| Piglet survival rate is one of the most important factors that influence the development of hog industry,and the immunoglobulin level in sow milk and its transportation to the newborn are crucial to piglet survival.The antibody level in piglet is nearly null at birth,therefore the immunity of infant pig predominantly depends on passive protection provided by the maternal antibodies from colostrum.Being the most abundant colostral Ig component in livestock animals, IgG must be transported across epithelial barriers through transcytosis when it is secreted from mammary gland to milk and when it is absorbed by the newborn animals,through a specific IgG transport receptor,namely the neonatal Fc receptor(FcRn).Research has shown that FcRn performs unique physiological functions,which not only plays a critical role in IgG transcytosis from mother to baby,but presents in adult through lifetime mentaining the homeostasis of serum IgG and albumin proteins as well,and it is also closely related with animal growth and development and immune response.Research on this receptor helps reveal IgG transfer mechnism and thus provides practical and theoretical guidance on piglet husbandry.Targeted on Landrace-Large White crossbred piglets from age of at birth to postweaning, tissues of liver,kidney,spleen,heart,slomach and different parts of intestine were collected at 12 age points and experiments were performed as follows.1.Expression of FcRn mRNA in different tissues of piglets was detected by RT-PCR and relative quantitation was determined and analvsed.The results indicated that FcRn was expressed in the tissues of liver,kidney,spleen,heart,stomach and all parts of intestine.The electrophoresis band density scanning analysis of FcRn/GAPDH showed that among the gastroinstestinal(GI) tract tissues the strongest signals existed at duodenum and jejunum;high level of FeRn gene transcription was detected in duodenum and jejunum from piglets at birth and reached its peak on 1d,decreased sharply from 4d on,and reached the lowest at pre-weaning(21d).At the ileum, FcRn expression was relatively low and no distinct changes observed.The minimum of FcRn expression was detected at colon and stomach,being relatively stable.The high level of FcRn expression in distal duodenum and adjacent jejunum implies that these two parts are the major sites of milk IgG transport to swine intestine.2.The quantitation of FcRn levels in pgilet liver and GI tract tissues was performed by quantitative real-time RT-PCR(RT-FQ-PCR) with primers and a fluorecent probe for FcRn heavy chain gene,withβ-actin chosen as house-keeping gene.The results showed a significant difference of FcRn levels in swine tissues.The most abundance of FcRn was present in liver(△Ct =4.95),followed by distal duodenum and adjacent jejunum,and then adjacent duodenum and distal jejunum,low expression levels were revealed in ileum and colon,whilst the lowest level was in stomach(△Ct =6.91).The trend of FcRn mRNA expression in GI tract was present accordly to age:Low level of FcRn was foound at birth with no significant difference between non-suckling(0 w) and suckling(0 y) piglets(P＞0.05),the expression markedlv increased on 1 d (P＜0.05),remained relatively stable and decreased at 21d,thereafter it increased sharply on 28d but dropped to previous level at 35d of age.3.The method of in situ hybridization(ISH) was effectively established for the expression of FcRn in the small intestine of piglets.A digoxin-labeled single-strand cDNA probe for detection of swine FcRn mRNA was plotted according to the conserved region of the gene for a chain of porcine FcRn published in GenBank.The ISH method was optimized through a series of trials such as the elimination of endogenous HRP,and fixation agent selection.Best procedureincluded:tissue slices with thickness of 14μm,fixed with 4%polyformal solution for 1h,pre-treated with 1%Triton X-100,hybridized with 1.5μg/mL of probe at 46.8℃for 18 hrs,colorated with DAB after being incubated with anti- DIG- HRP fragment at 4℃for 16 hrs,and finally the slices were counter-stained with hematoxylin.4.FcRn mRNA localization in swine intestinal sections was analysed by means of ISH and the change of expression level was depicted.Limpid hybridization signals were detected in the small intestinal epithelial cells and submucosa of duodenum,jejunum and ileum,among which large amount of positive signals were detected in the intestinal epithelia with the most predominant in jejunum,followed by distal duodenum,whereas the expression in adjacent duodenum and ileum was faint.The relative quantitation results of swine intestinal FcRn kinetics showed a high consistency with that of RT-FQ-PCR detection.5.Titration of serum IgG levels in piglets was taken by radioimmunoassay(RIA).The RIA results of IgG level changes in swine showed that the piglets at birth presented no IgG in the body before suckling whereas the lgG level was promptly increased after having sucked the colostrum, reaching peak concentration at 1d,dropping sharply at 3d,but recovering to the 2nd peak at 10d, then a distinct decrease occurred at 10d followed by a period of relative stable levels,and then the IgG titer reached maximum at 35d with a concentration of 4.74 mg/mL.Generally,both the qualitative,quantitative and the positioning detection of piglet FcRn expression indicated that FcRn mRNA was predominantly expressed at distal duodenum and adjacent jejunum,consistence with the primary nutrition absorption sites.Therefore it mplied that FcRn was involved in intestinal IgG transportation and played a role of transfer receptor for IgG. The high performance of FcRn mRNA in piglets within 3d age was consistant with the timing of their peak absorption of colostrum and that of "gut-closure",and was also testified by the detection of changes of serum IgG level in piglet showing a close correlation with FcRn expression,which illustrated the critical importance of FcRn presence to the acquisition of effective passive immunity in newborn animals.
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