Dietary Chinese herbal formula supplementation improves yolk fatty acid profile in aged laying hens

Abstract Chinese herbal formula (CHF) has the potential to improve the performance of aged laying hens through integrated regulation of various physiological functions. The present study aimed to investigate the effects of dietary CHF supplementation on the yolk fatty acid profile in aged laying hens. A total of 144 healthy 307-day-old Xinyang black-feather laying hens were randomly allocated into two groups: a control group (CON, fed a basal diet) and a CHF group (fed a basal diet supplemented with 1% CHF; contained 0.30% Leonurus japonicus Houtt., 0.20% Salvia miltiorrhiza Bge., 0.25% Ligustrum lucidum Ait., and 0.25% Taraxacum mongolicum Hand.-Mazz. for 120 days). The fatty acid concentrations in egg yolks were analyzed using a targeted metabolomics technology at days 60 and 120 of the trial. The results showed that dietary CHF supplementation increased (p < .05) the concentrations of several saturated fatty acids (SFA, including myristic acid and stearic acid), monounsaturated fatty acids (MUFA, including petroselinic acid, elaidic acid, trans-11-eicosenoic acid, and cis-11-eicosenoic acid), polyunsaturated fatty acids (PUFA, including linolelaidic acid, linoleic acid, γ-linolenic acid, α-linolenic acid, 11c,14c-eicosadienoic acid, eicosatrienoic acid, homo-γ-linolenic acid, arachidonic acid, and docosapentaenoic acid), and fatty acid indexes (total MUFA, n-3 and n-6 PUFA, PUFA/SFA, hypocholesterolemic/hypercholesterolaemic ratio, health promotion index, and desirable fatty acids) in egg yolks. Collectively, these findings suggest that dietary CHF supplementation could improve the nutritional value of fatty acids in egg yolks of aged laying hens, which would be beneficial for the production of healthier eggs to meet consumer demands.


Introduction
Egg yolks have high nutritional value and are one of the most important sources of essential fatty acids in daily diet, which are of great significance for the growth and development of the human body, protection from diseases, and immunity regulation (Kovacs-Nolan et al. 2005).Different fatty acids induce specific physiological effects on human health.However, due to the great intensification of poultry breeding, several metabolic disorders occur in laying hens after the peak-laying period.Metabolic disorders during the laying period lead to poor digestibility, absorption, and utilization of nutrients, abnormal accumulation of liver and abdominal fat, and the declined reproductive system function, thus inducing a negative influence on the health, performance, and egg quality of laying hens (Liu et al. 2018;Gu et al. 2021;Dai et al. 2022).In addition, the late laying period occupies a high proportion of time in the total reproduction cycle of the laying period of hens.Therefore, maintaining the production performance and improving the quality and nutritional value of egg yolks are important strategies to ameliorate the productivity of aged laying hens.
Recently, the application of Chinese herbs as antibiotic alternatives in poultry production has received special attention.Studies have confirmed that Chinese herbs and their extracts have the potential to improve the organism health, production performance, and product quality in poultry (Obianwuna et al. 2022;Chen et al. 2023), with the advantages of comparatively higher safety and lower side effects (Hou and Jiang 2013).Although most of the previous findings partially proved the effectiveness of a single Chinese herb supplementation in diets (Abbood et al. 2017;Lv et al. 2019;Zhang et al. 2023), considering Chinese herbal formula (CHF) as a whole can regulate various physiological functions in multiple ways and achieve integrated effects to confirm the complexity of actual production conditions according to the guidelines of traditional Chinese medicine theory (Wang et al. 2021).Meanwhile, the application of Chinese herbs as additives was limited due to low utilization rate, poor palatability, and limited expected outcome.The ultrafine pulverization technology helps to fully dissolve the bioactive components in the cells of Chinese herbs and improve the drug efficacy, thus reducing the cost (Liu et al. 2022).However, only a few studies have investigated the effects of dietary ultrafine CHF on egg yolk quality.
Our previous studies indicated that dietary ultrafine CHF supplementation significantly improved the egg production rate and eggshell strength, increased the plasma estradiol level, and enhanced hepatic anti-oxidation ability during the late laying period (Han, Lin, et al. 2023;Han, Lan, et al. 2023); thus, prolonging the 'service period' of laying hens.However, the effects of dietary CHF on the nutritional values of eggs remained unknown.Therefore, we hypothesized that CHF supplementation in laying hen's diet might enhance the nutritional value of eggs.Thus, the present study evaluated the effects of dietary CHF supplementation on egg yolk fatty acid profile in aged laying hens, aiming to provide a theoretical reference for the application of CHF in poultry production.

Animals and experimental treatments
A total of 144 healthy 307-day-old Xinyang blackfeather laying hens were randomly allocated into two groups, with eight replicates per group and nine hens per replicate.The birds in the control group were fed a basal diet (CON group), and the birds in the experimental group were fed a basal diet supplemented with 1% CHF (CHF group) according to our previous findings (Han et al. 2023), after seven days of acclimation.The composition and nutrient levels of the basal diet were as described by the Xinyang black-feather laying hens management guidelines (Shanghai Poultry Breeding Company, Ltd., Shanghai, China) and presented in Table 1.Total fatty acid composition of control and CHF diets is shown in Table 2.The animal experiment was carried out at the Fuyang Hui Siyuan Ecological Agriculture Co., Ltd., Anhui, China.During the trial, the laying hens were fed twice daily (09:00 and 15:00), and water was available ad libitum.The laying hens were subjected to a 16-h light per day and kept in a temperature (18 − 23 °C) and humidity (55%−65%) controlled room.The experiment lasted 120 d.
The animal study was approved by The Ethics Committee of the Institute of Subtropical Agriculture, Chinese Academy of Sciences (No. ISA-2018-071).

Sample collection
At days 60 and 120 of the trial, eight laying hens from each group (one hen per replicate) with similar body weight were selected for sampling after 12 h fasting.Blood samples (10 mL) were collected from the wing vein, heparinized, and centrifuged at 3500 × g for 10 min at 4 °C to obtain plasma and then stored at −80 °C until further analysis of biochemical parameters.Meanwhile, eight eggs from each group  (one egg per replicate) were randomly selected, broken to collect egg yolk samples, and then immediately transferred at −80 °C until further analysis.

Determination of plasma biochemical parameters
The plasma concentrations of total protein (TP), albumin (ALB), total cholesterol (TC), and triglyceride (TG) were measured by a Roche automatic biochemical analyzer (Cobas c311; F. Hoffmann-La Roche Ltd., Basel, Switzerland) using the commercially available kits (F.Hoffmann-La Roche Ltd., Basel, Switzerland).

Determination of total cholesterol in egg yolks
The TC content in egg yolks at days 60 and 120 of the trial was measured using the total cholesterol kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) following the manufacturers' instructions.

Targeted metabolomics analysis for medium-and long-chain fatty acids profiles in egg yolks
Egg yolk samples (100 mg) were placed in 2 mL centrifuge tubes, homogenized for 1 min with 800 µL 50% acetonitrile water, and centrifuged at 10,000 × g for 15 min at 4 °C to obtain the supernatant.
The LC-MS analysis was performed on Waters Acquity Ultra-high performance LC (Waters Corporation, Milford, MA, USA) and AB SCIEX 5500 QTRAP -MS (AB Sciex Pte., Ltd., Framingham, MA, USA).The Acquity UPLC HSS T3 column (1.80 µm, 2.10 × 100 mm; Waters Corporation, Milford, MA, USA) was used to carry out the chromatographic separation of all the samples, with the column temperature maintained at 40 °C.The chromatographic separation conditions were as follows: mobile phase A, ultra-pure water with 0.01% formic acid; mobile phase B, acetonitrile; flow rate, 0.30 mL/min; and injection volume, 2 μL.The gradient conditions of the metabolic processes were 60%, 20%, 1%, 1%, 60%, and 60% at 1, 5, 16, 17, 18, and 20 min, respectively.The MS parameters were as follows: curtain gas flow rate, 20 Arb; collision gas flow rate, 9 Arb; ion spray voltage, −4200 V; ion source temperature, 450 °C; ion source gas 1 flow rate, 35 Arb; and ion source gas 2 flow rate, 35 Arb.The multiple reaction monitoring (MRM) modes were selected for MS analysis.The data were obtained for further metabolomics and statistical analysis.

Targeted metabolomics data analysis
The methodology of metabolomics data analysis was conducted as previously described with some modifications (Liu et al. 2022;Zhu et al. 2022).Briefly, the raw data were collected from the LC-MS analyzer and pre-processed using the Compound Discoverer software (Thermo-Fisher Scientific, Waltham, MA, USA) to normalize the sum of the peak area before analysis.Then, the standard curves were established to calculate the concentration of each item using the Excel 2022 software according to the interior label.Principal component analysis (PCA) was performed using the SIMCA software (V14.1;Sartorius Stedim Data Analytics AB, Umea, Sweden) to visualize the distribution of the samples.Orthogonal projection to latent structures-discriminant analysis (OPLS-DA) was applied to visualize group separations and identify significant metabolites.The p value <.05 was considered as statistical significance.The Kyoto Encyclopedia of Genes and Genomes (KEGG) and MetabolAnalyst databases were applied for pathway enrichment analysis (http://www.genome.jp/kegg/).

Statistical analysis
All experimental data were analyzed by two-way analysis of variance (ANOVA) using the general linear model procedure of SPSS statistical software (SPSS 22.0; IBM Inc., Chicago, IL, USA).The statistical model includes the effects of diets, time, and their interactions.Differences were considered significant if p < .05.Tukey's multiple comparison tests were used to differentiate significantly different among treatments according to the interaction of the main effects (p < .05).Data are presented as means with standard error of the mean (SEM).

Effects of dietary CHF supplementation on plasma biochemical parameters of aged laying hens
The effects of dietary CHF supplementation on the plasma biochemical parameters of aged laying hens are presented in Table 3. Dietary CHF supplementation did not affect (p > .05)plasma ALB, TC, TG, and TP levels at days 60 and 120 of the trial compared with the CON group.In addition, there were no significant time and diet interactions (p > .05)for plasma ALB, TC, TG, and TP levels.

Effects of dietary CHF supplementation on total cholesterol level in egg yolks
The TC level in egg yolks is shown in Figure 1.Dietary CHF supplementation decreased (p < .05)TC level in the egg yolk at day 60 of the trial compared with the CON group.The TC level in the egg yolk of the CHF group was increased (p < .05)with age.There was an interaction between time and diet on the TC level in the egg yolk.

Effects of dietary CHF supplementation on mediumand long-chain fatty acids profiles in egg yolks
After searching the corresponding fragment and peak area and exporting the raw data, the negative ion mass spectrum identified the characteristics of 52 species of targeted MCFA and LCFA.The unsupervised PCA (Figure 2(A,B)) and supervised OPLS-DA (Figure 2(C,D)) showed that the fatty acids distribution of the CHF and CON groups was significantly separated at days 60 and 120 of the trial, indicating that MCFA and LCFA metabolome profiles of the samples were different after dietary CHF supplementation.The results of the permutation test showed that the OPLS-DA model was successfully established (Figure 2(E,F)).
The KEGG database was used for metabolic pathway enrichment analysis of differential metabolites to explore which metabolic pathways were affected by dietary CHF supplementation (Figure 5).At day 60 of the trial, dietary CHF supplementation enriched three differential metabolic pathways, including biosynthesis of unsaturated fatty acids, arachidonic acid metabolism, and fatty acid biosynthesis compared with the CON group.At day 120 of the trial, dietary CHF supplementation enriched six differential metabolic pathways, including biosynthesis of unsaturated fatty acids, linoleic acid metabolism, fatty acid biosynthesis, arachidonic acid metabolism, fatty acid degradation, and fatty acid elongation compared with the CON group.
To evaluate the nutritional value of the fatty acids profile in egg yolks, we classified the various fatty acids and calculated the health-related indicators (Table 5).The SFA, MUFA, PUFA, and PUFA/SFA levels were increased (p < .05)with the increased age of laying hens.Dietary CHF supplementation increased  (p < .05) the MUFA, PUFA, and PUFA/SFA levels compared with the CON group.The values of SFA, MUFA, PUFA, and PUFA/SFA in the D120-CHF group were higher (p < .05)than those in the D60-CON, D60-CHF, and D120-CON groups.Moreover, the n-3 and n-6 PUFA concentrations were increased (p < .05)with the age of laying hens and CHF treatment.The n-6 PUFA concentration was higher (p < .05) in the D120-CHF group than that in the other three groups, as well as the n-6/n-3 PUFA concentration than that in the D120-CON group.
Regarding healthy indexes (Table 5), there was an increase (p < .05) in HH ratio and DFA at day 120 of the trial.Dietary CHF supplementation increased the HH ratio, HPI, and DFA while decreasing AI level when compared with those fed with a basal diet (p < .05).There was an interaction (p < .05) between experimental time and CHF treatment for the HH ratio and DFA concentration.The HH ratio and DFA concentration in the D120-CHF group were higher (p < .05)than those in the other three groups.

Discussion
Numerous metabolic disorders occur during the late laying period, which enormously affects the body health and egg quality of laying hens, resulting in economic losses.Several factors are related to fatty acid in yolks, such as the age of laying hens and diet composition (Koppenol et al., 2014).Different nutritional strategies have been used to improve the Different letters indicate significant differences among the four groups (p < .05).COn: birds fed a basal diet; CHF: birds fed a basal diet supplemented with 1% CHF; SFa: saturated fatty acids; MuFa: monounsaturated fatty acids; PuFa: polyunsaturated fatty acids.
body health and egg quality of laying hens during the late laying period over the previous few decades.Therefore, the present study evaluated the effects of dietary CHF supplementation on the plasma biochemical parameters and yolk fatty acids profiles of Xinyang-black feather laying hens at 54 and 62 weeks of age (in the late laying period), and the results showed that dietary CHF could significantly improve the nutritional value of the egg yolk.The egg yolk contains more than 60% lipids (including fatty acids) of its dry weight, which are the main components of egg yolk (Golzar Adabi et al. 2013).The composition and concentrations of fatty acids in egg yolks affect the physical and chemical properties and flavor of eggs and are important indicators of the nutritional value of eggs (Nielsen 1998).In the present study, dietary CHF supplementation increased the total PUFA and n-3 PUFA (including α-linolenic acid and 5Z,8Z,11Z,14Z,17Zeicosapentaenoic acid) in egg yolks compared with the CON group at days 60 and 120 of the trial.Meanwhile, the concentrations of n-6 PUFAs such as linoleic acid, γ-linolenic acid, docosatrienoic acid, and arachidonic acid in egg yolks in the CHF group were higher compared with the CON group at day 120 of the trial, especially arachidonic acid.The γ-linolenic acid can be converted into docosahexanoate, known as 'brain gold' , improving brain health, reducing blood lipids, and delaying aging (Horrocks and Yeo 1999).Linoleic acid can be converted to γ-linolenic Figure 5. the scatter plots of the impact and enrichment of the metabolic pathway for all the matching significant mediumand long-chain fatty acids in egg yolks of aged laying hens between the Chinese herbal formula (CHF) group and the COn group at days 60 (a) and 120 (B) of the trial (n = 8).Metabolic pathway enrichment bubble plot: the vertical coordinate with the bubble indicates the P-value of the enrichment analysis, taking the negative logarithm of the natural number e as the base (i.e. for the -ln p-value, darker colors indicate a smaller p-value and a more significant enrichment).the larger the bubble size, the larger the influence factor; the darker the color, the smaller the p-value, and the more significant the enrichment degree.acid in the human body, which in turn becomes arachidonic acid.In addition, linoleic acid also reduces the serum levels of cholesterol and low-density lipoprotein cholesterol, which could affect lipoprotein metabolism and reduce the incidence of cardiovascular disease (Marangoni et al. 2020).Arachidonic acid can produce eicosanoidin, a bioactive inflammatory substance that produces inflammatory mediators, thus regulating the inflammatory response (Burns et al. 2018).A previous study showed that the top 20 pathways of KEGG functional enrichment analysis of TM included arachidonic acid metabolism (Liu et al. 2022).Consistent with these results, our pathway enrichment analysis in metabolomics confirmed the effects of dietary CHF supplementation on the biosynthesis of unsaturated fatty acids (UFA) and arachidonic acid metabolism.Therefore, the TM may partly contribute to the promising potential of CHF for the regulation of PUFA in egg yolks.
MUFAs are the main components of TG, cholesterol esters (cellular membrane components and precursors of steroid hormones and bile acids), wax esters, and phospholipids comprising cellular membranes (Ravaut et al. 2020).In the present study, dietary CHF supplementation significantly increased the total amount of MUFA in a time-dependent manner and several MUFA concentrations, including elaidic acid, petroselinic acid, and petroselaidic acid.Moreover, dietary CHF supplementation significantly decreased SFAs concentrations, including myristic acid, pentadecylic acid, and palmitic acid in egg yolks at day 60 of the trial, whereas increased the concentrations of these SFAs in egg yolks at day 120 of the trial.The SFAs are considered a predisposing factor for cardiovascular disease, associated with higher cholesterol, hyperlipidemia, and obesity (Virtanen et al. 2016).Nevertheless, other studies have reported that SFAs play a significant role in the growth and metabolism of cells (Rioux and Legrand 2007).However, the inconsistencies of these results on SFAs concentrations in the present study might be attributed to the treatment time and the age of the laying hens.
The source, synthesis, and transportation of fatty acids in egg yolks are closely related to the liver and ovary.Polysaccharides, alkaloids, flavonoids, UFA, and other bioactive components in Chinese herbs can play crucial roles in lipid-lowering and related regulating metabolism.Ajugol, the LJ extracts, ameliorates hepatic steatosis via mTOR-TFEBmediated lysosomal biogenesis (Zhang et al. 2021).Oleanolic acid in LL has hypoglycemic, hypolipidemic, and antioxidant effects on diabetic rats and protects liver function by inhibiting alloxan-induced damage (Gao et al. 2009).The TM extracts have been found useful for hypolipidemic effects due to their down-regulated expression of fatty acid synthase and the inhibiting activity of acetyl-coenzyme A carboxylase through the phosphorylation of AMPactivated protein kinase (Liu et al. 2014).In vivo and in vitro studies showed that SM extracts, such as tanshinone IIA and salvianolate, induce anti-oxidative and anti-inflammatory effects (Ren et al. 2019).The regulatory effects of dietary CHF on fatty acids in egg yolks may be attributed to the characteristics of Chinese herb ingredients as mentioned earlier not given by differences in the composition of the diets actually provided (Table 2).These bioactive ingredients in the CHF might be involved in regulating the liver and ovarian performance of laying hens, which warrants further in-depth study.
The nutritional values of egg yolk are attributed to its fat quality and fatty acid composition, and a balanced intake of fatty acids is essential for reducing the risk of atherosclerosis, cardiovascular disease, and other related diseases (Shahidi and Ambigaipalan 2018).The n-6 and n-3 PUFA and n-6/n-3 PUFA are the crucial fatty acids that control the hypocholesterolemic index.The n-3 PUFA plays a major role in regulating the thrombogenic index, whereas the n-6 PUFA is dominant for the atherogenic.Moreover, the n-6/n-3 PUFA ratio < 4.0 can prevent cardiovascular diseases (Wołoszyn et al. 2020), as also indicated in the present study.The European Health Agency recommended ratio of PUFA/SFA is higher than 0.45 in a daily diet (Scollan et al. 2006).The PUFA/SFA ratio in the CHF groups was higher than 0.45, even up to 0.70 at day 120 of the trial, whereas it was lower than 0.45 in the CON group.These results suggest that dietary CHF supplementation improved the egg fatty acids profiles.
The AI and TI indexes reflect trends in platelet aggregation (Chen and Liu 2020), which commonly recommended for human consumption are less than 0.5 and 1.0, respectively, in food products (Wołoszyn et al. 2020).In the present study, dietary CHF supplementation decreased the AI index in egg yolks from 0.68 to 0.52.Also, the TI index ranged from 0.71 to 0.81 in the CHF groups, which was within the recommended range.The HH ratio is related to cholesterol metabolism; generally, a higher HH value is associated with better health (Dernekbaşı and Karayücel 2021).Moreover, HPI and DFA indexes are beneficial for human health (Medeiros et al. 2014;Hanuš et al. 2018).In the present study, dietary CHF supplementation significantly increased the HH ratio, HPI, and DFA in egg yolks.These results indicate that dietary CHF supplementation could enhance the nutritional values of egg yolks.Leonurin isolated from LJ and tanshinone I, tanshinone IIA, cryptotanshinone, salvianolic acid B, ferulic acid, and salvianolic acid A isolated from SM, showed protective effects on the cardiovascular disease (Wojtyniak et al. 2013;Zhang et al. 2022).These components in the CHF may play a potential role in fatty acid changes in egg yolks.

Conclusion
In summary, dietary CHF supplementation could increase the concentrations of fatty acids and optimize the fatty acid composition in egg yolks without affecting the plasma biochemical parameters in aged laying hens.Dietary CHF supplementation increased MUFA, PUFA/SFA, n-6/n-3 PUFA, HH, and DFA while decreased AI and TI indexes in the egg yolk.These findings will provide the guiding significance of using CHF as feed additives in poultry production to improve the nutritional value of eggs.

Figure 1 .
Figure 1.effects of dietary supplementation with Chinese herbal formula (CHF) on total cholesterol (tC) concentration in the egg yolk of aged laying hens.Data are shown as means with their SeM (n = 8).a−c Different letters indicate significant differences among the four groups (p < .05).COn, birds fed a basal diet; CHF, birds fed a basal diet supplemented with 1% Chinese herb formula.

Figure 2 .
Figure 2. PCa and OPlS-Da models to evaluate fatty acids data in the egg yolk of aged laying hens (n = 8).the principal component analysis (PCa; a and B) and orthogonal projections to latent structures-discriminant analysis (OPlS-Da; C and D) score plots of the fatty acids in egg yolks and permutation test of the OPlS-Da model (e and F) for the CHF vs. COn groups at days 60 and 120 of the trial.COn, birds fed a basal diet; CHF, birds fed a basal diet supplemented with 1% Chinese herb formula.CHF: Chinese herbal formula.

Figure 3 .
Figure 3. Volcano plots showing the changes in medium-and long-chain fatty acids profile in the egg yolk of aged laying hens (n = 8).(a and B) represent the results of Volcano plots between the Chinese herbal formula (CHF) group and the COn group at days 60 and 120 of the trial, respectively.COn, birds fed a basal diet; CHF, birds fed a basal diet supplemented with 1% CHF.

Figure 4 .
Figure 4. analysis of differential fatty acids in egg yolks of aged laying hens (n = 8).Heatmap of differential medium-and long-chain fatty acids in egg yolks of aged laying hens at days 60 (a) and 120 (B) of the trial.COn, birds fed a basal diet; CHF, birds fed a basal diet supplemented with 1% Chinese herbal formula.

Table 1 .
Composition and nutrient levels of the basal diet, % air-dried.
a the premix provided the following per kg of diets: vitamin a 7,000 iu, vitamin D 3 2,500 iu, vitamin e 24 mg, vitamin Κ 3 9 mg, VB 1 3.15 b Crude protein and ether extract were measured values, while the others were calculated values.

Table 3 .
effects of dietary supplementation with Chinese herbal formula (CHF) on plasma biochemical parameters of aged laying hens.

Table 4 .
effects of dietary supplementation with Chinese herbal formula (CHF) on medium-and long-chain fatty acid composition in egg yolks of aged laying hens. a−d

Table 5 .
effects of dietary supplementation with Chinese herbal formula (CHF) on medium-and long-chain fatty acid indexes in egg yolks of aged laying hens.