Effect of dietary supplementation of a phytogenic blend containing Schisandra chinensis, Pinus densiflora, and Allium tuberosum on productivity, egg quality, and health parameters in laying hens

Objective This study was conducted to investigate the supplementary effect of a phytogenic blend (SPA: a mixture containing fermented Schisandra chinensis pomace, fermented Pinus densiflora needle extract, and Allium tuberosum powder in the ratio of 2:2:1) on egg production, egg quality, blood constituents, and visceral organs in laying hens. Methods A total of 135 Hy-line brown laying hens (48-wk-old) were randomly allocated to three dietary treatments with 5 replicates of 9 hens. The control group (CON) was fed a basal diet (no exogenous SPA addition) and the experimental groups were fed the basal diet containing SPA at the level of 0.1% and 0.3% for 6 weeks. Results The feed intake was significantly improved in SPA supplemented groups as compared with the control (p<0.05). However, egg production, daily egg mass, and feed conversion ratio were not different among the dietary treatments (p>0.05). For egg quality traits, only Haugh unit (HU) was significantly improved in SPA (0.3%) (p<0.05) as compared with other groups. However, HU was not affected during 4-wk of storage at 18°C among the dietary treatments (p>0.05). Furthermore, SPA supplementation did not affect the blood biochemical constituents except for the phosphate content, which was significantly higher in SPA groups than the CON group (p<0.05). There were no significant differences in visceral organ characteristics and immune indicators (immunoglobulin A [IgA], IgG, and IgM) in SPA or CON groups. Conclusion This study suggested that the supplementation of SPA may have beneficial effects on feed intake and egg quality in laying hens.


INTRODUCTION
Antibiotics have been routinely used in farm animal production since mid1950s to pro mote their wellbeing and growth. However, the prolonged and unregulated use of antibiotics have raised a concern of the emergence of antibiotic resistance in the animals' bacteria and from there to bacteria infecting humans [1]. The poultry industry has grown substantially in the direction of improving the productivity with the use of alternative antibiotics to improve growth and feed conversion efficiency as well as to prevent infections [2]. In this context, phytogenic feed additives (PFA) comprising a wide variety of herbs, spices, and plantderived extract have received considerable attention in poultry diets over other com monly used nonantibiotic growth promoters, such as organic acids and probiotics due to their safe and environmentally friendly nature [36]. PFA contain several bioactive compounds such as polyphenols, es sential oils, saponins, and terpenoids [7], which are reported to have digestion stimulating effects [3], antimicrobial and antioxidant properties [5], and antiinflammatory potential [8]. Several recent studies indicated that the dietary supple mentation of PFA improves productive performance, egg quality, digestibility of nutrients, some blood biochemical constituents, and immune activity in laying hens [36,8]. How ever, the efficacy of these additives is influenced by several factors such as plant species, extract composition, applica tion method and levels, poultry age and breed, and housing conditions [3,9].
Schisandra chinensis (S. chinensis) Baill. of Schisandraceae family is native to Far East of Russia, South of China, and Korea [10]. The fruit of S. chinensis, commonly called as a "fiveflavored berry", is rich in lignans, with dibenzocyloocta diene lignans (type A) being the most abundant (80%). In addition, the berries are rich in phytoestrogens, minerals, vitamins, and essential oils [11]. Research on Schisandra fruit revealed that its active lignan possesses antioxidant, antimi crobial, hepatoprotective, and antiinflammatory functions [10,12]. The inclusion of Schisandra meal in broiler diets was shown to improve meat quality [13] and some blood anti oxidant parameters [14].
Pine needles have been traditionally used as supplements in various foods and folk medicine in Asia, specifically in Korea and China [15]. Pine needles exhibit several biological activities, including antimicrobial, antioxidant, antimutagenic, antitumor, and anticholesterol [1517]. These beneficial properties may be related to their bioactive compounds such as phenolics, flavonoids, and tannins [17]. Recently, several studies indicated the potential of pine needles as natural feed additives in poultry production via their stimulatory effects on the antioxidant status of birds [16,1821]. However, it was also indicated that the high content of condensed tannins in pine needles might affect the nutrient absorption and protein digestibility in animals [22].
Chinese chive (Allium tuberosum) is widely used for cu linary purposes and its application in folk medicine has also increased its popularity in Asia [23]. Chinese chive has been reported to demonstrate antioxidant [23], antimicrobial [24], hepatoprotective [25], and antiinflammatory [23] activities. The beneficial properties of Chinese chive are attributed mainly to organosulfur compounds, polyphenols, and sa ponins [26].
To our knowledge, little research reported on the effect of these additives in laying hens. Therefore, the current study was designed to evaluate the effect of Schisandra fruit pomace, pine needles, and Chinese chive supplementation to layer diets on laying performance, egg quality, egg storage, blood constituents, and visceral organs.

Preparation of feed additive
A phytogenic blend was prepared by mixing fermented S. chinensis pomace, fermented pine needle extract, and Chinese chive powder in the ratio of 2:2:1 (SPA) based on the prelimi nary experiments. The S. chinensis fruit pomace was procured from a juicemaking plant (Mungyeongsi, Gyeongsangbuk do, Korea), sundried for 24 to 48 h and stored at 4°C until use. The fruit pomace was then fermented using an indige nous isolate, Wickerhamomyces sp. SK1819 (1%, v/v) in yeast malt broth (Difco Laboratories, Detroit, MI, USA) at 1:1 ratio at 30°C and 100 rpm for 16 h. The fresh pine needles (Pinus densiflora) were collected from the region of Bonghwagun, Gyeongsangbukdo, Korea. The needles were then washed, airdried, and spontaneously fermented in a medium con taining equal amounts of water and sugar for a period of 1 year. The supernatant of this spontaneously fermented pine needles was used as a fermented pine needle extract. The Chinese chive was purchased from a local market (Seoul, Korea) and juice was squeezed using a juicer (Angeljuicer, Busan, Korea). Chinese chive powder was prepared by mix ing its juice with soybean meal (used as excipient) in the ratio of 7:3 and dried at 40°C for 36 h in a dry oven (WFO600S, Eyela Singapore Pte. Ltd., Singapore). Table 1 presents the proximate chemical composition of the tested SPA, includ ing moisture, crude protein, crude fat, crude fiber, ash, calcium, and available phosphorus [27].

Experimental animal and design
A total of 135 Hyline brown laying hens was randomly as signed to 3 treatment groups with 5 replicates of 9 birds each. The experimental house was a 2tier batterycage facility and hens were housed 3 per cage of the dimension of 43 cm length, 45 cm deep, and 42 cm height. A cornsoybean meal basal diet was formulated to meet or exceed the 1994 National Re search Council recommendations [28] ( Table 2). The levels of SPA supplied to the basal diet was as follows: 0%, 0.1%, and 0.3%. The appropriate amount of SPA was added to the basal diet and mixed for 5 min using a feed mixer (DKM

Egg productivity
The number of eggs produced was recorded daily at 13:00 h including those were broken. Egg production rate (%) was calculated from the total number of eggs laid in 1 wk divided by the total number of hen days in that week on a replicate basis. Average egg weight was obtained by dividing the total weight of collected eggs by the number of normal eggs. The daily egg mass was calculated by multiplying the egg pro duction rate by the average egg weight. We recorded feed intake weekly for each replicate. The feed conversion ratio (FCR) was calculated as grams of feed intake per gram of daily egg mass produced.

Egg quality
On a weekly basis, fifteen eggs per treatment (3 eggs per replicate) were randomly collected for the egg quality mea surements including egg weight, egg breaking strength, Haugh unit (HU), eggshell color, yolk color, and eggshell thickness. The egg breaking strength was measured using egg break ing strength tester (FHK, Fujihira Co. Ltd., Tokyo, Japan). HU, a measure of the height of the albumen of the eggs broken out on a flat surface, was calculated using the for mula 100×log (H+7.57-1.7W0.37), where H is the height of the egg white (mm) and W is the weight of the egg (g). Egg shell color was measured using an eggshell color fan (Samyang Co., Ltd., Seoul, Korea). Egg yolk color was measured us ing an egg yolk color fan of Roche. Egg shell thickness was measured at central part of the eggshell fragments without eggshell membrane using a Digimatic micrometer (Series 29333030, Mitutoyo Corporation, Kawasaki, Japan).

Egg storage
Four days before the end of the experiment, all the normal eggs were collected to measure the HU according to the stor age period of the eggs. Collected eggs were stored at 18°C for 4 wk, and a total of 45 eggs, 3 eggs with similar egg weight per replicate, were selected at 1, 2, and 4 wk to measure the HU as described above.

Blood parameters
After 6 wk of feeding, 8 hens were selected from each treat ment with a standard weight of 2.1±0.1 kg to measure the blood parameters. About 10 mL of blood was collected from the carotid artery and serum was separated by centrifugation at 1,500 rpm for 20 min. The separated serum was sent to Seoul Clinical Laboratories (Giheunggu, Yonginsi, Gyeonggi do, Korea) to analyze the levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), triglyceride (TG), total cholesterol (TC), albumin, globulin, creatinine, calcium, phosphate, amylase. The analysis was performed with an automated analyzer (Hitachi 7600, Tokyo, Japan). High density lipoprotein (HDL) cholesterol was measured using an HDL diagnostic kit (HDLcholesterol kit, Youngdong Medical Corporation, Seoul, Korea). Afterthat, lowdensity lipoprotein (LDL) and verylowdensity lipoprotein (VLDL) cholesterol were precipitated by adding sedimentation re agent (dextran sulfate, phosphotungstic acid) to the serum and the remaining estertype cholesterol was decomposed with esterase and released. Subsequently, an enzyme solu tion (cholesterol oxidase, peroxidase) was added to form a red quinoid pigment, and the absorbance was measured at

Visceral organ properties
After collecting blood, liver, spleen, gizzard, heart, abdomi nal fat, jejunum, ileum, cecum and ovary were collected and expressed in terms of relative weight per 100 g of body weight. The length of jejunum and ileum were measured after re moving the mesentery and expressed as relative length per 100 g of body weight. After that, the contents of jejunum and ileum were collected, and the pH of the contents was mea sured using a digital pH meter (735P, iStek Inc., Seoul, Korea).

Statistical analysis
Data are analyzed in a completely randomized design using the PROC mixed procedure of SAS version 9.4 (SAS Insti tute, Cary, NC, USA). The data of productivity performance and egg quality were analyzed by considering replicate as the experimental unit. For the measurements of blood and organ properties, individual bird was considered as the experimen tal unit. The significance of difference among the treatments was assessed using the Tukey's test. Means were considered statistically different at pvalue < 0.05. Results were presented as least squares means±standard error.

Effect on laying performance
The effects of dietary supplementation of SPA on laying per formance in hens from 50 to 56 wk of age are shown in Table  3. During this period, the SPA supplementation into layers' diet had no effects on egg production, egg weight, feed con version, and egg mass when compared to the control group (p>0.05). However, a linear significant increase in the feed intake was observed with the increase in SPA levels (p = 0.03).

Effect on egg quality
The effects of dietary supplementation of SPA in laying hens on HU, eggshell color, egg yolk color, eggshell breaking strength and eggshell thickness are shown in Table 4. Dietary treatments had a significant effect (p<0.05) on HU. The SPA (0.3%) group had higher HU as compared with control and SPA (0.1%) groups (p = 0.001). However, dietary supple mentation of SPA had no effects on eggshell color, egg yolk color, eggshell breaking strength, and eggshell thickness (p>0.05).

Haugh unit change according to egg storage period
The effect of dietary supplementation of SPA in laying hens on HU according to egg storage period is shown in Figure 1. During storage, the HU decreased in the case of all the di etary treatments. However, the supplementation of SPA had no effects on HU of eggs stored for 4 wk at 18°C (p>0.05).

Impact on blood parameters
The effects of SPA inclusion in diets on blood biochemical parameters of layers at the end of the experiment (56 wk of age) are presented on Table 5. Serum levels of AST, ALT, TG, TC, HDL, HDL (%) LDL+VLDL, albumin, globulin, creati nine, calcium, and amylase did not differ among the dietary treatments (p>0.05). However, blood phosphate content was significantly higher in SPA supplemented groups than the control group (p = 0.02). Table 6 shows the effects of dietary supplementation of SPA on laying hens on the Ig content in the blood. Ig including IgA, IgG, and IgM did not differ significantly in all the treat ments (p>0.05).

Impact on visceral organs properties
A summary of visceral organ weights of the experimental birds is shown in Table 7. After 6 wk of dietary SPA supple mentation, there were no statistically significant changes in the relative weights of liver, spleen, heart, gastrointestinal tracts (gizzard, jejunum, ileum, and cecum), and abdominal fat in comparison to the control birds (p>0.05). The relative weight of the liver, however, was reduced (p = 0.09) in the supplemented treatment groups as compared with that of the control group. The pH and length of the jejunum and cecum remained unaffected among the dietary treatments (p>0.05) ( Table 8).

DISCUSSION
Several studies have supported the nutritional value of plant extracts as natural feed additives in layer diets [35,8]. In this study, egg production, egg weight, egg mass, and FCR were not affected with or without dietary supplementation of SPA, suggesting that there was no toxicity of the phytogenic used up to the level of 0.3 g/kg of diet. The absence of adverse effects on performance traits has also been reported in broiler Values are means ± standard error. SPA, fermented S. chinensis fruit pomace, fermented pine needle extract, and Chinese chive powder in the ratio of 2:2:1. 1) CON, control, basal diet; SPA (0.1%), basal diet+0.1% SPA; SPA (0.3%), basal diet+0.3% SPA. a,b Means within a same row with different letters differ significantly at p < 0.05. breeds when fed with pine (Pinus brutia) needle powder (10 g/kg of diet) [18] and S. chinensis powder (5, 10, or 20 g/kg of diet) [13]. Alternatively, Kim et al [29] observed that the egg production rate was significantly improved when the mixtures of Artemisia campestris (A. campestris), Ca mellia sinensis (C. sinensis), S. chinensis, and Viscum album var. coloratum were fed to laying hens. Similarly, Ma et al [30] observed the beneficial effects of the S. chinensis on the Values are means ± standard error. SPA, fermented S. chinensis fruit pomace, fermented pine needle extract, and Chinese chive powder in the ratio of 2:2:1; Ig, immunoglobulin. 1) CON, control, basal diet; SPA (0.1%), basal diet+0.1% SPA; SPA (0.3%), basal diet+0.3% SPA. Values are means ± standard error. SPA, fermented S. chinensis fruit pomace, fermented pine needle extract, and Chinese chive powder in the ratio of 2:2:1. 1) CON, control, basal diet; SPA (0.1%), basal diet+0.1% SPA; SPA (0.3%), basal diet+0.3% SPA. egg production traits of laying hens during heat stress. The inconsistencies in these results could be related to the vari ation in animal breeds and their health status, dose of herbs, and bioactive constituents in the used plant extracts as well as the experimental conditions. In a concentrationdepen dent manner, the SPA used in this study improved the feed intake. It is assumed that SPA might have improved the pal atability of feed due to their aromatic characteristics and thereby could promote feed consumption when added to layers' diets. This result was consistent with previous reports those indicated increased feed intake in broilers fed pine (Pinus yunnanensis) needle powder at 50 g/kg diet [19] and onion (Allium cepa) powder at 30 g/kg diet [31]. Due to the nonavailability of the information related to layers, the pro ductive performance results of this study were also compared with the research that examined the supplemental effects of some PFA on the growth performance of broilers. This study showed no significant difference in yolk color and eggshell properties (color, breaking strength, and thick ness) among the dietary treatments. This is consistent with the previous reports that indicated no alteration in most of the egg quality traits in laying hens fed S. chinensis [32] and pine needles [33]. Li et al [32] reported that egg weight, egg shell thickness, egg white height, HU, and eggshell breaking strength had not changed in a study that fed 24wkold Lohmann Brown laying hens with 0.5% to 1% Chinese herbal mixture (containing 70% pine needles and 30% Artemisia annua) in the diet. Kim and Paik [33] also indicated no changes in eggshell strength, eggshell thickness, eggshell color, egg yolk, and HU when 66wkold Hyline Brown hens were fed 0.2% plant extracts, including turmeric, Angelica gigas, S. chinensis, Glycyrrhiza glabra, and Levisticum officinale. On the other hand, Kim et al [29] demonstrated that HU and egg yolk color were significantly increased compared to control, but eggshell thickness, and eggshell breaking strength were not changed when a mixture of A. campestris, C. sinensis, S. chinensis, and Viscum album var. coloratum was fed to laying hens. The discrepancies in the results of egg quality may be ascribed to the variability in animal breed, age, dosage, and environmental conditions. The literature relating Chinese chive supplementation to layers is scarce. HU is the "gold standard" of internal egg quality determi nation. Eggs are graded based on their HU values: AA, 72 or more; A, 71 to 60; and B, <60 [34]. Herein, HU in all the dietary treatments were above 72, suggesting that the eggs produced in this study are of good quality. Moreover, the inclusion of SPA at 3 g/kg of diet in laying hen diets signifi cantly increased HU. These improvements in HU may be attributed to the antioxidant property of bioactive constituents in S. chinensis, pine needles and Chinese chive. In particu lar, the antioxidant properties of these plant extracts have been reported in previous studies [14,18,23], which might have reduced the lipid and protein oxidation in eggs. In addition, the bioactive constituents of plants were shown to protect magnum and uterus, as well as enhance the al bumen secretion in laying birds [35], however, this needs to be specifically studied. HU also acts an important indi cator of egg freshness and it is related to shelf life [36]. In the current study, HU decreased with the storage time as expected, but the effect of dietary SPA inclusion was not observed in the HU of eggs during storage.
Blood biochemical constituents can be indicative of the health status of birds. In the current study, except serum phosphate levels, no changes in blood biochemistry were observed. Phosphorus is an essential nutrient for laying hens, which plays an important role in bone formation and maintenance, energy storage, cytoskeletal maintenance, and egg production [37]. The use of exogeneous phytase to im prove the phosphorus availability and blood phosphorus content of monogastric animals has been shown to be an ideal approach and is actively studied [37,38]. However, few studies have been conducted on the effects of dietary plant extracts supplementation on the blood phosphorus content in laying hens. The dietary SPA used in this study resulted in an increase in serum phosphate, which might be caused due to the increased digestibility of phosphorus or due to the presence of higher amount of available phos phorus in SPA supplemented diets. The previous reports of Amad et al [39] and Hafeez et al [40] observed the improve ment in the ileal digestibility of nutrients including phosphorus in broilers by a dietary PFA and attributed it to the stimu lation of endogenous digestive enzymes and to an increased absorption surface area in the intestine.
The changes in size and structure of internal organs are important for predicting the effect of diet and its components on the development and function of organs in laying hens. Generally, as the size of the organ increases, the energy re quired to maintain the organ increases, which in turn decreases the amount of energy input to productivity [4]. Our results revealed that dietary SPA supplementation did not affect rel ative weights of internal organs (liver, spleen, heart, gizzard, jejunum, and cecum), intestinal length, intestine pH, and abdominal fat, which indicated that dietary supplementation with SPA up to 3 g/kg of diet could have no observed adverse effects on organ development. Research on the effects of supplementing PFA in layer diets on internal organ charac teristics are limited.
Ig content plays an essential role in immune regulation. Three main classes of Ig exist in poultry: IgA, IgM, and IgY. IgY is a counterpart of mammalian IgG [41]. It has been thought that the antioxidant constituents of S. chinensis, pine needles, and Chinese chive might have a role in the develop ment of immune response in birds. In this study, dietary treatments did not affect serum IgA, IgM, and IgG contents, suggesting no effects of SPA on humoral immune status of laying hens. In contrast, Ma et al [30] indicated that supple mentation with S. chinensis at 10 g/kg of diet significantly elevated antibody responses against Newcastle disease virus in laying hens during heat stress. These inconsistent results could be explained by the fact that healthy poultry reared under clean and ideal environmental conditions usually do not respond to the feed additives [42]. Another explanation could be the higher dosage of SPA may be needed to stimu late humoral immune responses. Further studies need to be conducted in laying hens under stress conditions to evaluate the effects of SPA on immune responses.
In conclusion, dietary supplementation of SPA containing a mixture of fermented S. chinensis pomace, fermented pine needle extract, and Chinese chive powder, positively influ enced feed intake, HU, and blood phosphorus in laying hens. However, most of the productivity and egg quality traits re mained unaffected by SPA inclusion in layers' diets. Moreover, no adverse effects on blood and organ characteristics were observed. Further research is required to assess the SPA ef fects on immune response indices under stress or pathogenic challenge conditions.

CONFLICT OF INTEREST
We certify that there is no conflict of interest with any financial organization regarding the material discussed in the manu script.