversion steps of ALA to DHA in liver. In short, ALA can be converted into EPA and DHA under the action of desaturases, extenders and β-oxidation. ALA = α-linolenic acid (C18:3); EPA = eicosapentaenoic acid (C20:5); DHA = docosahexaenoic acid (C22:6).

During chicken follicle selection, the very low-density lipoprotein receptor (VLDLR) plays a crucial role in facilitating the oocyte's uptake of liver-synthesized yolk protein. Without VLDLR, oocytes cannot progress into the rapid growth phase of follicular development (Nimpf et al., 1989; Stifani et al., 1990; Schneider, 2009). The development of chicken follicles is primarily divided into 3 stages: 1) the slow growth stage, transitioning from small white follicles to small yellow follicles; 2) the selection stage, spanning from small yellow follicles to 12 to 15 mm grade follicles (typically denoted as F6 follicles); 3) the rapid growth and development stage, occurring from F6 follicles to ovulatory follicles. Furthermore, yolk deposition is notably active during the F6 stage. Proteomic studies have indicated a significant increase in various proteins associated with yolk incorporation during this phase, including apolipoprotein B (APOB), prostate stem cell antigen (PSCA), coagulation factor X (F10), vitellogenin-1 (VTG1), and vitellogenin-3 (VTG3) (Chen et al., 2020).

In previous experiments, we observed that Dwarf Layer have a higher capacity for depositing n-3 PUFA in eggs. This article aims to investigate the factors contributing to variations in n-3 PUFA deposition among different breeds, focusing on intestinal morphology, liver lipid metabolism, and follicular lipid transport. The purpose of the present study is to elucidate the molecular mechanisms responsible for the superior synthesis and deposition performance of n-3 PUFA in Dwarf Layer.

MATERIALS AND METHODS

Ethics Statement

The animal experiments in this study followed the Guidelines for Experimental Animals provided by the Animal Care and Use Committee of China Agricultural University, with permit number AW10803202-1-2. The experiments also complied with the Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines.

Animals and Management

One hundred and fifty 24-wk-old White Leghorn were divided into 2 groups: 1) the control group (CW), fed with basal diet; 2) the experimental group (EW), fed a diet supplemented with 2.5% flaxseed oil in addition to the basal diet. Similarly, 150 twenty-four-wk-old Dwarf Layer chickens were divided into 2 groups: 1) the control group (CD), fed a basal diet; 2) the experimental group (ED), which was fed a diet supplemented with 2.5% flaxseed oil along with the basal diet. The flaxseed oil used in the study was sourced from Ningxia Qianhoufu Trading Co., Ltd. (Yinchuan, Ningxia, China). The experimental populations were generated through pedigree mating, with each generation resulting from a random breeding process.

The basal diet used in this study was formulated to comply with the nutritional standards recommended by the National Research Council (1994) and the feeding standards for chickens (NY/T 33-2004). The nutritional components of the diet are detailed in Supplementary Table 1, while the fatty acid composition of the groups is presented in Supplementary Table 2. The chickens were housed in individual cages, one chicken per cage, during the 28-d study period. Recommended feeding times is twice a day, with egg collection once a day. The nipple drinkers are used to provide free drinking water to hens. The lighting system consists of a morning and evening light supplement system, with a light duration of 16h per day and a light intensity of 10 Lx. It is important to check the hens daily to ensure they have adequate feed and water supplies and to monitor their health. During the experiment, production data, including average daily feed intake and egg production for each group of animals was recorded. Production performance parameters for different groups are delineated in Supplementary Table 3.

Sample Processing and Analyses

Samples Collection

After 28 d of feeding, 30 eggs and 10 chickens were randomly selected from each group. Blood samples were collected from each chicken for biochemical analysis. Their livers (4 g for total RNA and protein extraction) and duodenal tissues and contents were quickly dissected and frozen in liquid nitrogen. Liver and duodenal tissue samples were utilized for real-time quantitative polymerase chain reaction (RT-qPCR) and Western Blot (WB) analyses, respectively. Duodenal contents were subjected to microbial analysis. The duodenum was incised using sterilized surgical scissors and forceps. The contents were gently scraped into sterilized centrifuge tube with a sterilized spoon, quickly frozen in liquid nitrogen, and stored at -80 ℃ for sequencing. The smallest hierarchical follicle (12-15 mm, F6) from each hen were quickly transferred to phosphate-buffered saline (Thermo Fischer Scientific, MA). The outermost layer of blood vessels was removed using tweezers, and the yolks were carefully extracted with the same. The remaining tissue, comprising the basement membrane, granulosa cell layer, yolk basement membrane, and oocyte plasma membrane, was washed with phosphate-buffered saline until no yolk residue remained. This remaining tissue was immediately frozen in liquid nitrogen for RT-qPCR and WB analysis. A plastic hand-held egg separator (Wuxi, Jiangsu, China) was applied to separate various egg components, such as yolk and albumen. After the albumen of all groups was eliminated, the yolks were weighed and stored at −20 ℃ until subsequent analyses were performed. The remaining liver samples were collected and stored at -20℃ for confirming n-3 PUFA enrichment.

Determination of Fatty Acid Composition and Content

Ten eggs and ten livers per group were utilized to determine the concentrations of n-3 PUFA. Yolks and livers were subjected to freeze-dried using a vacuum freeze dryer at −80℃ for 72 h and subsequently weighed. The freeze-dried livers and yolks were then crushed into powder, and fatty acids were determined using gas chromatography (Agilent 6890, Agilent Technologies Inc., Santa Clara, CA), following the national standard GB-5009.168-2016. Briefly, 0.5 g of each sample was accurately weighed into a screw-top glass tube. Toluene and a 10% solution of acetyl chloride in methanol were added to the tube. After thorough mixing, the samples were incubated in an 80℃ water bath for 2 h. Subsequently, the reaction solution was transferred to a centrifuge tube, and the glass tube was rinsed with sodium carbonate solution. Finally, 100 µL of the supernatant was extracted, filtered using a membrane, and subjected to analysis using a gas chromatograph to determine the content of fatty acids.

In accordance with the methodology outline in El-Zenary's study, the efficiency of n-3 PUFA synthesis and deposition was calculated by dividing the total liver content of n-3 PUFA (mg) by the dietary intake of ALA (mg) and then multiplying by 100 (El Zenary et al., 2022). Likewise, following the approach detailed in Elkin's research, the conversion and deposition efficiency of ALA into n-3 PUFA was determined by dividing the total yolk content of n-3 PUFA (mg) by the dietary intake of ALA (mg), and then multiplying by egg production, and finally by 100 (Elkin and Harvatine, 2023).

Intestinal Morphometry

A 5 cm sample was excised from the middle of the duodenum and fixed in 10% formalin. Duodenal samples were subsequently serially in increasing concentrations of ethanol. Ethanol was then removed with a hydrophobic scavenger, and the samples were embedded in molten paraffin. The blocks were sectioned into small pieces to obtain 5 μm transverse section of the duodenum. These sections were mounted on microscope slides and stained with hematoxylin and eosin. Subsequently, a Scanning machine 3D Panoramic MIDI was used to examine the slides, and digital images were captured for morphological analysis. The image analysis software Slide Viewer was employed to measure the villus height from the tip to the base of the lamina propria and the crypt depth from the villus base to the mucosa. Each chicken was measured at least 5 times for each trait, and the average value per chicken was used for statistical analysis. The villus-to-crypt ratio was calculated by dividing the villus length by the crypt depth (Kettunen et al., 2001).

Microbial Analysis of Duodenal Contents

Majorbio Bio-Pharm Technology Co. Ltd. (Shanghai, China) was commissioned to perform 16S ribosomal RNA (rRNA) microbial analysis on duodenal contents samples. DNA was extracted from the duodenal content samples, resulting in a total of 32 DNA samples. The 16s rRNA was amplified using the 16S amplicon sequencing method, and the amplified 16s rRNA genes were sequenced using Illumina MiSeq system. Illumina official connector sequences were added to the outer ends of the target area through PCR, followed by gel recovery to isolate and recover the PCR product. Subsequently, elution was performed with Tris HCl buffer, and the product was detected using 2% agarose electrophoresis. Finally, sodium hydroxide was utilized to denature and generate single stranded DNA fragments to complete the construction of the Illumina library. To ensure data quality, the Fastp software was employed for raw data quality control, followed by the use of FLASH to merge the data that passed quality control. The readings were clustered into operational classification units (OTUs) with a cut-off value of 97% similarity using UPARSE (v7.1). Subsequently, an RDP classifier (v2.2) was utilized to annotate the classification of representative sequences for each OUT, employing a 16S rRNA database (Silva v138) as a reference with a confidence threshold of 0.7.

Real-Time Quantitative PCR

Total RNA was extracted from livers, duodenal tissue, and chicken ovarian follicles, which were also used for gene expression analysis. Total RNA was extracted from each liver, duodenal tissue, and ovarian follicles sample using the RNA Easy Fast Tissue/Cell Kit (Tiangen Biotech, Beijing, China) according to the manufacturer's instruction. Synthesis of the cDNA was performed using FastKing gDNA Dispelling RT SuperMix (Tiangen Biotech, Beijing, China) according to the manufacturer's protocol. RT-qPCR for the mRNA expression levels of ELOVL1, ELOVL2, ELOVL5, FADS1, FADS2, peroxisome proliferator-activated receptor alpha (PPAR), Acyl-CoA synthetase (ACSL), CPT1, ACOX1, FABP1, FATP1 and VLDLR was performed using the Talent qPCR PreMix (SYBR Green) (Tiangen Biotech, Beijing, China) with primers listed in Supplementary Table 4 on an ABI7500 fluorescence quantitative analyzer (Applied Biosystems).The quantitative primer for FADS1, FADS2, ELOVL2, ELOVL5, LCPT1, PPAR- ɑ, ACOX1 and β-actin refer to the primer sequences published by Shahid (Shahid et al., 2020). The quantitative primers for ACSL and FABP1 refer to the primer sequences published by Neijat (Neijat et al., 2017). The VLDLR quantitative primers refer to the primer sequences published by Chen (Chen et al., 2020). RT-qPCR was carried out under the following conditions: predenaturation at 95°C for 3 min; 40 cycles of denaturation at 95°C for 10 s; annealing at the primer-specific temperature for 30 s, and extension at 60°C for 30 s. A melting curve was used to verify primers specificity. Each sample was run in triplicate, and the results were normalized to the expression levels of β-actin. The relative mRNA expression levels were calculated using the 2−ΔΔCT relative quantification method.

Western Blot

Total protein was extracted from liver tissue and F6 using a RIPA lysis buffer supplemented with phenylmethyl sulfonyl fluoride (Servicebio, Wuhan, China) at a ratio of 100:1. Proteins were separated on 10% SDS-PAGE gels and then transferred to polyvinylidene difluoride (PVDF) membranes (ISEQ00010, Millipore, Danvers, MA). The membranes were blocked with QuickBlock Blocking Buffer for 15 min. After blocking, the membranes were washed 3 times with Western Wash Buffer (10 min/time) and then incubated with primary antibodies for overnight at 4°C. Following primary antibody incubation, the membranes were washed 3 times with Western Wash Buffer and incubated with secondary antibody conjugated with HRP (Proteintech, Jiangshu, China) for 1 h at room temperature. Signal detection was enhanced by ECL Plus (Biosharp), and images were captured using Photoshop CS6. Quantification of protein bands was characterized by densitometry using Image J. To normalize protein levels, β-actin was used as a loading control. The relative expression level of the target protein in each sample was determined by dividing the target protein grayscale values by the β-actin grayscale values. Primary antibodies against ELOVL5 were obtained from Affinity (Jiangsu, China). Primary antibodies against FADS1, VLDLR, and β-actin were sourced from Proteintech (Wuhan, China). The secondary antibody used was goat anti-Rabbit-IgG from Proteintech.

Statistical Analyses

Statistical analyses of the results were performed using SPSS version 19.0 (IBM, Chicago, IL). The fatty acid composition, deposition efficiency of n-3 PUFA, qPCR results, and WB results were analyzed using a general linear model. The fixed factors included breeds and diet, with a significance set at P < 0.05. Post hoc testing was conducted using Duncan's test. Serum biochemical indicators and intestinal indicators were assessed using student t-tests. Graphics were generated using GraphPad Prism 5. Results were presented as Mean ± SD of 10 replicates, with statistical significance set at P < 0.05.

RESULTS

Fatty Acid Composition and Deposition of Liver and Eggs in Different Breeds

Supplementary Table 5 shows the results of fatty acid composition (mg/100g) of livers from different breeds fed flaxseed oil. There were no significant differences in the content of ALA, EPA, DHA, and total n-3 PUFA in the livers of the different breeds when fed a basal diet. After feeding flaxseed oil, the content of ALA, DHA and n-3 PUFA in the liver of different breeds were significant increased. Specifically, the content of ALA (82.42 mg/100g vs. 77.65 mg/100g), DHA (145.30 mg/100g vs. 134.69 mg/100g), and n-3 PUFA (233.74 mg/100g vs. 220.50 mg/100g) in the liver of Dwarf Layer were higher than those in White Leghorn, although the difference was not statistically significant. The results of the fatty acid composition (mg/yolk) of eggs from different breeds fed flaxseed oil are presented in Figure 2. There were no significant differences in the content of ALA, EPA, DHA, and total n-3 PUFA in the eggs of the different breeds when fed a basal diet. Similarly to livers, after feeding flaxseed oil, the contents of ALA, DHA, and n-3 PUFA in eggs of different breeds significantly increased. After feeding flaxseed oil, the content of ALA (163.15 mg/yolk vs. 139.76 mg/ yolk), DHA (74.34 mg/ yolk vs. 62.77 mg/ yolk), and n-3 PUFA (240.45 mg/ yolk vs. 206.22 mg/ yolk) in the egg of Dwarf Layer were significantly higher than those in White Leghorn.

Figure 2.

Figure 2

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Fatty acids composition (mg/yolk) of eggs from different breeds fed flaxseed oil. Results are shown as mean ± SD, n = 10; “a, b, c” means with different superscripts indicate significant differences (P < 0.05), and different groups of the same substance were compared. ALA = α-linolenic acid (C18:3); DHA = docosahexaenoic acid (C22:6); ∑n-3 PUFA = ALA + EPA + DHA; CD = the control group of Dwarf Layer control group, fed with basal diet; CW = the control group of White Leghorn, fed with basal diet; ED = the experimental group of Dwarf Layer, which fed with 2.5% flaxseed oil + basal diet; EW = the experimental group of White Leghorn, fed with 2.5% flaxseed oil + basal diet.

The deposition efficiency of dietary ALA into n-3 PUFA in egg yolk and liver of different breeds is shown in Figure 3. There were no significant differences in the deposition efficiency of n-3 PUFA in yolk of the different breeds when fed a basal diet. After feeding flaxseed oil, the deposition efficiency of n-3 PUFA into the yolk of different breeds did not show significant increases or decreases. The deposition efficiency of n-3 PUFA into the yolk of Dwarf Layer (17.98 vs. 14.48) was significantly higher than that in White Leghorn after feeding flaxseed oil. The deposition efficiency of n-3 PUFA into the liver of Dwarf Layer (0.41 vs. 0.53) was significantly lower than that in White Leghorn when fed a basal diet. After feeding flaxseed oil, the deposition efficiency of n-3 PUFA into the liver of different breeds significantly decreased. There were no significant differences in the deposition efficiency of n-3 PUFA into the liver of the different breeds when fed flaxseed oil.

Figure 3.

Figure 3

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Deposition efficiency of n-3 PUFA in yolk and liver of different breeds. (A) Deposition efficiency of n-3 PUFA in yolk. (B) Deposition efficiency of n-3 PUFA in liver. Results are shown as mean ± SD, n = 10; “a, b, c” means with different superscripts indicate significant differences (P < 0.05). CD = the control group of Dwarf Layer control group, fed with basal diet; CW = the control group of White Leghorn, fed with basal diet; ED = the experimental group of Dwarf Layer, which fed with 2.5% flaxseed oil + basal diet; EW = the experimental group of White Leghorn, fed with 2.5% flaxseed oil + basal diet.

Serum Lipid Metabolites

The effect of flaxseed oil on serum biochemical profiles of different breeds is presented in Supplementary Table 6. Flaxseed oil and breeds did not have significant effects on cholesterol (CHO), free fatty acids (NEFA), low-density lipoprotein (LDL), and estrogen (E2). When fed a basal diet, the triglycerides (TG) of the Dwarf Layer were significantly higher than those of White Leghorn, while there was no significant difference after feeding with flaxseed oil.

Intestinal Morphometry

The intestinal morphometric analysis of different breeds is illustrated in Supplementary Table 7. After feeding flaxseed oil, there was no significant effect on the villus height, crypt depth, and villus-to-crypt ratio of the layer. There was no significant difference in the villus height, crypt depth, and villus-to-crypt ratio in the duodenum, jejunum, and ileum between the 2 breeds fed with basal diets. After feeding flaxseed oil, there were no significant differences in the villus height, crypt depth, and villus-to-crypt ratio in the jejunum and ileum between the 2 breeds, but the villus-to-crypt ratio in the duodenum of Dwarf Layer was significantly higher than that of White Leghorn (P < 0.05).

Comparison of Microbial Diversity Between Groups

The microbiota of duodenal contents was analyzed using 16S rRNA gene sequencing. A total of 2,474,187 high-quality reads were obtained from 32 content samples, averaging 77,318 reads per sample. There was no significant difference in the total microbial abundance between groups in duodenal contents (CD vs. ED vs CW vs. EW, Shannon index median, 2.46 vs. 2.27 vs. 1.87 vs. 2.65) (Figure 4A). The microbial diversity in the duodenal contents of CD (Chao index median, 419.6) was significantly higher than that in EW (Chao index median, 249.1) (P < 0.05) (Figure 4B). CD, ED, CW presented a distinct clustering of microbial community structure, while the EW group had a similar structure to that of the CD and ED (Figure 4C). There are 340 species shared by the 4 groups (Figure 4D).

Figure 4.

Figure 4

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Comparison of microbial α diversity and β diversity in duodenum between breeds. (A) Microbial community diversity (measured by Shannon index). (B) Microbial community abundance (measured by Chao index). (C) Principal component analysis (PCA) plots of Bray–Curtis dissimilarities between breeds. (D) Venn between breeds. CD = the control group of Dwarf Layer control group, fed with basal diet; CW = the control group of White Leghorn, fed with basal diet; ED = the experimental group of Dwarf Layer, which fed with 2.5% flaxseed oil + basal diet; EW = the experimental group of White Leghorn, fed with 2.5% flaxseed oil + basal diet.

At the phylum level, Firmicutes, Actinobacteriota and Campilobacterota were the dominant phyla both in CD and ED samples. Firmicutes, Actinobacteriota, and Proteobacteria were dominant in both CW and EW samples. The relative abundance of Firmicutes, predominantly Gram-positive bacteria, was higher in both the Dwarf Layer (CD vs. ED, 80.97% vs. 71.01%) and White Leghorn (CW vs. EW, 51.63% vs. 75.22%). In contrast, Actinobacteriota was more abundant in the White Leghorn (CW vs. EW, 34.15% vs. 15.65%) compared to the Dwarf Layer (CD vs. ED, 4.00% vs. 0.78%). Campilobacterota (Gram-negative) showed higher abundant in the Dwarf Layer (CD vs. ED, 10.82% vs. 25.22%) compared to the White Leghorn (CW vs EW, 1.29% vs. 0.64%). Proteobacteria (Gram-negative) exhibited higher abundant in the White Leghorn (CW vs. EW, 12.11% vs. 4.58%) compared to the Dwarf Layer (CD vs. ED, 2.11% vs. 2.00%) ​(Figure 5A). At the genus level, the relative abundance of Lactobacillus was similar between the Dwarf Layer (CD vs. ED, 71.16% vs. 67.30%) and the White Leghorn (CW vs. EW, 47.71% vs. 68.26%). The relative abundance of Helicobacter was higher in the Dwarf Layer (CD vs. ED, 10.81% vs. 24.36%) compared to the White Leghorn (CW vs. EW, 1.26% vs. 0.64%). Additionally, the relative abundance of Rhodococcus was higher in the White Leghorn (CW vs. EW, 33.30% vs. 13.53%) compared to the Dwarf Layer (Figure 5B). The linear discriminant analysis effect size (LEfSe) analysis showed that the relative abundance of Pathogenic bacteria, including Rhodococcus, Brucella and Pandoraea, was significantly higher in duodenum content of CW compared to CD (Figure 5C). Smilarly, potentially Pathogenic bacteria such as Rhodococcus and Brucella, were significantly higher in duodenum content of EW compared to ED (Figure 5E). Furthermore, LEfSe analysis showed that the relative abundance of the bacteria capable of converting long-chain fatty acids into short chain fatty acids, including Romboutsia, Subdoligranulum and Lachnoclostridium, was significantly higher in duodenum content of CD compared to ED (Figure 5D). Additionally, some potentially beneficial bacteria such as Clostridium, were significantly higher in duodenum content of CW compared to EW (Figure 5F).

Figure 5.

Figure 5

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Differences in abundance and microbial composition. (A) Microbial community composition of duodenum content at the phylum level. (B) Microbial community composition of duodenum content at the genus level. (C) Differentially abundant taxa of content microbiota between breeds fed with basic diet. LDA score ≥ 2.5. (D) Differentially abundant taxa of content microbiota in Dwarf Layer between CD and ED. LDA score ≥ 2.5. (E) Differentially abundant taxa of content microbiota between breeds fed with flaxseed oil. LDA score ≥ 2.5. (F) Differentially abundant taxa of content microbiota in White Leghorn between CW and EW. LDA score ≥ 2.5. CD = the control group of Dwarf Layer control group, fed with basal diet; CW = the control group of White Leghorn, fed with basal diet; ED = the experimental group of Dwarf Layer, which fed with 2.5% flaxseed oil + basal diet; EW = the experimental group of White Leghorn, fed with 2.5% flaxseed oil + basal diet.

Effects of Breeds on the mRNA Levels of Lipid Metabolism-Related Genes

To discern disparities in gene expression associated with lipid metabolism across different breeds, we employed qPCR to scrutinize tissues such as liver, duodenum, and F6 follicle. Initially, under basal dietary conditions, no significant differences were observed in the expression levels of ELOVL5, FADS1, and FATP1 in the liver, as well as VLDLR in the follicles, between the 2 breeds. However, distinct differences emerged in the expression patterns of ELOVL2 and PPAR in the liver, and FATP1 in the duodenum of the Dwarf Layer compared to the White Leghorn. Specifically, the Dwarf Layer exhibited significantly higher expression levels of ELOVL2 and PPAR in the liver, and FATP1 in the duodenum, in contrast to the White Leghorn. Conversely, other genes displayed notably lower expression levels in the Dwarf Layer compared to the White Leghorn under basal dietary conditions. After feeding flaxseed oil, the expression of ELOVL5, FADS1, and FABP1 in the liver of both breeds showed an increasing trend. Our results showed that after feeding flaxseed oil, the expression levels of ELOVL5, FADS1, and FATP1 in the liver of Dwarf Layer were significantly higher compared to those of White Leghorn (Figures 6A, 6B, and 6D). Furthermore, the expression of β-oxidation related genes in the liver of Dwarf Layer was significantly lower than that of White Leghorn (Figure 6C). There was no significant difference in ELOVL2 and FADS2 between breeds (Figures 6A and 6B).

Figure 6.

Figure 6

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Effects of breeds on the mRNA levels of lipid metabolism-related genes. (A) The expression of extending enzyme related genes in the liver; (B) The expression of desaturase enzyme related genes in the liver; (C) The expression of β-oxidation related genes in the liver; (D) The expression of transport proteins related genes in the liver; (E) The expression of transport proteins related genes in the duodenum; (F) The expression of VLDLR in the F6. Results are shown as mean ± SD, n = 6; “a, b, c” means with different superscripts indicate significant difference (P < 0.05), and different groups of the same gene were compared. CD = the control group of Dwarf Layer control group, fed with basal diet; CW = the control group of White Leghorn, fed with basal diet; ED = the experimental group of Dwarf Layer, which fed with 2.5% flaxseed oil + basal diet; EW = the experimental group of White Leghorn, fed with 2.5% flaxseed oil + basal diet.

Differential Protein Expression

Based on the RT-qPCR results (Figures 6A, 6B, and 6F), we selected one desaturase and one elongation enzyme in the liver, namely FADS1 and ELOVL5, as well as the key protein VLDLR that transports nutrients to the follicles. Employing WB analysis, we compared the expression levels of these 3 proteins across different breeds. Under basal dietary conditions, there were no notable differences in the expression of FADS1 in the liver and VLDLR in the follicles between the 2 breeds. However, upon supplementation with flaxseed oil, a significant increase in the expression of ELOVL5 and FADS1 was observed in the liver of the Dwarf Layer, while no significant change was observed in the White Leghorn. After feeding with flaxseed oil, there was a significant difference in the expression of ELOVL5 in the liver and VLDLR in the follicles of both breeds. Additionally, after feeding flaxseed oil, the levels of ELOVL5 in the liver and VLDLR proteins in the follicles of the Dwarf Layer were significantly higher than those of the White Leghorn. However, there was no significant difference in the expression of FADS1 protein in the liver between the 2 breeds (Figure 7).

Figure 7.

Figure 7

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Effects of Breeds on the protein Levels of Lipid Metabolism-related Genes. (A) Representative immunoreactive bands of FADS1, ELOVL5, and VLDLR; (B) The relative expression levels of target proteins were normalized by β-actin. Results are shown as mean ± SD, n = 3; “a, b, c” means with different superscripts indicate significant differences (P < 0.05), and different groups of the same protein were compared. CD = the control group of Dwarf Layer control group, fed with basal diet; CW = the control group of White Leghorn, fed with basal diet; ED = the experimental group of Dwarf Layer, which fed with 2.5% flaxseed oil + basal diet; EW = the experimental group of White Leghorn, fed with 2.5% flaxseed oil + basal diet.

DISCUSSION

In this experiment, feeding flaxseed oil significantly increased the content of ALA, EPA, DHA, and total n-3 PUFA in the liver and egg, consistent with other articles. Feeding flaxseed and other substances rich in n-3 PUFA significantly increased n-3 PUFA in egg yolk and liver (Gatrell et al., 2015; Neijat et al., 2017; Elkin et al., 2018; Huang et al., 2020; El Zenary et al., 2022). Additionally, we also compared the differences in n-3 PUFA content in the yolk and liver of different breeds. After feeding flaxseed oil, a significant difference in ALA, DHA, and n-3 PUFA was observed in the yolk between breeds. While, there was no significant difference in n-3 PUFA in the liver between breeds, which differs from the difference observed in yolk. Furthermore, n-6/n-3 in the liver decreased from 16.69 to 4.80. A higher n-6/n-3 promotes the pathogenesis of many diseases, including cardiovascular disease, cancer, inflammation, and autoimmune diseases. Reducing the n-6/n-3 ratio can suppress the occurrence of these diseases (Simopoulos, 2008).

To better compare the differences in n-3 PUFA deposition among different breeds, we calculated the deposition efficiency of n-3 PUFA in eggs and livers. Consistent with the results of El Zenary's research (El Zenary et al., 2022), feeding flaxseed oil significantly reduced the deposition efficiency of n-3 PUFA in livers. The deposition efficiency of n-3 PUFA in the liver can reach 0.53% when fed with a basic diet, while it decreases to 0.22% when fed with flaxseed oil. Similarly, to the deposition pattern of the liver, feeding substances rich in ALA can significantly reduce the deposition efficiency of n-3 PUFA in eggs (Elkin et al., 2018; Elkin and Harvatine, 2023). In our experiment, White Leghorn (14.48 vs. 15.52) also showed a certain downward trend in the deposition efficiency of n-3 PUFA in eggs after feeding flaxseed oil, but it was not significant. The decrease in deposition efficiency may be related to the complex physiology of the body. As dietary ALA levels increase, the low conversion of ALA to n-3 PUFA is speculated to be related to the saturation of desaturase activity involved in ALA metabolism in the liver (Cachaldora et al., 2008). There is ample literature indicating that compared to ALA, direct feeding of EPA or DHA is easier to achieve the enrichment of these substances in eggs (Cachaldora et al., 2008; Elkin et al., 2015; Neijat et al., 2017). This may be due to the lower efficiency of chicken liver's ability to synthesize longer-chain n-3 PUFA. However, Dwarf Layer (17.97 vs. 16.98) shows a certain upward trend. Currently, the addition of flaxseed oil in research and previous studies has led to a greater enrichment of n-3 PUFA in tissues or eggs. However, this enrichment situation can result in lower conversion efficiency. In the future, when enriching n-3 PUFA, the enrichment efficiency should be considered to determine a reasonable amount of addition and reduce waste. Additionally, this experiment found that the deposition efficiency of n-3 PUFA in eggs from Dwarf Layer (17.97 vs. 14.48) was significantly higher than that of the White Leghorn after feeding flaxseed oil. The differences between these breeds may be related to their own fatty acid metabolism, further research is needed on the regulatory mechanisms involved.

Blood lipids are the general term for all lipid substances in the blood, mainly including TG, CHO, phospholipids, free fatty acids, etc. TG is the main form of energy storage in mammals, and studies have shown that increasing dietary energy levels can increase TG levels in dog serum (Romsos et al., 1978). The levels of serum CHO, LDL, and TG all increase with the increase of dietary lipids (Li et al., 2023). However, studies have also shown that feeding flaxseed oil can decrease CHO, TG, and LDL in serum (Shahid et al., 2020). In our experiment, the levels of CHO, TG, and LDL in the serum decreased after feeding flaxseed oil. This decrease may be related to the role of n-3 PUFA in inhibiting TG, apolipoprotein synthesis, higher clearance of very low density lipoprotein (VLDL) by liver peripheral tissues, and elimination of more bile through feces, leading to a decrease in serum TG concentration (Shahid et al., 2019). In this experiment, TG decreased in Dwarf Layer and White Leghorn and CHO and LDL in Dwarf Layer showed a downward trend. Lipids in the body are mainly transported in the form of plasma lipoprotein. The role of HDL is to input cholesterol from outside the liver for decomposition, while the role of LDL is exactly the opposite, delivering endogenous cholesterol to various tissues outside the liver(Sheridan, 1988). The NEFA produced by the decomposition of triglycerides can be absorbed and metabolized by muscle and liver tissues. E2 increases the expression of its target gene ELOVL5 by downregulating the expression of miR-218-5p in the liver of laying hens, thereby enhancing the synthesis of n-3 and n-6 PUFA in the liver (Zhang et al., 2017). Research has found that feeding a diet rich in n-3 PUFA significantly increases the levels of E2 in eggs (de Haas et al., 2017). Similar to previous research, it showed an increasing trend in E2 and NEFA in Dwarf Layer and White Leghorn in our experiment. After feeding with flaxseed oil, the LDL of Dwarf Layer was lower than that of White Leghorn. When fed with a normal diet, the NEFA level in White Leghorn was higher than that in Dwarf Layer.

Research on poultry has shown that the intestinal microstructure of laying hens can affect the efficiency of n-3 PUFA incorporation into eggs (Nain et al., 2012). A larger duodenal surface area/villus or higher villus length and villus-to-crypt ratio can increase nutrient absorption and promote the conversion of medium chain fatty acids to long-chain fatty acids. Recent studies have shown that in broiler chickens fed with enzymatic flaxseed, the villus height, villus width, and crypt depth of the jejunum significantly increase (Apperson and Cherian, 2017). Adding flaxseed to the diet can significantly increase the height and width of duodenal and jejunal villi in brown layer(Westbrook and Cherian, 2019). Similar to previous studies, flaxseed oil improved the morphology of the intestine, increased the villous height and the ratio of villous height to crypt depth of the duodenum, and reduced crypt depth (Westbrook and Cherian, 2019). However, there was no significant effect on the villous height, crypt depth, and the ratio of villous height to crypt depth of the jejunum and ileum. There was significant difference in villus-to-crypt ratio in the duodenum between Dwarf Layer and White Leghorn after feeding flaxseed oil. This may be due to differences in the breed of chickens and the source of n-3 PUFA. The crypt is associated with increased epithelial cell turnover, and shortened villous and deeper crypt depth can lead to poor nutrient absorption, increased gastrointestinal secretion, and decreased production performance (Xu et al., 2003). The ratio of villous height to crypt depth is considered a useful measurement method for evaluating the digestive capacity of the small intestine, as a high ratio indicates that well differentiated intestinal mucosa has higher digestive and absorption capacity (Montagne et al., 2003). Therefore, as observed in this study, feeding flaxseed oil resulted in a significantly higher ratio of villous height to crypt depth (4.78 vs. 3.60) for Dwarf Layer compared to White Leghorn, which may lead to better nutrient absorption for Dwarf Layer.

There are significant differences in the composition of gut microbiota among different species (Ley et al., 2008; Davenport et al., 2017), and there are also certain spatial differences in the community structure of gut microbiota among the same species (Yeoman et al., 2012; Suzuki and Nachman, 2016; Tropini et al., 2017). The gut microbiota of chickens also varies by breed, strain, and gender. Pandit et al. found significant differences in the structure of cecal microbiota between 42-days-old Ross and Cobb commercial broiler breeds and 42-days-old Indian local breeds (Pandit et al., 2018). Schokker et al. found significant strain differences in the gut microbiota of broiler chickens at 0, 4, and 16 d of age (Schokker et al., 2015). Yang et al. observed significant differences in fecal microbiota composition between the 40th generation of antibody titer bidirectional breeding strains of domestic chickens at Virginia Tech as experimental materials (Yang et al., 2017). Rhodococcus can cause pathogenicity (Bell Ks, 1998), Brucella organisms are responsible for one of the most widespread bacterial zoonoses, named brucellosis (Suarez-Esquivel et al., 2020; Roop et al., 2021). There are also some genera of bacteria that are currently unrecognized, but this seems not uncommon because nearly 70% of the bacteria in the intestine are currently unrecognized, also known as dark matter in the intestine. Romboutsia (Bojovic et al., 2020), Subdoligranulum is almost non-existent in obese and diabetic populations, but systematically present in healthy populations (over 70% of the population). Further animal research has found that this bacterium can reduce sugar and body weight, and has strong anti-inflammatory effects. Therefore, the lack of this bacterium may promote the occurrence of diabetes and obesity (Le Roy et al., 2022). These substances have a positive effect on the growth of intestinal epithelial cells and the enhancement of intestinal barrier function, as well as anti-inflammatory effects (Liu et al., 2022). Studies have shown that after supplementing with n-3 PUFA, the inflammatory bowel disease group detected an increase in the abundance of Escherichia coli, Faecalibacterium, Streptococcus, and Veillonella, and a decrease in the abundance of Bacteroides and Oscillospira (Santoru et al., 2017). In mice, high levels of n-3 PUFA in tissues are associated with differences in gut microbiota, such as Bifidobacteria and Lactobacilli (Han et al., 2015; Buendia et al., 2024). According to research, Bifidobacterium may be the main bacterial genus regulating the utilization of n-3 PUFA by microorganisms. However, in our study, there was no significant difference in these bacteria caused by flaxseed oil, which may be related to different gut choices and species factors. In high-fat diets, there was a downward trend in Bacteroides, Clostridium and Eubacteria genera (Agans et al., 2018). Whole oat oil can reduce bacteria associated with hyperlipidemia, such as Lachnochlostrium, Blautia, Lachnospiraceae UCG-006, Roseburia, Faecalibacula, Colidextribacter, and Desulfovibrio) (Duan et al., 2021). Romboutsia may be a specific bacterial group of non-alcoholic fatty liver disease in the Asian population (Oh et al., 2021), the abundance of this bacterium in the intestine was decreased after ingesting flaxseed (Yang et al., 2023). Our results showed that the abundance of these bacterial genera (Romboutsia, Lachnoclostridium, Clostridium) was reduced by flaxseed oil, which may lead to less degradation of long-chain fatty acids and more ALA being absorbed.

Only 25% of the fatty acids required for yolk deposition are provided by the diet, while the remaining fatty acids are produced by the liver (70%) and adipose tissue (5%), although this balance is influenced by diet (Van Eck et al., 2023). The lipids in the feed are first digested and converted into monoglycerides, diglycerides, and most triglycerides (TG) in the digestive tract. Due to their water insoluble, lipids are transformed into the portal system, where they are termed portomicrons (Hermier, 1997) or chylomicrons in mammals (Van Eck et al., 2023). TG is further processed in the liver and bind to lipoproteins for transport to other organs, including follicles, adipose tissue, muscle, and heart tissue. VLDL targeted for yolk deposition specifically (VLDLy) contains a high ratio of apoVLDL – II to ApoB100, which can bind to oocytic receptors (Walzem et al., 1999). Figure 8 provides an overview of the formation process and transport of n-3 PUFA to follicles.

Figure 8.

Figure 8

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Overview of the formation process and transport of n-3 PUFA to follicles. In short, with the intake of n-3 PUFA rich substances, these substances are first digested by the digestive tract, and then fatty acids are transported to the liver through fatty acid binding proteins (FABP) and fatty acid transporters (FATP) for further conversion. After fatty acids enter the liver, they pass through β-oxidation, desaturation, and extension ultimately forming longer chains of n-3 PUFA, which are then transported by fatty acid binding proteins and fatty acid transporters and internalized by VLDLR into follicles to form yolk.

The conversion of ALA to long-chain n-3 PUFA involves a series of elongation and desaturation steps, catalyzed by enzymes such as the elongation enzymes of extremely long chain fatty acids (ELOVL2 and ELOVL5) (Schackmann et al., 2015) and fatty acid desaturases (FADS1 and FADS2) (Shahid et al., 2020), as well as their involvement β- Oxidizing enzymes (PPARα,ACOX1, CPT1) (Ding et al., 2003; Yang, 2010; Gamboa-Gomez et al., 2014). The transport and deposition of fatty acids rely on fatty acid transport related proteins (FABP1 and FATP1) and VLDLR (Diaz et al., 2011).

The CPT1 is considered a main regulatory enzyme of β-oxidation catalyzes the conversion of cytoplasmic fatty acyl-CoA to fatty acyl-carnitine and enters mitochondria (Yang, 2010). The peroxisome proliferator-activated receptor alpha (PPARα) can regulate and regulate the expression of genes encoding various mitochondrial and peroxisomal fatty acids β-oxidation enzymes (Aoyama et al., 1998; Gamboa-Gomez et al., 2014). The ACOX1 is a peroxisome that limiting the rate of β-oxidation (Ding et al., 2003). In our study, PPAR in the liver of Dwarf Layer was observed α significantly low expression after feeding flaxseed oil, consistent with previous findings (Shahid et al., 2020). However, PPAR in the liver of White Leghorn is significantly overexpressed after feeding flaxseed oil, and ACOX1 is significantly downregulated. Feeding basal diet resulted in significantly lower expression of CPT1 and ACOX1 in Dwarf Layer compared to White Leghorn. After feeding flaxseed oil, the expression of PPAR, CPT1 and ACOX1 in Dwarf Layer is significantly lower than that of White Leghorn. This indicates that the degree of β- oxidation in the Dwarf Layer is significantly lower than that of White Leghorn, which leads to a significantly lower degree of de novo synthesis of fatty acids in Dwarf Layer. This result also explains that gender linked dwarf genes may reduce the ability of de novo fatty acid biosynthesis (Burghelle-Mayeur et al., 1989), which may be related to the significant low expression of β-oxidation related genes in Dwarf Layer.

ELOVL1 is involved in the elongation of saturated and monounsaturated long-chain fatty acids, while ELOVL2 and ELOVL5 are involved in the elongation of polyunsaturated fatty acids (Jakobsson et al., 2006; Schackmann et al., 2015). There are also certain differences in the function of elongation enzymes among different species. ELOVL2 only has activity against C20 and C22 PUFAs and EPA is mainly converted to 24:5 through ELOVL2 metabolism in Chicken, while rats and mice also have certain activity against C18 (Leonard et al., 2002; Gregory et al., 2011). ELOVL5 can prolong C18-22 PUFA in chickens, but it does not prolong C22 PUFA in rats and humans (Leonard et al., 2000; Gregory et al., 2011). The ELOVL5 enzyme can extend Docosapentaenoic acid (DPA) in chickens, while the mammals mainly rely on ELOVL2 enzyme to extend DPA (Gregory et al., 2011; Gregory et al., 2013; Gregory and James, 2014). Our study found that after feeding flaxseed oil, both breeds showed a certain degree of high expression of elongation enzymes in the liver, this is consistent with the results of previous articles (Shahid et al., 2020). When fed with a basic diet, the expression of ELOVL2 in the Dwarf Layer was significantly higher than that of White Leghorn, while ELOVL5 showed no significant difference. This means that Dwarf Layer can convert more C20-C22 PUFA into DHA, but due to the limited amount of C20-C22 PUFA obtained from the diet, there is no significant difference in DHA content among different breeds. After feeding with flaxseed oil, ELOVL2 had no significant difference between 2 breeds, and ELOVL5 in Dwarf Layer was significantly higher than White Leghorn. This means that Dwarf Layer can convert more C18-C22 PUFA from the diet into DHA, which explains why the DHA content in eggs and liver from Dwarf Layer is significantly higher than that in White Leghorn.

FADS1 and FADS2 play a crucial role in the conversion of ALA to DHA. In our experiment, feeding with flaxseed oil resulted in significantly higher expression of FADS1 in Dwarf Layer, which is similar to previous studies (Shahid et al., 2020). After feeding flaxseed oil, the expression of FADS1 in Dwarf Layer was significantly higher than that of White Leghorn, while there was no significant difference in FADS2. This means that Dwarf Layer can convert more C20:4 into EPA, thereby producing more DHA. The difference between breeds has also been mentioned in previous articles, where (Rodriguez et al., 2017) feeding the same diet resulted in higher expression of FADS1 and FADS2 genes in the liver of slow-growing chickens, as well as higher levels of n-3 PUFA in the breast meat (Boschetti et al., 2016).

A study has found that the addition of EPA to duck liver cells cultured in vitro has no significant effect on the expression of FABP (Liu et al., 2011). Moreover, direct feeding of ALA did not significantly affect FABP1 in the liver of laying hens (Neijat et al., 2017). However, some studies have found that high levels of dietary fat can increase the expression of FATP and FABP mRNA in the liver (Yuan et al., 2012), as well as the expression of FABP in the intestine (Katongole and March, 1980). In our experiment, feeding flaxseed oil resulted in high expression of FABP1 and FATP1 in liver and duodenum. This may be related to different chicken breeds and sources of n-3 PUFA. After feeding with flaxseed oil, the expression of FATP1 in the liver of Dwarf Layer was significantly higher than that of White Leghorn, and FABP1 in the duodenum was significantly higher than that of White Leghorn, allowing more fatty acids to be transported to the liver for conversion. This may also lead to a significantly higher ALA in the liver of Dwarf Layer than that in White Leghorn.

In the process of chicken follicle selection, VLDLR plays a crucial role in the absorption of yolk protein by oocytes. Without VLDLR, oocytes cannot enter the rapid growth phase of follicle development (Nimpf et al., 1989). During the development of small white follicles, VLDLR migrates to the follicle wall, allowing vitellogenin to be internalized into the yolk (Diaz et al., 2011). In our study, both flaxseed oil and breeds had no significant effect on the expression of VLDLR.

The efficiency of converting ALA to EPA is only 0.2%, conversion to DPA is only 3.65%, and conversion to DHA is 37% (Pawlosky et al., 2001). Based on qPCR, we selected genes with significant differences between breeds for validation. And found that there was no significant difference in FADS1 protein in the liver of Dwarf Layer after feeding flaxseed oil compared to those in White Leghorn; ELOVL5 is significantly higher than White Leghorn, and the protein of VLDLR in follicles of Dwarf Layer is significantly higher than White Leghorn. This once again confirms the important role of ELOVL5 in the synthesis of n-3 PUFA.

Based on the results of RT-qPCR and WB, we have identified the effects of FADS1, ELOVL5, and VLDLR genes and proteins on the synthesis and deposition of n-3 PUFA in breeds. Meanwhile, it was explained that the ability of Dwarf Layer to absorption, synthesize and deposit more n-3 PUFA, may be due to significantly higher ratio of villus height to crypt depth in the duodenum, the significant expression of ELOVL5 enzyme in the liver and VLDLR in the follicles. Higher ratio of villus height to crypt depth in the duodenum leads more absorption from diet. High expression of ELOVL5 leads to more conversion of ALA to DHA, while high expression of VLDLR leads to more deposition of n-3 PUFA into the yolk.

CONCLUSIONS

After feeding flaxseed oil, the ratio of villus height to crypt depth in the duodenum of Dwarf Layer was significantly higher than that of White Leghorn, showing higher absorption capacity. In addition, it can be seen that Dwarf Layer regulate the metabolism and deposition of fatty acids by upregulating the mRNA level of FADS1 to promote carbon chain desaturation, ELOVL5 to promote carbon chain elongation, and FATP1 to promote fatty acid transport in the liver, as well as the protein level of ELOVL5 in the liver and VLDLR in the follicles. In conclusion, Dwarf Layer exhibits unique performance in the absorption, synthesize and deposition of n-3 PUFA.

DISCLOSURES

The authors declare no conflicts of interest.

ACKNOWLEDGMENTS

This work was financially supported by National Key Research and Development Program of China (2021YFD1200803), National Key Research and Development Program of China (2022YFD1300100), the China Agriculture Research Systems (CARS-40), and Jinan Introductory Innovation Team Project (No. 202228037).

Footnotes

Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.psj.2024.104016.

Appendix. Supplementary materials

mmc1.doc (127KB, doc)

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