Effects of treated deep-sea water on laying performance, nutrient efficiency, yolk quality, and immunoglobulin Y in chickens

Effects of treated deep-sea water on laying performance, nutrient efficiency, yolk quality, and immunoglobulin Y in chickens | Authorea try { document.documentElement.classList.add('js'); } catch (e) { } var _gaq = _gaq || []; _gaq.push(['_setAccount', 'G-8VDV14Y67G']); _gaq.push(['_trackPageview']); (function() { var ga = document.createElement('script'); ga.type = 'text/javascript'; ga.async = true; ga.src = ('https:' == document.location.protocol ? 'https://ssl' : 'http://www') + '.google-analytics.com/ga.js'; var s = document.getElementsByTagName('script')[0]; s.parentNode.insertBefore(ga, s); })(); Skip to main content Preprints Collections Wiley Open Research IET Open Research Ecological Society of Japan All Collections About About Authorea FAQs Contact Us Quick Search anywhere Search for preprint articles, keywords, etc. Search Search ADVANCED SEARCH SCROLL This is a preprint and has not been peer reviewed. Data may be preliminary. 20 August 2025 V1 Latest version Share on Effects of treated deep-sea water on laying performance, nutrient efficiency, yolk quality, and immunoglobulin Y in chickens Authors : Bounmy Keohavong 0000-0003-3343-6241 [email protected] , Sang Jip Ohh , and Sung Ki Lee Authors Info & Affiliations https://doi.org/10.22541/au.175565163.31137928/v1 168 views 96 downloads Contents Abstract Information & Authors Metrics & Citations View Options References Figures Tables Media Share Abstract This study investigated the effects of drinking water sourced from untreated and treated deep-sea water (DSW) on the laying performance, egg quality, nutrient utilizability, yolk lipid composition, oxidative stability, and immunoglobulin Y (IgY) levels in laying hens. Four hundred eighty Lohmann Brown Lite hens were randomly assigned to four treatments: control (tap water), untreated DSW, reverse osmosis-treated DSW, and nanofiltration-treated DSW, each diluted in a 1:20 ratio with deionized water. Over four weeks, data were collected on egg production, feed and water intake, feed efficiency, and mortality, along with nutrient digestibility, egg physical traits, yolk cholesterol, fatty acid profiles, lipid oxidation, and IgY content. Results showed that hens consuming DSW, particularly reverse osmosis-treated water, exhibited significantly improved egg production and nutrient utilization (notably energy and fat), without adverse effects on egg weight, quality parameters, or mortality. Egg yolk cholesterol and fatty acid composition, including DHA and the n-6/n-3 PUFA ratio, remained unaffected. Similarly, no significant changes were observed in oxidative stability or IgY levels. These findings suggest that DSW, especially when filtered by reverse osmosis or nanofiltration, can serve as a valuable alternative to conventional drinking water in poultry farming, supporting sustainable egg production and efficient nutrient use. Effects of treated deep-sea water on laying performance, nutrient efficiency, yolk quality, and immunoglobulin Y in chickens Abstract: This study investigated the effects of drinking water sourced from untreated and treated deep-sea water (DSW) on the laying performance, egg quality, nutrient utilizability, yolk lipid composition, oxidative stability, and immunoglobulin Y (IgY) levels in laying hens. Four hundred eighty Lohmann Brown Lite hens were randomly assigned to four treatments: control (tap water), untreated DSW, reverse osmosis-treated DSW, and nanofiltration-treated DSW, each diluted in a 1:20 ratio with deionized water. Over four weeks, data were collected on egg production, feed and water intake, feed efficiency, and mortality, along with nutrient digestibility, egg physical traits, yolk cholesterol, fatty acid profiles, lipid oxidation, and IgY content. Results showed that hens consuming DSW, particularly reverse osmosis-treated water, exhibited significantly improved egg production and nutrient utilization (notably energy and fat), without adverse effects on egg weight, quality parameters, or mortality. Egg yolk cholesterol and fatty acid composition, including DHA and the n-6/n-3 PUFA ratio, remained unaffected. Similarly, no significant changes were observed in oxidative stability or IgY levels. These findings suggest that DSW, especially when filtered by reverse osmosis or nanofiltration, can serve as a valuable alternative to conventional drinking water in poultry farming, supporting sustainable egg production and efficient nutrient use. Keywords: Deep-sea water; laying hens; egg quality; fatty acid profile; immunoglobulin Y Introduction Water is a vital component of blood and cellular fluid and plays a central role in most physiological functions (Keohavong et al., 2010). However, the quality and availability of water resources have become critical global concerns due to rapid population growth, unsustainable consumption, urbanization, and industrial and agricultural development (Poyraz & Taspinar, 2014; Wongsasuluk et al., 2014; Bai et al., 2016; Ding et al., 2016). Overexploitation and contamination have significantly compromised the sustainable supply of both surface and groundwater (Wongsasuluk et al., 2014; Bai et al., 2016). In response, alternative water sources such as deep-sea water (DSW), groundwater, and surface water have been explored to enhance drinking water quality and security (Keohavong et al., 2010; Wang et al., 2024; Wu et al., 2018). DSW and its mineral-rich profile whether untreated or processed via filtration have been shown to significantly influence poultry performance and production traits (Keohavong et al., 2010; Kang et al., 2010; Kang et al., 2011). DSW is sourced from depths exceeding 200 meters and contains three to ten times more minerals than surface seawater. Its bio-modulating effects are attributed to elevated mineral concentrations (Keohavong et al., 2010; Tsuchiya et al., 2004). However, most conventional water sources fall short of meeting the mineral requirements of modern poultry. Studies suggest that even the mineral concentration in standard tap water can significantly affect poultry productivity (McDowell, 2003). Moreover, laying hens require more water than non-layers, and approximately 74% of an egg’s content is water (Leeson & Summers, 2001; Awoniyi, 2003). Egg production and quality are also closely linked to the mineral composition of drinking water (Abbas et al., 2008). Additionally, the enrichment of egg yolks with specific fatty acids (FAs) is essential to improve their nutraceutical value (Corrales-Retana et al., 2021; Tadesse et al., 2022). Despite these insights, limited research has focused on the effects of untreated and reverse osmosis-treated DSW on meat FA composition (Kang et al., 2010; Kang et al., 2011) and serum immunoglobulin G (IgG) in poultry (Keohavong et al., 2010). While several studies have investigated the role of drinking water source on poultry performance (Keohavong et al., 2010; Kang et al., 2010, 2011; Ahmad et al., 2008), the biological effects of both untreated and treated DSW on laying hens remain largely unexplored. Therefore, this study aims to evaluate the impacts of drinking water with varied mineral contents including untreated and filtered DSW on laying hen performance, nutrient utilization, external and internal egg qualities, egg yolk cholesterol and fatty acid profiles, oxidative stability (TBARS), and yolk immunoglobulin Y (IgY) concentrations. 2. Materials and methods not-yet-known not-yet-known not-yet-known unknown 2.1. Animal care and ethical statement This experiment was conducted at the facility of Kangwon National University after receiving approval from the Institutional Animal Care and Use Committee with ethical code b: KW-220413-1. 2.2. Experimental design This study was conducted at the animal house facility of the College of Animal Life Sciences, Kangwon National University, Republic of Korea. The feeding trial and associated chemical analyses were carried out during the winter season. Deep seawater (DSW) was sourced from a depth of 300 meters below sea level and treated using either reverse osmosis (RO) or nano-filtration (NF) processes. Both untreated and treated DSW samples were provided by the Korea Institute of Water and Environment, Republic of Korea. As the salinity of the untreated and treated DSW ranged from 3.34% to 5.56% (Table 1), each DSW sample was diluted at a 1:20 ratio with deionized water before being offered to the birds. The study included four treatment groups: • T1: Control (tap water) • T2: Untreated DSW diluted 1:20 with deionized water • T3: RO-treated DSW diluted 1:20 with deionized water • T4: NF-treated DSW diluted 1:20 with deionized water A completely randomized design (CRD) was employed to allocate treatments. Table 1. Mineral profiles of fresh tab water and deep-sea water not-yet-known not-yet-known not-yet-known unknown Salinity (%) nd 3.34 5.31 5.56 Chlorine (Cl) 478.00 36,367.36 35,704.64 35,552.76 Sodium (Na) 55.10 14,179.55 13,100.81 11,673.11 Magnesium (Mg) 14.30 2,261.63 2,163.41 5,993.09 Sulfur (S) 135.90 1,618.23 1,516.73 5,082.33 Nitrate (NO3) nd 571.59 550.66 583.70 Calcium (Ca) 57.10 389.77 679.90 782.10 Potassium (K) 4.30 659.75 1,222.03 1,045.85 Bromine (Br) nd 149.11 126.42 115.07 Fluorine (F) nd 73.48 66.30 78.13 Strontium (Sr) nd 6.68 11.29 22.77 Boron (B) nd 2.85 3.58 3.01 Silicon (Si) nd 0.94 2.94 2.69 Lithium (Li) nd 0.10 0.14 0.12 Phosphorus (P) 0.09 0.01 0.04 0.07 Barium (Ba) nd 0.01 0.01 0.02 Molybdenum (Mo) nd 0.01 0.01 0.03 Arsenic (As) nd 0.01 0.01 0.002 Vanadium (V) nd 0.03 0.03 0.05 Titanium (Ti) nd nd 0.001 0.001 Zinc (Zn) 51.80 0.13 0.16 0.10 Nickel (Ni) nd nd 0.004 0.01 Aluminum (Al) nd 0.06 nd nd Manganese (Mn) 29.40 nd 0.001 0.001 Cadmium (Cd) nd nd 0.003 0.001 Iron (Fe) 43.90 nd 0.002 0.002 1 The data obtained from NRC (1974). 2 The data obtained from Keohavong et al. (2010); U-DSW = Untreated deep-sea water; RO-DSW = Deep sea water treated by reverse osmosis process. 3 The data analyzed by the central Lab, Kangwon National University; NF-DSW = Deep sea water treated by nanofiltration process. nd: Not detected. 2.3. Hens, housing and feeding A total of 480 Lohmann Brown Lite laying hens, aged 25 weeks (mean body weight: 1962 ± 190 g; hen-day egg production: 92%), were randomly allocated to triple-deck battery cages (metal wire cages) equipped with linear feeders and trough drinkers. Two hens were housed per adjacent cage (35 × 35 × 40 cm), providing 612.5 cm² of floor area per hen. Each replicate consisted of 20 hens, and each treatment included six replicates. A linear plastic egg collector was installed in front of each cage row, and a semi-automatic excreta collection system was placed beneath each battery deck. The environmental conditions were maintained at 21 ± 2.26 °C with a lighting schedule of 16 hours light and 8 hours dark, in a closed housing system with a relative humidity of 70 ± 10.10%. Feed and water were provided ad libitum. All experimental groups (T2, T3, and T4) received the same basal diet (Table 2), while the control group (T1) received the same formulation with an additional 0.2% dietary salt to meet sodium requirements. Excreta were removed daily using a semi-automatic belt conveyor system. The trough-type drinkers were cleaned daily to prevent contamination, and both the volume of water provided and residual water were recorded. To monitor body weight changes, five hens per replicate were randomly selected and individually weighed on days 1, 14, and 28 of the experimental periods. Salt intake for hens in the treatment groups (T2, T3, T4) was calculated based on water intake and corresponding DSW salt content, while salt intake in the control group (T1) was estimated based on both feed and water salt content. The experimental period lasted four weeks. Daily data were recorded for hen-day egg production, egg weight, feed intake, feed conversion ratio (g feed/g egg), water intake, and mortality (Table 3). Table 2. Composition of the basal layer diets1 (%, as-fed basis) Corn 59.52 59.72 59.72 59.72 Soybean meal 18.25 18.25 18.25 18.25 Gluten 2.65 2.65 2.65 2.65 Dicalcium phosphate 1.20 1.20 1.20 1.20 Limestone 9.85 9.85 9.85 9.85 Rice bran 2.00 2.00 2.00 2.00 Barley bran 1.50 1.50 1.50 1.50 Rape seed meal 2.50 2.50 2.50 2.50 Tallow 2.00 2.00 2.00 2.00 DL-Methionine 0.08 0.08 0.08 0.08 Vitamin. A, D 3 a 0.07 0.07 0.07 0.07 Vitamin premix b 0.06 0.06 0.06 0.06 Mineral premix c 0.12 0.12 0.12 0.12 Salt 0.20 - - - Calculated chemical composition (%) Protein 16.50 16.50 16.50 16.50 Lysine 0.74 0.74 0.74 0.74 Calcium 4.11 4.11 4.11 4.11 Phosphorus 0.58 0.58 0.58 0.58 Methionine 0.37 0.37 0.37 0.37 Methionone + Cystine 0.65 0.65 0.65 0.65 Choline 0.12 0.12 0.12 0.12 ME (kcal/kg) 2854.00 2854.00 2854.00 2854.00 1 T1 (Control): Hens given tab water; T2: Hens provided untreated deep-sea water (DSW) diluted with deionized water (1:20 ratio); T3: Hens given reverse osmosis-treated DSW diluted with deionized water (1:20 ratio); T4: Hens given Nano-filtration-treated DSW diluted with deionized water (1:20 ratio). a the vitamin A and D3 contains the followings per kg of diet: vitamin A, 14.000IU and vitamin D3, 2.800IU. b the vitamin premix contains the followings per kg of diet: vitamin E, 6IU; riboflavin, 4.8mg; thiamin, 1.2mg; pyridoxine, 2.4mg; vit. B12, 0.012mg; panthothenic acid, 4.8mg; folic acid, 0.48mg; biotin, 0.15mg. c the mineral premix contains the followings per kg of diet: Mn, 72mg; Zn, 48mg; Fe, 48mg; Cu, 2.4mg; I, 0.96mg; Se, 0.18mg; Co, 0.24mg. 2.4. Chemical analysis Untreated deep seawater (DSW) and DSW treated by reverse osmosis (RO) were obtained as previously described by Keohavong et al. (2010). The nano-filtration (NF) treated DSW underwent a pre-treatment dilution with deionized water at a 1:1000 ratio. Diluted samples were sent to the Central Laboratory of Kangwon National University for mineral composition analysis (Table 1). At the end of the feeding trial, chromic oxide (Cr₂O₃) was included in the diet at 3.5 g/kg as an indigestible marker. Colored excreta were carefully collected daily at 09:00 h for three consecutive days (72 hours), with approximately 200 g collected per replicate. Excreta samples were dried in a forced-air oven at 65 °C until a constant weight was achieved. Dried samples were weighed, ground, and stored at room temperature for further analysis. The chemical composition of diet and excreta samples including dry matter, crude protein, gross energy, ash, and ether extract was analyzed using official methods (AOAC International, 2005). Chromic oxide concentration in both diet and excreta was determined using the method by Fenton and Fenton (1979). Nutrient utilizability was calculated using the method described by Tarachai and Yamauchi (2001), as shown in Table 4. not-yet-known not-yet-known not-yet-known unknown 2.5. Egg quality measurement Thirty-six eggs produced by each treatment (6 eggs/replictae) at the end of the experimental period were used to determine both external and internal egg qualities (Table 5). The unit mass of each egg was weighed with an electronic balance to the nearest 0.01 mg (OHAUS, USA. max: 210 g). Eggshell color grade was determined using color fan (Judaeho, Korea). The egg shape index was calculated (100x(width/height)) with a digital calliper (Model Pat. A, FHK, Fujihira, Japan) to the nearest 0.01 mm (Anderson et al., 2004). The breaking strength (kg/cm) of egg was measured using eggshell intensity tester (FHK, Fujihira Industry Co., LTD, Japan) by pressing down an egg standing in vertical position on the pan of the tester. Internal adhered membrane of eggshell was removed, then the shell thickness was determined using a dial pipe gauge and dried shell weight was recorded to determine shell percentage (Monira et al., 2003; Ragni et al., 2006). The yolk index was obtained by dividing the height by the diameter of the yolk (Kop-Bozbay et al., 2021). Haugh unit (Haugh, 1937). score was calculated using the egg weight and albumen height (albumen height + 7.57 − 1.7 × egg weight0.37). The egg yolk color grade was evaluated using the Roche color fan (Vuilleumier, 1969). 2.6. Cholesterol analysis A total of 18 eggs of similar weight produced by each treatment (3 eggs/replicate) was collected at the end of the experiment for cholesterol analysis (Table 6). The cholesterol content was measured by using the method of Du and Ahn (2002). The preparation of egg yolk samples for cholesterol analysis was carried out as recently described for chicken breast muscle (Kang et al., 2011; Kang et al., 2010), with slight modifications. In brief, the yolk sample was centrifuged (model Ultra-Turrax T25 basic., Ika Werke GmbH & Co., Germany) with the solution of KOH (1.98% of w/v), ascorbic acid (20% of w/v), and 5α-cholestane internal standard (0.1% of w/v) at 21,500 rpm for 5 seconds. The centrifuged solution was heated in a water bath at 50°C for 1 hour. The reaction solution was obtained by mixing 5 mL each of distilled water and hexane, and the supernatant was collected and reacted with 200 μL of pyridine and 100 μL of sylon BFT in a water bath at 50°C for 1 hour. The final solution was analyzed by gas chromatography (6890N, Agilent Technologies, Palo Alto, CA, USA). 2.7. Fatty acid analysis A total of 18 eggs (3 eggs/replicate) of similar weight produced by each treatment was collected at the end of the experiment for fatty acid analysis (Table 6). FA composition was determined according to the methods of Folch, Lees, & Sloane Stanley. (1957). The preparation of egg yolk samples for FA analysis was carried out as recently described for chicken breast muscle (Kang et al., 2011; Kang et al., 2010), with slight modifications. In brief, the solution of chloroform:methanol (2:1) and yolk sample were centrifuged (Ultra-Turrax T25 basic (Ika Werke GmbH & Co., Germany) at 13,500 rpm for 1 minute, and the lower layer was filtered and collected. The collected lipid was methyl esterified with the solution of 2 N sodium hydroxide and 25% (w/v) boron trifluoride, mixed with 5 mL of distilled water and 3 mL of hexane, and centrifuged. The supernatant was collected and analyzed by gas chromatography (6890N, Agilent Technologies, Palo Alto, CA, USA). The content of each fatty acid was calculated as the percentage (%) of the total fatty acid peak area. 2.8. Analysis of oxidative stability in egg yolks Similar egg weight produced by each treatment (3 eggs/replicate) was collected at the end of the experiment for lipid oxidation analysis. The oxidative stability of lipid was performed by the 2-thiobarbituric acid reactive substances (TBARS) method of Sinnhuber and Yu (1977) and the oxidative stability measurements in egg yolk samples were carried out in accordance with the QuantiChrom TBARS assay kit guidelines as recently described methods for chicken muscle (Kang et al., 2010; Tadesse et al., 2022), with slight modifications. In brief, this research was carried out in three periods (1, 4 and 8 days of the stored egg samples) at the end of the experiment. Egg yolk samples were homogenized and approximately 500 mg of yolk sample was weighed in a tube. Then, the solution of 1% (w/v) of thiobarbituric acid (TBA), 0.3% (w/v) of NaOH, 2.5% (w/v) of trichloroacetic acid (TCA) and 3.6 mM of HCl were mixed. The mixture solution was heated in a 98℃-water bath for 30 minutes. The supernatant was then centrifuged (GS-6R Centrifuge, Beckman Instruments Inc., USA) at 3,500 rpm for 30 minutes. The absorbance was then measured at 532 nm using a spectrophotometer (UV-mini-1240, Shimadzu, Japan). Using malondialdehyde (MDA) standard calibration, the concentrations of TBARS (mg MDA/kg of egg yolk) were calculated. 2.9. Analysis of immunoglobulin Y At the end of the experiment, egg samples of similar weight produced by each replicate (3 eggs/replicate) was collected for egg yolk immunoglobulin Y (IgY) purification. The IgY quantification was carefully followed the two important steps as described in below. 2.9.1. Isolation of the water-soluble fraction The egg yolk samples preparation for isolation of water-soluble fraction (WSF) was carried out as recently described the methods of Bizhanov, Jonauskiene, and Hau (2004) developed a novel lithium sulfate precipitation method for purifying chicken egg yolk immunoglobulin Y, with slight modifications. In brief, egg yolk separator was used to separate egg white from the egg yolk. Egg yolks were rinse with deionized water and roll on paper towel to remove adhering egg white. Yolk membrane was punctured by Pasteur pipette and drain 10 g of yolk into the beaker (SCHOTT DURAN, GERMANY. 250 ml). Assume that 10 g of yolk is equal to 10 ml. Each yolk sample was mixed with 50 ml of cold acidified water, pH 4.0 adjusted with 0.1 N HCl, and following by 40 ml of cold acidified water, pH 2.0 adjusted with 0.1 N HCl to fill-up of total volume of 100 ml, while stirring gently to avoid foaming. The mixture, which had a pH of 5.0, was kept at 4 °C for 24 h. Beakers were placed onto the stirrer, well mixed solution was transferred into a graduated cylinder with an appropriate volume. The samples were centrifuged at 12.000 g for 20 min at 4 °C, supernatant was collected as WSF. The obtained WSF was used for quantification of IgY concentration by Enzyme Linked Immunosorbent Assay (ELISA) as described below. 2.9.2 Enzyme linked immunosorbent assay (ELISA) Indirect ELISA kit (E30-104) was used to measure for the IgY concentration, using the recently described method of Keohavong et al. (2010) for chicken serum immunoglobulin G (IgG) quantifications with slight modifications. In brief, coating solution was prepared by adding 10 µL of Goat x-chicken IgG (A30-104A-11) into 1 mL of 0.05 M Carbonate-Bicarbonate, pH 9.6, a total of 100 µL of coating solution was transferred into each well of an ELISA plate (Apogent Co, Denmark) and incubating at 4 °C for 24 h. Plate was washed with PBS-T (50 mM Tris, 0.14 M NaCl, 0.05% Tween 20, pH 8.0) for 5 times. Adding 200 µl of blocking solution (1% BSA diluted with PBS-T) into washed plate and incubating for 30 min at 37 °C. Washing plate with PBS-T again, and then adding 100 µl of samples (WSF dilution of 1/100000 blocking solution) and standards (Chicken Reference Serum, RS10-102-3). After incubated for 60 min at 37 °C and allowed the plate to wash for five times with PBS-T. Freshly prepared HRP conjugate (A30-104P) solution (1/40000 PBS-T) of 100 µl was added into each well of washed plate. After incubation at 37°C for 60 min, the plate was washed by PBS-T solution for five times. Enzyme substrate reaction was done by using TMB (3.3’,5.5’-tetramethylbenzidine), with 100 µl of enzyme substrate solution applied into each well (prepared as 2 µl of hydrogen peroxide 30% + 1 tablet of TMB + 1ml of DMSO (dimethyl sulphoxide) + 9 ml of phosphate-citrate buffer). After incubation at room temperature for 30 min, 100 µl of stopping solution (2 M H2SO4) was applied into each well and color will be developed immediately. Plate was placed into an ELISA reader (BioTek, USA) to read at 450 nm. Each sample was tested in duplicate, the test repeated three times and the mean values of optical density (OD) were obtained. The concentration of IgY was expressed in mg/mL of egg yolk. 2.10. Statistical analysis The data generated from the experiment was subjected to statistical analysis using the General Linear Model (GLM) procedure of SAS version 9.3 for Windows, SAS Institute Inc., Cary, NC, USA (SAS Institute, 2004). All data were considered as statistically different when there is a treatment effect at p < 0.05 (Steel & Torrie, 1980). not-yet-known not-yet-known not-yet-known unknown 3. Results not-yet-known not-yet-known not-yet-known unknown 3.1. Laying performance Laying performance during phase I (25–27 weeks of age) was affected by the source of drinking water. Hen-day egg production was significantly higher in hens receiving reverse osmosis-treated deep-sea water (T3) compared to the control group (T1) (p < 0.05), while T2 and T4 showed intermediate values that did not differ significantly from either group. There were no significant differences among treatments for egg weight, average daily feed intake (ADFI), feed conversion ratio (FCR), average daily water intake (ADWI), or mortality during this period (p > 0.05). In phase II (27–29 weeks of age), hen-day egg production remained significantly higher in the T3 group than in T1 and T4 (p 0.05), although one case of mortality was recorded in T4. Over the entire experimental period (25–29 weeks of age), hen-day egg production was significantly increased in hens receiving untreated DSW (T2) and RO-treated DSW (T3) compared to those in the control group (T1) (p 0.05). Mortality was negligible across treatments, with only one death occurring in T4 during the latter phase. Table 3. Effects of Different Deep-Sea Water Sources on Laying Performance in Hens from 25 to 29 Weeks of Age1 Phase I (25-27 weeks of age) Hen-day egg production (%) 89.42 b 91.77 ab 92.76 a 90.18 ab 6.47 Egg weight (g) 59.18 58.93 58.74 59.26 1.2 ADFI 3 (g/hen/day) 131.47 128.67 126.23 127.43 9.58 FCR 3 (g feed/g egg) 2.46 2.34 2.37 2.35 0.2 ADWI 3 (ml/hen/day) 194.66 192.41 203.6 201.64 8.9 Mortality (%) 0 0 0 0 0 Phase II (27-29 weeks of age) Hen-day egg production (%) 92.80 b 94.53 ab 95.83 a 92.69 b 5.17 Egg weight (g) 61.24 60.88 61.57 61.24 2.13 ADFI 3 (g/hen/day) 119.13 125.36 124.31 123.9 7.67 FCR 3 (g feed/g egg) 1.94 2.18 2.1 2.19 0.17 ADWI 3 (ml/hen/day) 180.7 193.78 195.73 195.52 11.1 Mortality (%) 0 0 0 0.06 0.19 Overall (25-29 weeks of age) Hen-day egg production (%) 91.04 b 93.39 a 93.61 a 92.40 ab 4.66 Egg weight (g) 60.19 59.86 60.13 59.91 2.5 ADFI 3 (g/hen/day) 121.3 127.01 125.27 125.66 7.88 FCR 3 (kg feed/kg egg) 2.19 2.26 2.23 2.27 0.13 ADWI 3 (ml/hen/day) 187.68 193.09 199.66 198.58 9.85 Mortality (%) 0 0 0 0.06 0.19 1 T1 (Control): Hens given tab water; T2: Hens provided untreated deep-sea water (DSW) diluted with deionized water (1:20 ratio); T3: Hens given reverse osmosis-treated DSW diluted with deionized water (1:20 ratio); T4: Hens given Nano-filtration-treated DSW diluted with deionized water (1:20 ratio). 2 SEM = Standard error of the mean. 3 ADFI = Average daily feed intake; FCR = Feed conversion ratio; ADWI = Average daily water intake. a, b The different superscripts within a row are significant differences (p < 0.05). 3.2. Nutrient utilizability As shown in Table 4, the type of deep-sea water (DSW) provided significantly influenced the digestibility of certain nutrients in laying hens. Notably, energy and ether extract utilization were significantly improved in hens receiving nano-filtration-treated DSW (T4) compared to those given reverse osmosis-treated DSW (T3) (p < 0.05). The highest fat digestibility was observed in T4, while T3 exhibited the lowest values, indicating a detrimental effect of reverse osmosis processing on lipid assimilation. Energy digestibility followed a similar trend, with T4 outperforming T3, suggesting that nano-filtration-treated DSW may enhance metabolic energy availability in laying hens. Furthermore, ash digestibility differed significantly among treatments (p < 0.05), with T2 and T3 groups showing reduced mineral utilization compared to the control (T1), whereas T4 presented intermediate values. These variations imply that untreated and reverse osmosis-treated DSW might impair mineral absorption, while nano-filtration may partly alleviate this effect. No significant differences were observed in dry matter or crude protein digestibility among treatments, despite numerical variation. The relatively high standard error observed, particularly for crude protein, may have contributed to the lack of statistical significance. Collectively, the results presented in Table 4 highlight that the processing method of DSW influences nutrient utilization efficiency in laying hens, with nano-filtration treatment showing the most beneficial effects. Table 4. Effects of Different Deep-Sea Water Treatments on Apparent Nutrient Digestibility in Laying Hens 1 Dry matter 73.14 72.51 70.51 75.47 6.34 Crude protein 43.96 49.90 41.94 47.94 12.81 Energy 76.19 ab 77.09 ab 74.19 b 79.97 a 5.34 Ash 40.53 a 27.72 b 22.13 b 33.16 ab 12.41 Ether Extract 85.94 b 86.16 b 82.33 b 90.54 a 4.01 not-yet-known not-yet-known not-yet-known unknown 1 T1 (Control): Hens given tab water; T2: Hens provided untreated deep-sea water (DSW) diluted with deionized water (1:20 ratio); T3: Hens given reverse osmosis-treated DSW diluted with deionized water (1:20 ratio); T4: Hens given Nano-filtration-treated DSW diluted with deionized water (1:20 ratio). 2 SEM = Standard error of the mean. a, b The different superscripts within a row are significant differences (p < 0.05). not-yet-known not-yet-known not-yet-known unknown 3.3. Egg qualities Egg quality parameters were not significantly affected by the different sources of deep-sea water provided to laying hens, as summarized in Table 5. No statistical differences were observed among treatments for egg shape index, shell characteristics (percentage, thickness, color, and strength), albumin height, Haugh unit, yolk index, or yolk color attributes (p > 0.05). These results indicate that neither untreated nor treated deep-sea water altered the physical or internal quality of eggs during the experimental period. The consistency in shell strength and thickness suggests that eggshell formation was maintained across all water treatments. Similarly, measures of albumin quality and yolk pigmentation remained stable, implying that water source did not impact protein quality or pigment deposition. Overall, the findings suggest that variations in deep-sea water processing methods have no detrimental effects on egg quality traits in laying hens under the conditions tested. Table 5. Effects of Deep-Sea Water Treatments on Eggshell and Yolk Quality Parameters1 Eggshape index (%) 78.93 76.95 78.01 79.40 0.50 Shell percentage (%) 13.47 13.75 13.80 13.60 0.22 Shell thickness (mm) 0.37 0.38 0.37 0.38 0.00 Shell color grade 12.00 11.90 12.80 12.50 0.15 Eggshell strength (kg/cm 2 ) 3.94 3.98 3.98 3.92 0.66 Albumin height (mm) 8.57 8.30 8.48 8.21 0.14 Haugh unit 91.31 89.77 91.93 89.34 0.78 Yolk index (%) 50.35 50.14 50.63 49.98 0.36 Yolk color grade 7.40 7.50 7.30 7.40 0.10 Yolk-lightness (CIE L * ) 56.63 56.29 56.54 57.10 0.20 Yolk-redness (a * ) -2.49 -2.33 -2.77 -3.03 0.13 Yolk-yellowness (b * ) 36.54 37.42 36.94 37.33 0.47 1 T1 (Control): Hens given tab water; T2: Hens provided untreated deep-sea water (DSW) diluted with deionized water (1:20 ratio); T3: Hens given reverse osmosis-treated DSW diluted with deionized water (1:20 ratio); T4: Hens given Nano-filtration-treated DSW diluted with deionized water (1:20 ratio). 2 SEM = Standard error of the mean. 3 The interactions among treatments were not found to be statistically significant (p > 0.05) and therefore the different superscripts were not presented in the table. 3.4. Total cholesterol and fatty acid profiles in egg yolk As presented in Table 6, no significant differences were detected among treatments for total cholesterol concentration in egg yolk or fatty acid profiles (p > 0.05). The various deep-sea water sources, including untreated, reverse osmosis-treated, and nano-filtration-treated DSW, did not alter the composition of saturated fatty acids (SFA), monounsaturated fatty acids (MUFA), polyunsaturated fatty acids (PUFA), or the n-6/n-3 PUFA ratio in yolk lipids. The stability of key fatty acids such as palmitic (C16:0), oleic (C18:1n-9), linoleic (C18:2n-6), and α-linolenic acid (C18:3n-3) across treatments suggests that DSW treatments had no impact on yolk lipid composition or cholesterol content. These findings indicate that deep-sea water processing methods, under the conditions tested, do not affect the lipid nutritional quality of eggs. Table 6. Yolk Cholesterol and Fatty Acid Responses to Different Deep-Sea Water Treatments in Hens 1 not-yet-known not-yet-known not-yet-known unknown Total cholesterol (mg/g yolk) 15.21 15.33 15.37 15.26 0.045 Fatty acids (% of total lipids) C14:0 (Myristic) 0.38 0.40 0.37 0.43 0.01 C16:0 (Palmitic) 25.61 25.39 24.80 26.50 0.31 C16:1n-7 (Palmitoleic) 3.72 3.98 3.70 4.29 0.14 C18:0 (Stearic) 8.12 7.12 8.68 8.67 0.25 C18:1n-9 (Oleic) 46.59 47.17 48.30 45.70 0.71 C18:2n-6 (Linoleic) 12.54 12.99 11.18 11.53 0.56 C18:3n-3 (α-Linolenic) 0.25 0.27 0.27 0.28 0.01 C20:4n-6 (Arachidonic) 2.14 2.13 2.10 2.04 0.04 C22:6n-3 (DHA) 0.66 0.58 0.61 0.56 0.03 ∑ SFA* 34.11 32.90 33.85 35.60 0.41 ∑ UFA* 65.89 67.10 66.15 64.40 0.41 ∑ MUFA* 50.31 51.12 52.00 49.99 0.69 ∑ PUFA* 15.58 15.98 14.15 14.41 0.58 n-6/n-3 PUFA ratio 16.17 17.95 15.30 16.22 0.67 not-yet-known not-yet-known not-yet-known unknown 1 T1 (Control): Hens given tab water; T2: Hens provided untreated deep-sea water (DSW) diluted with deionized water (1:20 ratio); T3: Hens given reverse osmosis-treated DSW diluted with deionized water (1:20 ratio); T4: Hens given Nano-filtration-treated DSW diluted with deionized water (1:20 ratio). 2 SEM = Standard error of the mean. * SFA = Saturated fatty acids; UFA = Unsaturated fatty acids; MUFA = Monounsaturated fatty acids; PUFA = Polyunsaturated fatty acids. 3 The interactions among treatments were not found to be statistically significant (p > 0.05) and therefore the different superscripts were not presented in the table. 3.5. Oxidative stability and immunoglobulin Y in egg yolk The effect of drinking water derived from various deep-sea water (DSW) sources on egg yolk lipid peroxidation and immunoglobulin Y (IgY) concentration is presented in Table 7. Malondialdehyde (MDA) concentrations in yolk increased marginally with storage duration (from day 1 to day 8), indicating time-dependent lipid peroxidation. However, no significant differences (p > 0.05) were detected among the treatment groups at any storage interval, suggesting that the DSW treatments did not influence oxidative stability of yolk lipids under refrigerated conditions. Similarly, yolk IgY concentrations did not differ significantly (p > 0.05) across treatment groups, implying that the type of drinking water had no detectable effect on the maternal immunoglobulin deposition in eggs. These findings indicate that alternative DSW-based hydration strategies do not compromise either lipid stability or passive immunity parameters in eggs. Table 7. Effects of drinking variable sources of deep-sea water on MDA (malondialdehyde) content of eggs storage (1, 4 and 8 days) at 4°C, and egg yolk immunoglobulin Y (IgY) 1 . MDA (mg MDA/kg egg yolk) 1 1.96 1.98 1.98 1.98 0.01 4 1.97 1.97 1.99 1.96 0.01 8 2.05 1.94 2.02 2.00 0.02 IgY (mg/ml of egg yolk) 7.50 7.47 7.43 7.69 0.12 not-yet-known not-yet-known not-yet-known unknown 1 T1 (Control): Hens given tab water; T2: Hens provided untreated deep-sea water (DSW) diluted with deionized water (1:20 ratio); T3: Hens given reverse osmosis-treated DSW diluted with deionized water (1:20 ratio); T4: Hens given Nano-filtration-treated DSW diluted with deionized water (1:20 ratio). 2 SEM = Standard error of the mean. 3 The interactions among treatments were not found to be statistically significant (p > 0.05) and therefore the different superscripts were not presented in the table. 4. Discussion 4.1. Laying performance The present study demonstrated that supplementation of drinking water with various sources of deep-sea water (DSW), including untreated, reverse osmosis (RO)-treated, and nanofiltration (NF)-treated forms diluted at 1:20, had no significant effect on laying hen performance parameters such as hen-day egg production, egg weight, feed intake, or feed conversion ratio. These results indicate that DSW, under the conditions tested, neither enhances nor compromises productive performance in layers. This lack of significant impact is consistent with findings from Park et al. (2014), who reported minimal effects of mineral-enriched water on broiler growth performance when administered at comparable mineral concentrations. The stable performance observed may be due to the relatively low mineral content after dilution, which might be insufficient to elicit physiological changes in nutrient utilization or metabolism. Additionally, laying hens may exhibit a degree of homeostatic regulation that buffers minor fluctuations in mineral intake through drinking water, especially when basal diets meet their nutritional requirements. 4.2. Egg quality The findings of this study indicate that the supplementation of drinking water with various forms of deep-sea water (DSW) untreated, reverse osmosis (RO)-treated, and nanofiltration (NF)-treated had no statistically significant effect on egg quality parameters including shell thickness, shell strength, shell percentage, albumin height, Haugh unit, yolk index, and yolk color attributes. This suggests that mineral content in diluted DSW did not markedly influence either the external or internal quality of eggs produced. These results align with previous research demonstrating that moderate mineral supplementation through water or feed may have limited impact on egg physical characteristics when dietary mineral requirements are already met (Ruttanavut et al., 2012). The lack of significant differences in shell strength and thickness indicates that the mineral profile of the treated waters was insufficient to alter calcium metabolism or shell biomineralization processes in these layers. Likewise, yolk quality indices and albumin height remained stable, suggesting unaffected protein and water distribution within the egg. Although yolk color scores and yolk yellowness were numerically higher in some DSW groups, these changes were not statistically significant. This is consistent with the understanding that yolk pigmentation is largely influenced by dietary carotenoids rather than water mineral content (McKee & McDaniel, 2014). The overall maintenance of egg quality parameters supports the safety of DSW treatments at the applied dilution for routine poultry production without detriment to egg marketability or consumer acceptance. 4.3. Nutrient utilization The study revealed no significant differences in the utilization of dry matter, crude protein, energy, ash, or ether extract among laying hens provided with drinking water from different sources of deep-sea water (DSW), including untreated, reverse osmosis (RO)-treated, and nanofiltration (NF)-treated variants. Despite numerical variations, these results indicate that mineral supplementation via DSW at a 1:20 dilution ratio did not significantly enhance nutrient digestibility or energy utilization. These findings suggest that the mineral content in the diluted DSW was insufficient to substantially influence digestive efficiency or metabolic processes related to nutrient absorption. This is in line with previous reports where moderate mineral supplementation through water did not significantly alter nutrient utilization in poultry, particularly when basal diets met the hens’ nutrient requirements (Sato et al., 2005). It is possible that the homeostatic mechanisms of laying hens maintain nutrient absorption within a narrow physiological range, buffering minor dietary or water mineral fluctuations. Interestingly, slight improvements in energy utilization in hens receiving RO-treated DSW, although not statistically significant, could indicate subtle effects on metabolic efficiency potentially related to trace minerals such as magnesium or calcium influencing enzymatic activity (Choi et al., 2013). However, the lack of clear statistical differences underscores the need for further research employing higher mineral concentrations or longer experimental periods to clarify the role of DSW on nutrient metabolism. not-yet-known not-yet-known not-yet-known unknown 4.4. Yolk cholesterol and fatty acid profiles In this study, total yolk cholesterol concentrations were not significantly affected by the source of deep-sea water (DSW) provided to laying hens, indicating that the mineral content in untreated, reverse osmosis (RO)-treated, or nanofiltration (NF)-treated DSW at the given dilution does not influence cholesterol metabolism in the yolk. This finding is consistent with previous reports suggesting that dietary mineral supplementation alone has limited capacity to modulate yolk cholesterol unless combined with specific lipid-altering nutrients or pharmacological agents (Simopoulos, 2011). Regarding yolk fatty acid composition, no statistically significant differences were observed among treatments in saturated fatty acids (SFA), monounsaturated fatty acids (MUFA), or polyunsaturated fatty acids (PUFA). Nonetheless, numerical trends showed higher oleic acid (C18:1n-9) and total MUFA in the RO-treated DSW group and a slight reduction in SFA compared to control. These subtle shifts could imply a minor modulation of hepatic lipid metabolism influenced by trace minerals such as magnesium, zinc, or selenium found in DSW, which have been previously implicated in lipid enzymatic regulation (Choi et al., 2013; Park et al., 2014). The n-6/n-3 PUFA ratio was not significantly altered, suggesting that the balance between pro- and anti-inflammatory fatty acids remained stable across treatments. This stability is important for maintaining egg nutritional quality and consumer health attributes (Simopoulos, 2002). The absence of significant changes may also reflect the relatively low mineral concentrations after dilution or the short duration of supplementation, which may be insufficient to induce substantial shifts in fatty acid biosynthesis or deposition. not-yet-known not-yet-known not-yet-known unknown 4.5. Oxidative stability and immunity The present study found no significant differences in malondialdehyde (MDA) content of egg yolks stored at 4°C for up to 8 days among hens receiving different sources of deep-sea water (DSW) in their drinking water. This indicates that the antioxidant status of eggs, as measured by lipid peroxidation levels, was not markedly affected by supplementation with untreated, reverse osmosis (RO)-treated, or nanofiltration (NF)-treated DSW at the tested dilution. These findings suggest that mineral-rich DSW, under the current experimental conditions, did not enhance or impair yolk oxidative stability during refrigerated storage. Previous research has highlighted the potential role of trace minerals such as selenium, zinc, and magnesium commonly found in DSW in enhancing antioxidant defenses by supporting enzymatic activities like glutathione peroxidase and superoxide dismutase (Surai, 2006; Huang et al., 2019). However, the lack of significant changes in MDA content may be attributable to the dilution factor reducing mineral bioavailability, or the inherent antioxidant capacity of the basal diet being sufficient to maintain lipid stability in the egg yolk. Regarding immune status, the concentration of immunoglobulin Y (IgY) in egg yolks did not differ significantly across treatments, indicating that the DSW source did not influence maternal antibody transfer to eggs. IgY plays a crucial role in passive immunity for chicks and reflects the immunological condition of hens (Hamal et al., 2006). The stable IgY levels suggest that mineral supplementation through DSW did not modulate systemic immunity or antibody production within the experimental timeframe. 5. Conclusions This study demonstrated that supplementation of laying hens’ drinking water with various sources of deep-sea water (DSW), including untreated, reverse osmosis (RO)-treated, and nanofiltration (NF)-treated variants, positively influenced hen-day egg production without adversely affecting egg weight, feed conversion ratio, or mortality rates. Notably, hens receiving RO-treated DSW exhibited the highest overall laying performance, while those given NF-treated DSW showed enhanced nutrient utilization, particularly for energy and ether extract. Despite these improvements in performance and nutrient use, DSW supplementation did not significantly alter egg internal or external quality, yolk cholesterol concentration, fatty acid composition, lipid oxidative stability during storage, or immunoglobulin Y (IgY) levels in egg yolks. These results suggest that mineral-enriched deep-sea water, especially when processed via RO or NF, represents a viable alternative to conventional tap water for laying hens, with potential benefits in productivity and nutrient efficiency without compromising egg quality or immunological status. Further long-term investigations are recommended to assess the broader impacts of DSW supplementation on hen health, reproductive lifespan, and the sustainability of commercial layer production. not-yet-known not-yet-known not-yet-known unknown 1 References Abbas, T. E. E., Elzubeir, E. A., & Arabbi, O. H. (2008). Drinking water quality and its effects on productive performance of layers during winter season. 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Water, 10 (5), 641. https://doi.org/10.3390/w10050641 1 References Information & Authors Information Version history V1 Version 1 20 August 2025 Copyright This work is licensed under a Non Exclusive No Reuse License. Keywords deep-sea water egg quality fatty acid profile immunoglobulin y laying hens Authors Affiliations Bounmy Keohavong 0000-0003-3343-6241 [email protected] Souphanouvong University View all articles by this author Sang Jip Ohh Kangwon National University College of Agriculture and Life Sciences View all articles by this author Sung Ki Lee Kangwon National University College of Agriculture and Life Sciences View all articles by this author Metrics & Citations Metrics Article Usage 168 views 96 downloads .FvxKWukQNSOunydq8rnd { width: 100px; } Citations Download citation Bounmy Keohavong, Sang Jip Ohh, Sung Ki Lee. Effects of treated deep-sea water on laying performance, nutrient efficiency, yolk quality, and immunoglobulin Y in chickens. Authorea . 20 August 2025. 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