45 2, (2018) Korean J. Poult. Sci. Vol.45, No.2, (2018) Effects of Dietary Organi

45 2, 97 107 (2018) Korean J. Poult. Sci. Vol.45, No.2, 97 107 (2018) https://doi.org/10.5536/kjps.2018.45.2.97 97 1 2 3 4 1 Effects of Dietary Organic Sulfur on Performance, Egg Quality and Cell-mediated Immune Response of Laying Hens Chun Ik Lim 1, Ho Seong Choe 2, Changwon Kang 3, Byoung Keon Lee 4 and Kyeong Seon Ryu 1 1 Department of Animal Science, Chonbuk National University, Jeonju 54896, Republic of Korea 2 Department of Animal Biotechnology, Chonbuk National University, Jeonju 54896, Republic of Korea 3 College of Veterinary Medicine, Chonbuk National University, Iksan 14058, Republic of Korea 4 Natural Eng Inc., Anyang 14058, Republic of Korea ABSTRACT This study aimed to evaluate the dietary effect of organic sulfur (OS) supplementation on performance, egg quality and serum constituents in laying hens. A total of 360 Lohmann brown laying hens at the age of 31 weeks were distributed into four treatments having five replicates of 18 hens each until 54 weeks. The hens were fed four levels (0.0, 0.1, 0.2 and 0.4%) of OS with basal diet. The number of eggs was investigated daily, and egg quality was confirmed every 8 weeks. Sulfur content in eggs, interleukin 2 (IL-2), T help cells (CD4+) and cytotoxicity cells (CD8+) were measured at the termination of the experiment. The result of the study showed that egg production tended to increase with 0.4% OS in diet after 39 weeks of age and, there was a significant effect (P<0.05) from 47 to 54 weeks of age. Egg quality traits of albumen height and haugh unit increased significantly (P<0.05) owing to the addition of OS to the diet. The polyunsaturated fatty acids in yolk were gradually increased while saturated fatty acids were decreased with increasing levels in OS (P<0.05). Total sulfur concentration in the eggs increased significantly (P<0.05) in treatments fed OS. Moreover, albumin, AST and HDL cholesterol levels in serum improved significantly (P<0.05) owing to the addition of OS. The IL-2 concentration and the ratio of CD4+ and CD8+ in blood were generally higher (P<0.05) at 0.4% OS. Therefore, it can be recommended that supplementary OS diet affected the performance, egg quality and stimulated immune response in laying hens. (Key words: egg quality, immune response, laying hens, organic sulfur, performance) methyl sulfonyl methane(msm),,., (Karabay et al., 2014),, (Van der Merwe and Bloomer, 2016)., (Lee et al., 2009; Karen, 2017), (Cho et al., 2005). (Karabay et al., 2014), (Notarnicola et al., 2016). (S),, To whom correspondence should be addressed : seon@jbnu.ac.kr

98 :, (Matin et al., 2013). 0.2%, (Shin et al., 2013), 0.03% (Hwang et al., 2017). 0.1%,, (Park et al., 2010).,. (Jacob, 1983),. 31 54,,. 31 0%, 0.1%, 0.2% 0.4%, 4 5 18 (2 9 ) 360 24. 31 40 2,800 kcal/kg, 17%, 41 54 2,750 kcal/kg, 16% (Table 1). 3 ( / : 70 cm 40 cm 50 cm /6 ),, 18 26 16., 8. Table 1. Basal diet composition Ingredients (%) 31 40 weeks 41 54 weeks Corn 65.14 66.08 Soybean meal 17.93 21.13 Corn gluten meal 5.78 1.74 Limestone 9.42 9.44 Calcium phosphate 0.92 0.87 Salt 0.38 0.37 L-lysine 0.07 - DL-methionine 0.03 0.04 Vitamin premix 1 0.18 0.18 Mineral premix 2 0.15 0.15 Chemical composition Total 100 ME (kcal/kg) 2,800 2,750 CP (%) 17 16 Lysine (%) 0.78 0.74 Methionine (%) 0.34 0.32 Calcium (%) 3.80 3.80 Avail. phosphorus (%) 0.32 0.32 1 Contains per kg: vit. A, 5,500 IU; vit D 3 1, 100 ICU; vit E, 11 mg; vit B 12, 0.0066 mg; vit K 3, 1.1 mg; riboflavin, 4.4 mg; pantothenic acid, 11 mg (calcium pantothenate: 1.96 mg); choline, 190.96 mg; folic acid, 0.55 mg; pyridoxine, 2.2 mg; biotin, 0.11 mg; thiamine, 2.2 mg; ethoxyquin, 125 mg. 2 Contains per kg: Cu, 10 mg; Fe, 60 mg; I, 0.46 mg; Mn, 120 mg; Zn, 100 mg., 1., 30 8. (QCM+; TSS, UK), (QC-SPA; TSS, UK), (FHK, Japan). 42 54 10

Lim et al. : Effects of Dietary Organic Sulfur on Performance, Egg Quality and Cell-mediated Immune Response of Laying Hens 99. 0.5 g methanol benzen 4:1 acetyl chloride 200 μl 100 1. 6% potassium carbonate hexane 2 ml, (3,000 rpm, 4, 15 ) hexane., split ratio 30:1 GC-MSD (6890N-5973, Agilent, USA). 4. 660 2, (HCl:H 2 O = 1:1) 12., ICP (Perkin-Elmer ICP-OES 2000DV, USA). 42 54 10 (3,000 rpm, 4, 15 ), (Automatic Biochemical Analyser, Thermo Scientitic, Konelab 20, Finland), AST,,,, HDL. Table 2. Primer used for the quantitative real-time PCR Cytokines IL-2 Forward Reverse Primer sequence 5 -GCTAATGACTACAGCTTAT GGAGCA-3 5 -TGGGTCTCAGTTGGTGTGTAGAG-3 chicken CD4+(abcom, Massachusetts, USA), CD3+ phycoerythrin-congugated mouse anti-chicken CD3+(abcom, Massachusetts, USA) CD8+ Cy5-congugated mouse antichicken CD8+(abcom, Massachusetts, USA), PBMC 1 10 6 cell/tube 4 30. (BD Accuri C6, MI, USA), FSC/SSC %. SAS(Statistical Analysis System, 9.2 Version, Cary, NC, 2002) GLM(general linear model) procedure, Duncan(1955) 0.05. IL-2 mrna 5 Table 2 IL-2 primer RT-PCR. Ribo-Ex(USA) mrna qpcr RT kit(revertra Ace, Japan) cdna. cdna primer Real time PCR(AB applied bio system, CA, USA) chicken GAPDH IL-2, Relative Quantification (RQ). IL-2 5 IL-2 ELISA (My Bio Source, MBS2508525, USA). 9 Ficoll PBMC(peripheral blood mononuclear cell) (Brunetti et al., 1995), CD3+, CD4+ CD8+. CD4+ FITC-conjugated mouse anti-,,, 1 Table 3. 31 38, 39, 47 54 0.4% 88.85% 85.72% (P<0.05)., 1. 0.2% (Shin et al., 2013; Jiao et al., 2017), 35 40 0.1%, (Park et al., 2010). 29 40 0.15% (Kim et al., 2013). 31 38, 39 54

100 :, Table 3. Effect of dietary organic sulfur on performance in laying hens Organic sulfur (%) Egg production (%) Feed intake (g) Egg weight (g) Daily egg mass (g) Feed conversion 31 to 38 weeks 0 94.13 112.58 62.28 58.61 1.922 0.1 93.95 112.80 62.13 58.38 1.935 0.2 94.31 112.30 62.19 58.64 1.916 0.4 95.67 111.52 61.39 58.72 1.900 SEM 0.50 0.30 0.15 0.29 0.012 P-value 0.63 0.48 0.13 0.98 0.790 39 to 46 weeks 0 90.61 112.92 63.07 57.16 1.976 0.1 90.83 114.13 63.05 57.28 1.996 0.2 91.67 112.65 63.00 57.76 1.951 0.4 93.52 113.00 62.48 58.43 1.934 SEM 0.45 0.43 0.18 0.28 0.012 P-value 0.08 0.67 0.66 0.38 0.300 47 to 54 weeks 0 85.72 b 102.64 60.69 52.03 1.974 0.1 86.55 b 104.74 61.26 53.03 1.976 0.2 87.57 ab 102.90 60.98 53.42 1.928 0.4 88.85 a 102.28 60.63 53.88 1.900 SEM 0.41 0.51 0.21 0.33 0.012 P-value 0.02 0.34 0.73 0.23 0.060 a,b Value with the different letters in the row are significantly different at 5% level.. (Jacob, 1983),., 38, 39 54, 39 0.4%.,, Table 4. (P<0.05), 96.93 97.23 3.49 (P<0.05). 38 4.37 4.81 kg/cm 2 4.12 kg/cm 2, 46 54.. Park et al.(2010) 0.1%, Ryu(2006)

Lim et al. : Effects of Dietary Organic Sulfur on Performance, Egg Quality and Cell-mediated Immune Response of Laying Hens 101 Table 4. Effect of dietary organic sulfur on egg quality in laying hens Organic sulfur (%) Albumen height (mm) Haugh unit Shell strength (kg/cm 2 ) Shell thickness (mm) 38 weeks 0 9.36 b 95.66 b 4.12 0.378 0.1 9.96 a 97.83 ab 4.81 0.374 0.2 10.02 a 98.44 a 4.37 0.375 0.4 10.08 a 98.76 a 4.57 0.376 SEM 0.09 0.42 0.10 0.003 P-value 0.02 0.03 0.08 0.960 46 weeks 0 8.80 b 92.58 b 4.19 0.352 0.1 9.30 a 95.07 a 4.16 0.352 0.2 9.31 a 95.21 a 3.90 0.345 0.4 9.41 a 95.84 a 4.01 0.362 SEM 0.08 0.41 0.07 0.003 P-value 0.04 0.03 0.45 0.130 54 weeks 0 8.76 b 91.55 b 4.02 0.336 0.1 10.09 a 98.79 a 4.34 0.351 0.2 9.79 a 97.14 a 4.31 0.353 0.4 9.73 a 96.74 a 4.22 0.343 SEM 0.12 0.61 0.10 0.003 P-value <0.01 <0.01 0.64 0.170 a,b Value with the different letters in the row are significantly different at 5% level.., (Karen, 2017),. (Lee et al., 2009),. Table 5. oleic acid (C18:1 n-9), palmitic acid(c16:0), linoleic acid(c18:2 n-6) stearic acid(c18:0). 42 linoleic acid(c18:2 n-6) 0.4% (15.04%) (10.92%) (P<0.05), (PUFA) 0.4% 17.84% 13.91% (P<0.05). 54 palmitic acid 0.4%, linoleic acid 0.2 (P<0.05). (MUFA) 45.16 46.18%, (PUFA) 0.4% (17.67%) (14.95%)

102 :, Table 5. Effect of dietary organic sulfur on yolk fatty acids (%) of laying hens Organic sulfur (%) C14:0 C16:0 C16:1 (n-7) C18:0 C18:1 (n-9) C18:2 (n-6) C18:3 (n-3) 42 weeks C20:1 (n-9) C20:4 (n-6) C22:6 (n-3) MUFA 1 PUFA 2 UFA 3 SFA 4 0 0.43 26.47 3.42 9.63 45.83 10.92 b 0.12 0.32 2.66 0.21 49.57 13.91 b 63.47 36.53 0.1 0.46 27.06 3.95 8.76 45.11 11.48 b 0.12 0.35 2.51 0.21 49.42 14.31 b 63.73 36.27 0.2 0.41 26.23 3.69 9.35 43.53 13.35 ab 0.12 0.35 2.78 0.22 47.55 16.47 ab 64.02 35.98 0.4 0.42 25.89 3.56 8.63 43.29 15.04 a 0.12 0.37 2.46 0.23 47.22 17.84 a 65.06 34.94 SEM 0.01 0.26 0.18 0.20 0.41 0.56 0.01 0.01 0.12 0.01 0.46 0.57 0.31 0.31 P-value 0.63 0.49 0.78 0.26 0.08 0.02 0.99 0.34 0.82 0.94 0.14 0.03 0.30 0.30 54 weeks 0 0.47 27.84 a 4.30 10.58 41.50 11.42 c 0.13 0.36 3.16 0.24 46.15 14.95 b 61.11 b 38.89 a 0.1 0.47 27.47 ab 4.17 10.06 41.59 11.94 bc 0.18 0.20 3.42 0.28 46.18 15.82 b 62.00 ab 38.00 ab 0.2 0.50 26.98 ab 3.67 10.17 41.84 12.99 b 0.14 0.35 3.10 0.25 45.87 16.48 ab 62.35 a 37.65 b 0.4 0.47 26.49 b 3.60 10.21 41.23 14.23 a 0.11 0.33 3.08 0.27 45.16 17.67 a 62.83 a 37.17 b SEM 0.01 0.18 0.15 0.10 0.18 0.30 0.01 0.02 0.11 0.02 0.23 0.32 0.19 0.19 P-value 0.73 0.04 0.26 0.32 0.71 <0.01 0.32 0.35 0.69 0.88 0.36 0.01 0.01 0.01 a c Value with the different letters in the row are significantly different at 5% level. 1 Monounsaturated fatty acid: C16:1+C18:1+C20:1, 2 Polyunsaturated fatty acid: C18:2+C18:3+C20:4+C22:6, 3 Unsaturated fatty acid: MUFA+PUFA, 4 Saturated fatty acid: C14:0+C16:0+C18:0. (P<0.05). (UFA) (P<0.05), (SFA) (P<0.05)., (Li et al., 2015), Hwang et al.(2017), Shin et al.(2013) 0.2% 0.6%.,. Fig. 1., (P<0.05), Fig. 1. Effect of dietary organic sulfur on sulfur concentration in egg. a,b Value with the different letters in the row are significantly different at 5% level. X-axis: organic sulfur addition level in basal diet (0, 0.1, 0.2 and 0.4%); Y-axis: sulfur concentration (%) in egg. 0.4% 0.2975%. (Hwang et al., 2010),

Lim et al. : Effects of Dietary Organic Sulfur on Performance, Egg Quality and Cell-mediated Immune Response of Laying Hens 103 (Hwang et al., 2017). Utterback et al.(2005),.,., AST,,,, HDL Table 6. 42 1.84 1.93 g/dl 1.64 g/dl (P<0.05), 54. AST, 0.2% (P<0.05). 42 5.53 5.64 g/dl 4.71 g/dl (P<0.05), 54., HDL 0.4% 4.7 mg/dl, (P<0.05). AST, AST (So et al., 2009)., AST 155.44 182.15 IU/L (P<0.05). 0.2% AST (Jiao et al., 2017),. HDL,, HDL 31.28% (Shin et al., 2013),, Table 6. Effect of dietary organic sulfur on blood composition in laying hens Organic sulfur (%) ALB 1 (g/dl) AST 2 (IU/L) Glucose (mg/dl) Protein (g/dl) TG 3 (mg/dl) CHOL 4 (mg/dl) HDL 5 (mg/dl) 42 weeks 0 1.64 b 182.15 a 239.91 4.71 b 1,367.33 124.12 4.08 b 0.1 1.93 a 172.24 ab 244.99 5.64 a 1,554.78 127.42 6.45 a 0.2 1.84 a 162.77 b 241.44 5.53 a 1,444.43 126.83 7.55 a 0.4 1.90 a 167.10 b 254.39 5.54 a 1,488.85 131.01 7.64 a SEM 0.04 2.69 3.23 0.13 66.45 4.80 0.48 P-value 0.01 0.04 0.42 0.01 0.82 0.97 0.01 54 weeks 0 1.92 173.89 a 271.69 5.92 1,824.67 158.07 6.49 b 0.1 2.07 164.91 ab 270.72 6.27 2,189.10 157.96 9.27 ab 0.2 2.14 164.96 ab 275.07 6.28 2,074.62 152.13 10.00 a 0.4 2.12 155.44 b 269.40 6.50 2,144.91 161.17 12.33 a SEM 0.03 2.20 2.45 0.12 119.45 5.26 0.67 P-value 0.08 0.02 0.89 0.31 0.74 0.95 0.01 a,b Value with the different letters in the row are significantly different at 5% level. 1 Albumin, 2 Aspartate amino transferase, 3 Triglycerides, 4 Cholesterol, 5 High density lipoprotein cholesterol.

104 :,. IL-2 mrna IL-2 Fig. 2 Fig. 3. IL-2 mrna IL-2 0.4% 2.5 1.5 (P<0.05). Table 7 Fig. 4 CD3+CD4+ 0.4% 64.34% 17% (P<0.05), CD3+CD8+ CD4+/CD8+ Table 7. Effect of dietary organic sulfur on CD4+ and CD8+ in blood of laying hens a c Organic sulfur (%) CD4+ and CD8+ defined subset (%) of CD3+ PBMC CD3+ (%) CD4+ (%) CD8+ (%) CD4+ /CD8+ 0 16.93 43.03 c 27.74 1.58 c 0.1 16.69 47.04 c 26.43 1.95 bc 0.2 14.92 54.13 b 24.54 2.23 b 0.4 17.24 60.34 a 22.60 2.70 a SEM 0.97 1.31 0.93 0.09 P-value 0.85 <0.01 0.23 <0.01 Value with the different letters in the row are significantly different at 5% level. PBMC: Peripheral blood mononuclear cell, CD3+: T cell, CD4+: T help cell, CD8+: Cytotoxicity cell. Fig. 2. Effect of dietary organic sulfur on IL-2 mrna in spleen of laying hens. a c Value with the different letters in the row are significantly different at 5% level. X-axis: organic sulfur addition level in basal diet (0, 0.1, 0.2 and 0.4%); Y-axis: IL-2 mrna level in spleen. Fig. 3. Effect of dietary organic sulfur on IL-2 in blood of laying hens. a,b Value with the different letters in the row are significantly different at 5% level. X-axis: organic sulfur addition level in basal diet (0, 0.1, 0.2 and 0.4%); Y-axis: IL-2 level in blood. 1.58 2.70 (P<0.05). T-lymphocyte CD4+, CD8+, CD4+/CD8+ (Shedlock and Shen, 2003), IL-2 CD4+ (Janeway et al., 1999). T-help IL-2., CD4+/CD8+ (Nishimura et al., 2014), IL-2, CD4+ CD4+/CD8+. (Jiao et al., 2017), 5 100 ml, 78% (DiSilvestro et al., 2008), (Droge and Breitkreutz, 2000)., 0.4% IL- 2 CD4+ CD4+/CD8+.

Lim et al. : Effects of Dietary Organic Sulfur on Performance, Egg Quality and Cell-mediated Immune Response of Laying Hens 105 Fig. 4. Representative photograph of dietary organic sulfur (0, 0.1, 0.2 and 0.4%) effects on percentage of T-lymphocyte CD3+, CD4+ and CD8+ using Flow cytometry. Distribution of CD3+ in the blood (CD3+: M2 level of A, C, E, G). Distribution to percentage of CD4+ and CD8+ of CD3+ in the control group (CD4+: Q2-LR level of B, D, F, H; CD8+: Q2-UL level of B, D, F, H). CD4+ fluorescently stained the FITC. CD8+ and CD3+ stained in the Cy5 and PE respectively. FITC: fluorescein isothiocyanate, PE: phycoerythrin, Cy5: cyanine5. 적 요 본 연구는 산란계 사료에 유기황의 수준별 첨가급여가 산 란계의 생산성, 계란품질 및 세포성 면역능에 미치는 영향 을 구명하고자 실시하였다. 31주령 로만브라운종에 분말 형 태의 유기황을 기초사료에 0%, 0.1%, 0.2%, 0.4% 수준으로 첨가 급여하였으며, 처리구당 5반복 반복당 18수씩 전체 360수를 수용하여 24주간 사양실험을 실시하였다. 산란 수 와 난중은 매일 조사하였고, 계란품질은 8주 간격으로 측정 하였다. 난황지방산과 혈액은 산란계 42와 54주령에 분석하 였으며, 계란의 황 함량, IL-2 및 CD4+와 CD8+은 사양실험 종료 후 분석하였다. 본 연구결과, 31 38주령에 산란율은 처리구 간에 차이가 없었지만, 39주령 이후 유기황의 급여 수준에 따라 대조구와 차이를 보였으며, 47 54주령의 산란 율은 0.4% 급여구에서 대조구에 비해 현저하게 증가하였다 (P<0.05). 계란의 난백높이와 호우유닛은 유기황 급여구에서 대조구에 비하여 유의적으로 개선되었다(P<0.05). 계란의 난황 내 다중불포화지방산은 첨가수준에 따라 현저하게 증 가하였다(P<0.05). 유기황 수준이 증대됨에 따라서 불포화 지방산은 증가하였고, 포화지방산은 감소되었으며, 이러한 현상은 54주령에서 더욱 현저하게 나타났다(P<0.05). 계란 에서 황 함량은 유기황 첨가 수준에 증대함에 따라서 증가 되었다(P<0.05). 또한 혈청 알부민은 42주령에서 유기황 급 여구에서 유의적으로 증가하였고(P<0.05), AST는 감소하였 다(P<0.05). 단백질은 42주령의 산란계에서 유기황 급여에 따라 현저히 증대되었고(P<0.05), HDL 콜레스테롤은 유기 황 첨가수준이 높아짐에 따라 유의적으로 상승하였다 (P<0.05). 비장과 혈중 IL-2 및 CD4+/CD8+도 유기황의 첨

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