| The objectives of the present study were 1) to evaluate the titer, specificity, stabilityand biological safety of chicken egg yolk antibody against pig adipose tissue plasmamembranes (AIgY) and the effect of dietary AIgY supplementation on pig growth and lipiddeposition, and 2) to reveal the cellular and molecular mechanisms involved in AIgY action,both in vivo and in vitro.1 Effect of AIgY on lipid deposition1.1 Preparation and stability of AIgYLaying-hens were intercutaneously immunized with adipose tissue plasma membraneprotein extracted from backfat of Erhualian pig. The titer and specificity of the harvestedyolk antibody (IgY) were determined with enzyme labeling linked immunosorbent assay(ELISA), while the stability of IgY against heating and storage period of IgY at roomtemperature were also researched. The titer of AIgY reached 1:12 800 determined byELISA, while weak cross reactivities of the AIgY with membrane of non-adipose tissuescould be detected. The processes of lyophilization had no influence in the titer of AIgY;AIgY was fairly heat resistant and could be stored for 300 d at room temperature.1.2 Biological safety evaluation for AIgYIn trial 1,150 female Sprague-Dawley rats weighting 110±10g were allotted randomlyinto five groups (basal diet adding 0, 75, 500, 1000 and 6000 mg/kg of yolk powdercontaining AIgY at the titer of 1:12 800). The trial lasted for 75 days and the clinicalexamination, body weight and feed/gain were determined. In trial 2, 160 crossbred(Duroc-Jersey×Landrace×Meishan) pigs, with initial live body weight of 27.5±2.4 kg,were treated with AIgY or non-immunized control yolk powder (NIgY) at the inclusionlevel of 75 mg·kg-1 diet for 104 d. The body weight, feed/gain, serum biochemicalparameter, visceral organ index and histopathological changes were determined. No death and toxicity were observed and all the toxicity indications in these two trials were negativeexcept that the spleen index of the boars was higher (P＜0.05). We demonstrated that AIgYwas safe for animal edibility.1.3 Effect of AIgY on growing performance and lipid deposition in pigsIn trial 1, 160 crossbred (Duroc-Jersey×Landrace×Meishan) pigs, weighingapproximately 27.5 kg (75 d old), were allotted randomly into two groups and fed dietssupplemented with 75 mg·kg-1 egg-yolk powder containing AIgY at the titer of 12 800 inexperimental group or non-immunized control egg yolk powder (NIgY) in control group for104 d. In trial 2, 80 crossbred (Duroc-Jersey×Landrace×Meishan) pigs weighingapproximately 47 kg (120 d old) were allocated randomly into treatment and control groups.The diet administration was as trial 1. In trial 1, AIgY enhanced average daily gain and feedefficiency by 13.03% (P＜0.01) and 7.49%, respectively. Dietary supplementation of AIgYincreased lean mass by 10.3% (P＜0.01) with unaffected dressing percentage. Backfatthickness between 6～7th rib was reduced by 24.14% (P＜0.01). The ratio of the perirenal,mesenteric and subcutaneous fat were reduced by 27.54% (P＜0.05), 20.42% (P＜0.01) and28.28% (P＜0.01), respectively. It also showed that the meat color was improved and thePH45, marbling and the intramuscular fat content of the longissimus muscle remainedunaffected. In trial 2, there was a trend of decrease on backfat thickness between 6～7th riband fat percentage of carcass. The results showed the dietary AIgY in early stage increasedthe growing performance and the lean mass percentage, while decreased the fat percentageof carcass and improved the meat quality in pigs.2 Preliminary Studies on involved mechanisms of AIgY2.1 Effect of AIgY on carcass composition and lipid metabolism-related hormones andenzymes in pigsAIgY treatment increased (P＜0.01) serum FFA and reduced serum urea-N, but did notalter serum levels of triglyceride (TG) and glucose. Serum concentrations of insulin andleptin were reduced by 30.81% (P＜0.05) and 30.74% (P＜0.05), respectively. Dietarysupplementation of AIgY reduced the size of adipocytes in all the three different adiposepads (P＜0.05). LPL activity in adipose tissue and the ratio of LPL activity between adiposetissue and skeletal muscle were reduced significantly (P＜0.05) whereas.ME activity wasunaffected. We demonstrated the oral effect of AIgY on fat deposition in the pig and thechanges of serum insulin and leptin levels as well as the tissue LPL activity may be involved in the acting mechanism.2.2 Effects of dietary supplementation of AIgY on apoptosis- related gene expressionin adipose tissue of pigsAIgY reduced DNA content of subcutaneous, perirenal and mesenteric fat depots by38.42% (P＜0.01), 39.92% (P＜0.05) and 39.16%, respectively. The relative abundance ofBcl-x1 mRNA in adipose tissue was decreased by 18.13% (P＜0.05) by AIgY treatment,while the relative abundance of Bax-αmRNA in adipose tissue was increased by 13.79%(P＜0.05) in treatment group. The results suggest that dietary supplementation of AIgYcaused the imbalance of Bax-αmRNA/Bcl-x1 mRNA ratio which could induce apoptosis inadipose tissue in pigs. Bcl-x1 gene down-expression and Bax-αgene up-expression indicatethat the apoptosis induced by AIgY is mediated through mitochondrial pathway.2.3 Effects of egg yolk antibody against adipose tissue plasma membranes on theproliferation and apoptosis of adipocytes in vitroThe 3T3-L1 preadipocytcs and differentiated adipocytes were treated with 0.1, 1.0,and 10μg/ml of AIgY for 12, 24, 48 and 72 hours, respectively. The proliferation activity of3T3-L1 preadipocytes was decreased obviously by the treatment of AIgY, whereascytotoxicity or complement-mediated cytotoxicity was not observed. Cytoplasmic fatcontent was reduced significantly. Apoptotic peak and typical morphological changes inapoptosis could be observed in cells exposed to 0.1, 1.0, and 10.0μg/ml of AIgY for 48h,and apoptotic rate determined by flow cytometry was significantly higher than that incontrol groups in a dose-related manner (P＜0.01). These results suggested that AIgY couldinhibit the proliferation of 3T3-L1 preadipocytes and affect lipid filling (hypertrophy) ofadipocytes, and induce the apoptosis of adipocytes in a dose-dependent manner.
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