ผลของอาหารแห้งเสริมของไคโตซาน humoral และเซลล์อักเสบในหย่านมถึงทรูดแบบ b s t r c tผลของอาหารแห้งเสริมของไคโตซานการ humoral และเซลล์อักเสบในการหย่านมถึงทรูดถูกสอบสวน หนึ่งร้อย และเอ้ทรูดหย่านมถึงที่ 28 d(Duroc×สีขาวขนาดใหญ่×แม่) ถูกกำหนดแบบสุ่มให้นวดอาหาร 5การทำซ้ำ 6 ในแต่ละทรีทเม้นต์ ทรูดในการรักษา 5 ถูกเลี้ยงในที่โรคอาหารเสริม ด้วย 0 (ควบคุม), 100, 500, 1000 และ 2000 mg ไคโตซานกิโลกรัมอาหาร ผลลัพธ์พบว่าไคโตซานขึ้นเซรั่มความเข้มข้น G (IgG) immunoglobulin ของทรูดในลักษณะขึ้นอยู่กับปริมาณกำลังสอง (P < 0.05), และเพิ่มเซรั่มเฉพาะ ovalbumin(OVA) IgG เนื้อหาในแบบเชิงเส้นหรือลักษณะขึ้นอยู่กับปริมาณกำลังสอง (P < 0.05) ในวันที่ 28ในขณะที่เซรั่ม immunoglobulin (อิกะ) และ immunoglobulin M (ระบาดของโรค) ความเข้มข้นไม่เปลี่ยนแปลงไป ด้วยการเพิ่มไคโตซาน immunoglobulin secretory (sIgA) ถูกปรับปรุงในพื้นผิว mucosal ileum ในลักษณะเชิงเส้น หรือกำลังสอง (P < 0.05) ในวันที่ 14 และปรับปรุง quadratically ในพื้นผิว mucosal jejunum ใน 28 วัน (P < 0.05) นอกจากนี้ ไคโตซานเซรั่มความเข้มข้นของ CD4 ละลายลดลง (sCD4) ในการกำลังสองยาขึ้นอยู่กับลักษณะ (P < 0.05) และ CD8 ละลาย (sCD8) ในลักษณะขึ้นอยู่กับยาเส้น หรือกำลังสอง(P < 0.05) ในวันที่ 28 ไคโตซาน quadratically เพิ่มความเข้มข้นของซีรั่มของ interleukin-1(IL-1) and interleukin-2 (IL-2) on day 14 as well as serum concentrations of tumor necrosisfactor-alpha (TNF-) on day 28 (P<0.05). These results implied that dietary supplementwith chitosan improved humoral and cellular immune responses of weaned piglets in adose-dependent manner, and in this experiment, the appropriate adding dose of chitosanmight be between 500 and 1000 mg/kg.Keywords: Cellular immunity, Chitosan, Humoral immunity, Weaned Piglets1. IntroductionThe immune system of piglet is underdeveloped fully at early age (Heugten et al., 1996), and the ability to resist diseasemainly depends on passive immunity from the sow during this time (Rooke and Bland, 2002). Early weaning not onlyinterrupts the supply of immunologically important factors from sow’s milk (Wu et al., 2004), but also impairs the productionof antibodies and compromises cellular immune functions (Touchette et al., 2002), which leads piglets to be infected moreeasily by pathogens. Traditionally, antibiotics were frequently used in the diets of newly weaned pigs for the prophylaxis ofinfections during the immediate post-weaning period in past decades (Bosi et al., 2011). However, there has been increasingTable 1 Composition and nutrient levels of the basal diet (air dry basis, %).The premix provides following nutrients per kg diet: Vitamin A, 16,000 IU; Vitamin D3, 2500 IU; Vitamin E, 60 IU; Vitamin K3, 4.5 mg; Vitamin B1,2.6 mg; Vitamin B2, 8.7 mg; Vitamin B6, 7.0 mg; Vitamin B12, 0.03 mg; vitamin C, 200 mg; Pantothenic acid, 13 mg; Nicotinic acid, 35 mg; Biotin, 0.47 mg;Folic acid, 0.85 mg; Iron, 155 mg; Copper, 35 mg; Zinc, 100 mg; Manganese, 25 mg; Iodine, 0.35 mg; Cobalt, 0.2 mg; Selenium, 0.25 mg; Choline chloride,750 mg; Phytase, 500 FTU.pressure on the livestock industry to decrease or discontinue these additions because of the potential development ofantibiotic resistance (Davis et al., 2004). Therefore, alternative additives that help develop the immune responses of weanedpiglets are highly recommended.Chitosan, a natural and nontoxic alkaline polysaccharide, is formed by the action of chitin deacetylases and is a keystructural component of helminths, arthropods and fungi (Synowiecki and Al-Khateeb, 2003). Porporatto et al. (2005) demon-strated that chitosan profoundly affected intestinal mucosal immunity by activating leukocytes. Our previous study foundthat chitosan improved the humoral and cellular immune functions in broilers (Li, 2009).In piglets, however, there were limited studies evaluating the effect of chitosan on immune function. Therefore, our studywas conducted to determine the effect of chitosan on the humoral and cellular immune function of weaned piglets and theappropriate chitosan supplemental level as an immuno-modulating agent.2. Materials and methodsAll procedures described in this experiment were approved by Animal Care and Use Committee of Inner MongoliaAgricultural University.2.1. Experimental design and animal managementA total of 180 piglets (Duroc × Large white × Landrace) with an initial average body weight of 7.6 kg were assignedrandomly to 5 treatments with 6 repetitions (3 pens of males and 3 pens of females) in each treatment, with 6 piglets in eachpen (4.0 m × 4.2 m). The formation of basal diets was showed in Table 1. All diets were offered in meal form. Five dietarytreatments supplemented with 0 (control), 100, 500, 1000 or 2000 mg chitosan/kg feed on the basal diet, respectively. Pigletswere weaned at the age of 28 d, penned in a temperature-controlled nursery building where temperature was maintained at26–27◦C and relative humidity was about 65–70%. The weaned piglets had one week of housing and management adaptationbefore the experimental phase. The experimental period was 28 d. Feed and water were freely available. Chitosan used inthis trial was provided by Jinan Haidebei Marine Bioengineering Limited Company (Jinan, China). The deacetylation degreeof chitosan was determined to be 85.09%, and the viscosity was 45 cps.2.2. Sample collectionOn day 14 and 28, one pig from each replicate of each treatment was randomly selected and blood samples were obtainedby puncturing the vena cava. The blood samples were centrifuged at 3000 × g for 20 min at 4◦C to yield serum. Serum wasstored at −20◦C until analysis of immunoglobulins, cytokines and sCD4, sCD8. At the beginning of trial, one piglet fromeach repetition of each treatment was selected randomly and injected with 1 mg ovalbumin/kg BW (Sigma, USA). Bloodsamples were collected by puncturing the vena cava on day 0 (before injection), 14 and 28 to test the specific OVA antibodyconcentrations in serum. The blood samples were centrifuged at 3000 × g for 20 min and stored at −20◦C until analysis.For determining the content of sIgA in small intestine mucosa, the pigs used to get blood samples were sacrificed, andthe jejunum and ileum were quickly removed, and then the samples were cut and washed with PBS (pH 7.2–7.4). Intestinalmucosa was gently scraped with slide, weighed 1 g and transferred to a centrifuge tube adding 9 mL saline then homogenizedby hand. Homogenates were centrifuged at 3000 × g for 20 min, and the supernatant was stored at −20◦C until analysis.Table 2 Effects of chitosan on the level of immunoglobulins and OVA-IgG in serum as well as the sIgA of jejunum and ileum mucosal in weaned piglet.Note: IgG = immunoglobulin G; IgM = immunoglobulin M; IgA = immunoglobulin A; OVA-IgG = ovalbumin IgG; SEM = standard error of the mean.2.3. Detection of immunoglobulins, cytokines, sCD4 and sCD8 in serumThe IgG, IgA and IgM were determined with a porcine ELISA kit (Bethyl Laboratories, Inc., USA) and the minimumdetectability was 15.6 mg/L. The IL-1, IL-2 and TNF- were measured with a commercially available porcine ELISA kit fromthe BioSource International, Inc. (Camarillo, USA). The minimum detectability of IL-1, IL-2 and TNF- were 0.003, 0.01 and0.03 ng/L, with 12–13%, 7–10% and 11–12% intra- and interassay CV, respectively. The sCD4 and sCD8 were measured witha commercial porcine ELISA kit from the BioSource International, Inc. (Camarillo, USA). The minimum detectable dose was0.1 U/mL with 9% and 15% intra- and interassay CV, respectively.2.4. Determination of specific ovalbumin antibody in serumOVA-IgG contents were determined with a porcine ELISA kit from the R & D Systems, Inc. (Minneapolis, USA). Briefly,standard samples were serially diluted and the final concentrations were 120, 80, 40, 20 and 10 g/mL, respectively. Sampleswere added to testing sample wells. The plate was incubated for 30 min at 37◦C then was washed and added HRP-conjugatereagent. Absorbance at 450 nm after adding stop solution and with in 15 min was read after coloring.2.5. Specific secretory immunoglobulin A analysisThe specific sIgA in small intestine mucosa was determined with a porcine ELISA kit (R&D Systems). Assay procedure wassimilar with the determination of OVA-IgG.2.6. Statistical analysisRegression analysis was conducted to evaluate linear and quadratic effects of chitosan on the various response criteriain piglets by using the SAS software 9.0. Trends were considered significant if probability values of P<0.05 were obtained.3. ResultsWith increasing addition of chitosan, serum IgG concentrations increased in a quadratic dose-dependent manner (P<0.05),while serum IgA and IgM concentrations were not altered (Table 2). Serum OVA-IgG average content of piglets in each groupwas about 30 g/mL and had no difference between treatments before the trial (Table 2). However, serum OVA-IgG wasenhanced by chitosan quadratically (P<0.05) on day 14 and improved linearly or quadratically (P<0.05) on day 28. Withincreasing chitosan, the sIgA was enhanced in ileum mucosa in a linear or quadratic manner (P<0.05) on day 14, and wasimproved quadratically in jejunum mucosa on day 28 (P<0.05) (Table 2).Table 3 Effects of chitosan on the concentration of sCD4, sCD8 and cytokines in serum of weaned piglets.
The result showed that chitosan decreased serum CD4 in a quadratic dose-dependent manner (P<0.05) and sCD8 in a linearor quadratic manner on day 28 (P<0.05) (Table 3). The maximum decrease of SCD4 in 1000 mg/kg chitosan group and sCD8 in500 mg/kg chitosan group were 13.71% and 13.79% on day 28, respectively. Chitosan quadratically enhanced serum IL-1andIL-2 on day 14 as well as serum TNF- in piglets on day 28 (P<0.05) (Table 3). In addition, piglets in 500 and 1000 mg/kgchitosan treatments had higher serum sIgA, IL-1, IL-2 and TNF- concentrations compar
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