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

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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

1 45 2, (2018) Korean J. Poult. Sci. Vol.45, No.2, (2018) 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

2 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 0%, 0.1%, 0.2% 0.4%, (2 9 ) ,800 kcal/kg, 17%, ,750 kcal/kg, 16% (Table 1). 3 ( / : 70 cm 40 cm 50 cm /6 ),, , 8. Table 1. Basal diet composition Ingredients (%) weeks weeks Corn Soybean meal Corn gluten meal Limestone Calcium phosphate Salt L-lysine DL-methionine Vitamin premix Mineral premix Chemical composition Total 100 ME (kcal/kg) 2,800 2,750 CP (%) Lysine (%) Methionine (%) Calcium (%) Avail. phosphorus (%) Contains per kg: vit. A, 5,500 IU; vit D 3 1, 100 ICU; vit E, 11 mg; vit B 12, mg; vit K 3, 1.1 mg; riboflavin, 4.4 mg; pantothenic acid, 11 mg (calcium pantothenate: 1.96 mg); choline, 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., (QCM+; TSS, UK), (QC-SPA; TSS, UK), (FHK, Japan)

3 Lim et al. : Effects of Dietary Organic Sulfur on Performance, Egg Quality and Cell-mediated Immune Response of Laying Hens g methanol benzen 4:1 acetyl chloride 200 μl % potassium carbonate hexane 2 ml, (3,000 rpm, 4, 15 ) hexane., split ratio 30:1 GC-MSD (6890N-5973, Agilent, USA) , (HCl:H 2 O = 1:1) 12., ICP (Perkin-Elmer ICP-OES 2000DV, USA) (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 cell/tube (BD Accuri C6, MI, USA), FSC/SSC %. SAS(Statistical Analysis System, 9.2 Version, Cary, NC, 2002) GLM(general linear model) procedure, Duncan(1955) 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, MBS , USA). 9 Ficoll PBMC(peripheral blood mononuclear cell) (Brunetti et al., 1995), CD3+, CD4+ CD8+. CD4+ FITC-conjugated mouse anti-,,, 1 Table , 39, % 88.85% 85.72% (P<0.05)., % (Shin et al., 2013; Jiao et al., 2017), %, (Park et al., 2010) % (Kim et al., 2013) , 39 54

4 100 :, 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 SEM P-value to 46 weeks SEM P-value to 54 weeks b b ab a SEM P-value a,b Value with the different letters in the row are significantly different at 5% level.. (Jacob, 1983),., 38, 39 54, %.,, Table 4. (P<0.05), (P<0.05) kg/cm kg/cm 2, Park et al.(2010) 0.1%, Ryu(2006)

5 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 b b a ab a a a a SEM P-value weeks b b a a a a a a SEM P-value weeks b b a a a a a a SEM P-value <0.01 < 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) %, (PUFA) 0.4% (17.67%) (14.95%)

6 102 :, 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 b b b b ab ab a a SEM P-value weeks a c b b a ab bc b ab ab ab b ab a b b a a a b SEM P-value < 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% %. (Hwang et al., 2010),

7 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 g/dl 1.64 g/dl (P<0.05), 54. AST, 0.2% (P<0.05) 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 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 b a b 1, b a ab a 1, a a b a 1, a a b a 1, a SEM P-value weeks a , b ab , ab ab , a b , a SEM P-value 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.

8 104 :,. IL-2 mrna IL-2 Fig. 2 Fig. 3. IL-2 mrna IL-2 0.4% (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+ /CD c c c bc b b a a SEM P-value 0.85 < <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 (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), ml, 78% (DiSilvestro et al., 2008), (Droge and Breitkreutz, 2000)., 0.4% IL- 2 CD4+ CD4+/CD8+.

9 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

10 106 :, (P<0.05). 0.4%,. ( :,,,, ) Brunetti M, Martelli N, Colasante A, Piantelli M, Musiani P, Aiello FB 1995 Spontaneous and glucocorticoid-induced apoptosis in human mature T lymphocytes. Blood 86: Cho JH, Min BJ, Kwon OS, Shon KS, Jin YG, Kim HJ, Kim IH 2005 Effects of MSM (Methyl Sulfonyl Methane) supplementation on growth performance and digestibility of CA and N in pigs. J Korean Soc of Food Sci and Nutr 34: DiSilvestro RA, DiSilvestro DJ, DiSilvestro DJ 2008 Methylsulfonylmethane (MSM) intake in mice produces elevated liver glutathione and partially protects against carbon tetrachloride-induced liver injury. The FASEB J 22: Droge W, Breitkreutz R 2000 Glutathione and immune function. Proc Nutr Soc 59: Duncan DB 1955 Multiple range and multiple F tests. Biometrics 11:1-42. Hwang JW, Cheong SH, Kim YS, Lee JW, You BI, Moon SH, Jeon BT, Park PJ 2017 Effects of dietary supplementation of oriental herbal medicine residue and methyl sulfonyl methane on the growth performance and meat quality of ducks. Anim Production Sci 57: Hwang YH, Jung HJ, Lee Y, Hwang BS 2010 Effect of MSM supplementation on growth performance in duck. Pages In: Proceeding of Korean Society of Poultry Science Conference. Jacob SW 1983 The current status of MSM in medicine. Am Acad Med Prev. Janeway CA, Traver P, Walport M, Shlomchik MJ 1999 Immunobiology: The Immune System in Health and Disease. Pages In: Fourth Education Elsevier/Garland, London. Jiao Y, Park JH, Kim YM, Kim IH 2017 Effects of dietary methyl sulfonyl methane (MSM) supplementation on growth performance, nutrient digestibility, meat quality, excreta microbiota, excreta gas emission, and blood profiles in broilers. Poult Sci 96: Karabay AZ, Aktan F, Sunguroglu A, Buyukbingol Z 2014 Methylsulfonylmethane modulates apoptosis of LPS/IFN-γactivated RAW macrophage-like cells by targeting p53, Bax, Bcl-2, cytochrome c and PARP proteins. Immunopharmacol and Immunotoxicol 36: Karen C 2017 List of foods high in sulfur. Livestrong.com, Kim KW, Yoon BS, No H, Ryu KS 2013 Effects of dietary Lactobacillus, blood meal, sulfur and total mixture supplementation on performance of broiler and laying hens. Pages In: Proceeding of Korean Society of Poultry Science Conference. Lee JI, Min HK, Lee JW, Jeong JD, Ha YJ, Kwack SC, Park JS 2009 Changes in the quality of loin from pigs supplemented with dietary methyl sulfonyl methane during cold storage. Korean J Food Sci Anim Res 29: Li Y, Hruby A, Bernstein AM, Ley SH, Wang DD, Chiuve SE, Hu FB 2015 Saturated fats compared with unsaturated fats and sources of carbohydrates in relation to risk of coronary heart disease: A prospective cohort study. J American College of Cardiol 66: Matin HRH, Dashtbin F, Salari J 2013 Absorption and macromineral interactions in broiler production. Global Vet 11: Nishimura R, Sato K, Miura Y, Jimbo T, Fukushima D, Yamaya H, Ohuchi N 2014 Serum albumin and neutrophils in patients with end-stage liver disease: Relationships with ATP levels in CD4-positive T cells. Page 714 In: Proceeding of the World Transplant Congress, The Transplantation Society and the American Society of Transplantation, America. Notarnicola A, Maccagnano G, Moretti L, Pesce V, Tafuri S, Fiore A, Moretti B 2016 Methylsulfonylmethane and boswellic acids versus glucosamine sulfate in the treatment of knee arthritis: Randomized trial. Inter J Immunopathol and Pharmacol 29: Park SM, Ahn IS, Hong SM, Kim DS, Kwon DY, Yang HJ 2010 The effects of the supplementation of opuntia

11 Lim et al. : Effects of Dietary Organic Sulfur on Performance, Egg Quality and Cell-mediated Immune Response of Laying Hens 107 humifusa water extracts and methyl sulfonyl methane on the laying productivity, egg quality and sensory characteristics. J Korean Soc Food Sci Nutr 39: Ryu MH 2006 Effects of dietary methylsulfontlmethane (MSM) on performance and egg quality in laying hens. MS. Dissertation. Pages in Konkuk Univ. SAS 2002 SAS/STAT Software for PC. SAS Institute Cary NC USA. Shedlock DJ, Shen H 2003 Requirement for CD4 T cell help in generating functional CD8 T cell memory. Sci 300: Shin JS, Kim MA, Lee SH 2013 Comparison of physiological changes in broiler chicken fed with dietary processed sulfur. Korean J Food Preserv 20: So HH, Jeon EO, Byun SH, Mo IP 2009 Early diagnosis of fatty liver-hemorrhagic syndrome using blood biochemistry in commercial layers. Korean J Poult Sci 36: Utterback PL, Parsons CM, Yoon I, Butler J 2005 Effect of supplementing selenium yeast in diets of laying hens on egg selenium content. Poult Sci 84: Van der Merwe M., Bloomer RJ 2016 The influence of methylsulfonylmethane on inflammation-associated cytokine release before and following strenuous exercise. J Sports Med 2016:1-9. Received May 15, 2018, Revised Jun. 25, 2018, Accepted Jun. 26, 2018

44 4, (2017) Korean J. Poult. Sci. Vol.44, No.4, (2017) Effects of Dietary Si

44 4, (2017) Korean J. Poult. Sci. Vol.44, No.4, (2017) Effects of Dietary Si 44 4, 267 274 (2017) Korean J. Poult. Sci. Vol.44, No.4, 267 274 (2017) https://doi.org/10.5536/kjps.2017.44.4.267 267 1 1 2 3 1 Effects of Dietary Silicate Based Complex Mineral on Performance, Egg Quality