Effect of chicken egg yolk antibody against adipose tissue plasma membranes on carcass composition and lipogenic hormones and enzymes in pigs

Livestock Science 107 (2007) 235 – 243 www.elsevier.com/locate/livsci

Effect of chicken egg yolk antibody against adipose tissue plasma membranes on carcass composition and lipogenic hormones and enzymes in pigs J.P. Jiang a,b , J. Zhou a , J. Chen a , X.H. Wei a , T.S. Lu a,c , H. Chi c , R.Q. Zhao a,⁎ a

Key Laboratory of Animal Physiology and Biochemistry, Ministry of Agriculture, Nanjing Agricultural University, Nanjing 210095, PR China b Lianyungang Academy of Agricultural Sciences, Lianyungang 222006, PR China c Walcom Bio-chemicals Industrial Limited, Shanghai 201206, PR China Received 15 April 2006; received in revised form 14 August 2006; accepted 15 September 2006

Abstract Chicken egg yolk antibody against pig adipose tissue plasma membranes (AIgY) was raised and used in the present experiment to evaluate the effect of dietary AIgY supplementation on pig growth and carcass composition. 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 egg yolk powder (NIgY) at the inclusion level of 75 mg/kg diet. Following a 104-day trial, the pigs were slaughtered for analyzing the carcass and meat quality traits. The perirenal, mesenteric and subcutaneous fat depots were weighed and the diameter of adipocytes from different fat depots was measured with histological methods. Serum concentrations of insulin and leptin as well as the activities of malic enzyme (ME) and lipoprotein lipase (LPL) in adipose tissue were measured. Dietary supplementation of AIgY enhanced average daily gain and feed efficiency by 13.03% (P b 0.01) and 7.49%, respectively, with no influence on feed consumption. AIgY increased the lean mass by 10.3% (P b 0.01) without affecting the dressing percentage. Backfat thickness at 6th–7th rib and the weights of perirenal, mesenteric and subcutaneous fat depots were reduced by 24.14% (P b 0.01), 27.27% (P b 0.05), 20.42% (P b 0.01) and 29.21% (P b 0.01), respectively. Dietary supplementation of AIgY reduced the size of adipocytes in all the three fat pads (P b 0.05). The meat color was improved whereas the marbling score, the intramuscular fat content, and pH45 of the longissimus muscle remained unaffected. Serum concentration of non-esterified fatty acids (NEFA) was significantly increased (P b 0.01) while urea-N content was reduced (P b 0.05). No alterations were detected for the serum levels of triacylglycerides (TG) and glucose. Serum concentrations of insulin and leptin were decreased by 26.19% (P b 0.05) and 26.53% (P b 0.05), respectively. LPL activity in adipose tissue was depressed significantly (P b 0.05) without affecting ME activity. This study demonstrates that dietary supplementation of AIgY can effectively improve growth and carcass composition of pigs and the changes of serum insulin and leptin levels as well as the tissue LPL activity may be involved in the acting mechanism. © 2006 Elsevier B.V. All rights reserved. Keywords: Yolk antibody (IgY); Adipose tissue plasma membrane (APM); Carcass composition; Pigs

1. Introduction

⁎ Corresponding author. Tel.: +86 2584395047; fax: +86 2584398669. E-mail address: [email protected] (R.Q. Zhao). 1871-1413/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.livsci.2006.09.020

Excessive fat deposition in livestock affects production efficiency, animal health, consumer perception, and marketability of meat products. Reduction of fat content in meat-producing animals is highly desirable for both

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consumers and producers. One approach to decreasing the fat content of domestic livestock is to destroy existing adipocytes with antibodies against adipocytes (or adipose tissue) plasma membranes (Flint, 1996). Previous studies showed that antisera raised against adipocyte (or adipose tissue) plasma membranes reduced lipid deposition in a variety of species including rats (Flint, 1998; Futter et al., 1992; Hu et al., 1992; Panton et al., 1990), rabbits (Dulor et al., 1990), sheep (Moloney, 1990; Monoley et al., 1998, 2002; Nassar and Hu, 1991), pigs (De clercq et al., 1997; Kestin et al., 1993) and chickens (Wu et al., 2000). In some studies the reduction of lipid deposition was accompanied by increases in lean mass producing favorable changes in body composition (Flint et al., 1986; Kestin et al., 1993; Panton et al., 1990). However, application of antisera against adipocyte (or adipose tissue) plasma membranes revealed certain limitations to their use. Up to now, production of antisera against adipose tissue plasma membranes was obtained by active immunization of other species, which is generally associated with multiple injections of antigens and adjuvants and repeated blood sampling procedures. Moreover, the quantity of collected antisera was also limited. Meanwhile, administration with considerable concentrations of antibody via a subcutaneous (s.c.) or intraperitoneal (i.p.) route caused side effects involving subdued behavior and reduced feed intake (Dulor et al., 1990). During the past 20 years, the use of chickens instead of mammals has increased since eggs from immunized chickens may provide a convenient and inexpensive source of antibodies. Oral administration of specific yolk antibody has been used successfully to prevent gastrointestinal infections (Crabb, 1998; Kuroki et al., 1994; Yokoyama et al., 1998) and could potentially be used against many frequently encountered diseases (e.g. common cold, cystic fibrosis,tonsillitis and caries)(Carlander et al., 2002; Kollberg et al., 2003; Kruger et al., 2004).Oral passive immunization with yolk antibody against pig adipose tissue plasma membrane (AIgY) may be a new promising strategy to reduce fat deposition in meat animals. Therefore, we conducted the present study to determine the effect of dietary supplementation of AIgY on growth and body composition of growing-finishing pigs. 2. Materials and methods 2.1. Production and characterization of AIgY The adipose tissue was extracted from backfat of Erhualian pigs. Plasma membrane was isolated (Kestin et al., 1993) and protein concentration was determined (Bradford, 1976). The plasma membrane preparation was diluted with buffer (0.1 mol/L of Na2HPO4, 0.02 mol/L of

EDTA, pH7.4) to 200 μg protein/ml and emulsified with an equal volume of Freund's complete adjuvant (sigma). 20 laying-hens were intercutaneously immunized with pig adipose plasma membrane preparation (80 μg of protein for one chicken) followed by booster injections using Freund's incomplete adjuvant (sigma) at intervals of 3 to 4 weeks. 20 additional laying-hens were similarly immunized using buffer instead of adipose tissue plasma membrane preparation to provide non-immunized control yolk antibodies (NIgY). The freshly laid eggs were collected throughout the antibody production phase and the egg yolk was separated, pooled and lyophilized. The crushed yolk power was eradiated with Co-60 for sterilization (radiant intensity: 8 KGY) before storage at −20 °C. Plasma membrane proteins were prepared from a range of pig tissues including liver, kidney, muscle, and erythrocytes using the same method as described for adipose tissue. Titer and specificity of the yolk antibodies to pig adipose tissue plasma membrane were determined by enzyme-linked immunosorbent assay (ELISA) as described previously (Hamada et al., 1989). 2.2. Animals and experimental design 160 crossbred (Duroc–Jersey × Landrace·Meishan) pigs (80 barrows and 80 gilts) born within 3 d of each other and weighing 27.5 ± 2.4 kg (approximately 75 d of age) at the start of the trial were divided randomly into 8 floor pens, with 10 gilts and 10 barrows in each pen (22.4 m2) as one experimental unit. The 8 pens of pigs were randomly allocated to one of two diet groups: AIgYtreated group fed the basal diet (Table 1) supplemented with 75 mg AIgY/kg diet, and control group fed the basal diet plus 75 mg NIgY/kg diet. The inclusion level of AIgY was determined by a preliminary trial on pigs, which suggested that 75 mg/kg of AIgY was effective in improving carcass traits. The pigs had free access to water from nipple drinkers and their respective diets were provided ad libitum. Daily feed intake was measured for each pen, initial and final weights were recorded individually. Pigs were fed their respective diets for 104 d before being transported 5 km to an abattoir. 12 pigs (6 barrows and 6 gilts) randomly selected from each treatment were slaughtered to determine the effect of oral administration of AIgY on body weight, carcass composition and meat quality. Perirenal, mesenteric and subcutaneous fat samples were excised and fixed in a 4% paraformaldehyde solution for morphological analysis. Organs (heart, kidneys, liver, and spleen) were removed and weighed, small portions were preserved in 4% paraformaldehyde for pathological analysis. Blood and tissue samples were taken and processed to meet with

J.P. Jiang et al. / Livestock Science 107 (2007) 235–243 Table 1 Composition of the basic diets used in this study Item

Growing period

Ingredient (%)

% of DM

Yellow dent corn Wheat Barley Wheat bran Soybean meal Colza meal Fish meal Dicalcium phosphate (DCP) Limestone Salt Premix

55.00 10.50 4.00 8.00 12.60 4.50 1.50 1.00 1.55 0.35 1.00a

50.00 11.00 11.50 8.00 10.50 5.00

13.19 15.9 0.94 0.60 0.62

13.23 14.7 0.92 0.60 0.47

24–45 kg

45–90 kg

1.00 1.60 0.40 1.00b

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hematoxylin and eosin (HE staining) for measuring the size of adipocytes. Fixed samples from different organs were embedded in paraffin, cut into 5-μm sections, and stained with HE for the assessment of pathological changes. 2.5. Hormone and metabolite determination Serum NEFA and TG concentrations were determined with respective commercial kits (Nanjing Jiancheng Biochemical Reagent Co., Nanjing, China). Serum glucose concentration was determined with a commercial kit (Shanghai Rongsheng Biotech, Ltd., Shanghai, China) following the instructed enzymatic spectrophotometric procedure. Serum urea N concentrations were determined

Calculated analysis, DM basis DE (MJ) Crude protein (CP) Calcium (Ca) Total Phosphorus (P) Lysine

a Contains per kg: vitamin A, 1200 × 103 IU; vitamin D3, 220 × 103 IU; vitamin E, 2000 IU; vitamin B1,120 mg; vitamin B2,500 mg; vitamin B12, 2.5 mg; Fe, 18 g; Cu, 18 g; Zn, 15 g; Mn, 3 g; I, 100 mg; Se, 3 mg. b Contains per kg: viatmin A, 300 × 103; vitamin D3, 50 × 103 IU; vitamin E, 1000 IU; Fe, 25 g; Cu, 25 g; Zn, 20 g; Mn, 3 g; I, 100 mg; Se, 25 mg.

the requirements for hormones and enzyme measurements. The experiment was undertaken following the guidelines of the regional Animal Ethics Committee. 2.3. Meat quality measurements The longissimus muscle at the 11th to 12th rib was removed from the left side of each carcass for meat quality measurements. After a 30-min bloom period at 4 °C, the 2.5-cm-thick longissimus muscle chops were visually evaluated for marbling (1 = devoid to 5 = moderately abundant or greater; NPPC, 1991) and color based on the American color standards (1 = pale, pinkish gray to 6 = dark purplish-red; NPPC, 1999) by a trained personnel. Samples of the longissimus muscle were obtained for intramuscular fat content (IMF) content evaluation according to Hovenier et al. (1992), using Soxhlet petroleum-ether extraction and expressed as the weight percentage of wet muscle tissue. 2.4. Histological studies Perirenal, mesenteric and subcutaneous adipose tissue samples were sectioned with a cryostat and stained with

Fig. 1. Immunological characteristics of AIgY. (A) Titer determination with enzyme-linked immunosorbent assay (ELISA). Serial dilutions (1/ 100 to 1/12800) of yolk are evaluated and titers represent the last dilution with positive reactivity to pig adipose tissue plasma membranes that are immobilized on the ELISA plate. The x-axis represents the dilution factor (e.g., 1 = 1/100 dilution of the egg yolk). Results are the average values of duplicate from at least four repeats of ELISA. (B) Specificity of AIgY determined by enzyme-linked immunosorbent assay (ELISA). Serial dilutions (1/100 to 1/12 800) of yolk are evaluated and the specificity is assessed by reactivity to plasma membrane preparations from various types of cell/tissue that are immobilized on the ELISA plate. The results shown are the average of duplicate from at least four repeats of ELISA. Yolk obtained from 5 different hens demonstrated similar results.

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Table 2 Effect of AIgY treatment on growth performance of pigs Parameter

TRT a

CON b

Initial weight c, kg Final weight c, kg Average daily gain c, g Feed/gain Dressing d, % Organ wt, % of live weight d Liver Kidney Spleen

27.5 ± 2.4 84.6 ± 3.8⁎ 548.9 ± 32.5⁎⁎ 3.21 73.73 ± 1.75

27.6 ± 2.6 78.0 ± 4.2 485.6 ± 39.1 3.47 73.96 ± 1.90

1.69 ± 0.16 0.34 ± 0.03 0.23 ± 0.04⁎

1.75 ± 0.18 0.32 ± 0.04 0.19 ± 0.04

⁎P b 0.05 differs from control. ⁎⁎P b 0.01 differs from control. a TRT = Animals that received yolk power containing antibody against pig adipose tissue plasma membrane. b CON = Animals that received non-immune yolk power. c Values are mean ± SD for 80 animals per group. d Values are mean±SD for 12 animals per group(6 barrows and 6 gilts).

ogy, Beijing, China. The detection limits for leptin and insulin were 0.2 ng/ml and 1.0 μIU/ml, respectively. The intra- and inter-assay coefficients of variation were 5% and 10%, respectively for both kits. The kits were validated for measuring porcine serum samples previously (Amoikon et al., 1995; Zhao et al., 2003). 2.6. Statistical analysis All data were expressed as mean ± SD. Differences were considered significant when P b 0.05 tested by t-test for independent samples or one way ANOVA with Statistical Packages for the Social Sciences 11.0 for Windows. 3. Results 3.1. Titer and specificity of AIgY

following the quantitative urease/Berthelot procedure (Nanjing Jiancheng Biochemical Reagent Co., Nanjing, China). ME activity of subcutaneous adipose tissue was measured according to a recent publication (Streckfuss et al., 2005). LPL activity in subcutaneous fat and longissimus dorsi muscle were determined using a commercial kit (Nanjing Jiancheng Biochemical Reagent Co., Nanjing, China) according to kit instructions. Serum leptin and insulin concentrations were measured with respective commercial multi-species RIA kits purchased from Beijing North Institute of Biotechnol-

Table 3 Effect of AIgY on carcass composition and meat quality in growingfinishing pigs (n = 12) a

Parameter

TRT

Backfat thickness, cm Perirenal fat c, % Subcutaneous fat c, % Mesenteric fat d, % Lean meat rate, % Loin eye area, cm2 Bone percentage, % Skin percentage, % Ham/carcass percentage, % pH45 Meat color Marbling Intramuscular fat rate, %

2.21⁎ ± 0.36 1.92⁎ ± 0.38 15.12⁎⁎ ± 2.02 1.13⁎⁎ ± 0.14 63.20⁎⁎ ± 1.43 30.64 ± 5.68 12.68 ± 1.02 9.17 ± 1.06 32.91 ± 1.10 6.39 ± 0.43 3.3 ± 0.5⁎ 3.0 ± 0.8 2.86 ± 0.35

CON

After immunized with pig adipose tissue plasma membrane preparations for 4 times, 5 laying-hens chosen randomly from the total flock of 20 produced high-titer yolk antibody that demonstrated high binding to immobilized pig adipose tissue plasma membrane at dilutions in excess of 1:12,800 (Fig. 1A). Although not entirely specific for adipose tissue plasma membrane, the yolk antibody displayed a remarkably high degree of reactivity to membranes of adipose origin compared with those isolated from a wide range of other tissues (Fig. 1B). Table 4 Effect of AIgY on metabolites, hormones and enzymes in growingfinishing pigs Parameter

TRT a

CON b

b

2.90 ± 0.48 2.64 ± 0.59 21.33 ± 2.90 1.42 ± 0.22 57.30 ± 2.07 26.31 ± 3.86 12.28 ± 1.14 8.96 ± 0.90 30.18 ± 1.68 6.32 ± 0.50 3.0 ± 0.4 2.6 ± 1.0 2.83 ± 0.22

a TRT = Animals that received yolk power containing antibody against pig adipose tissue plasma membrane. b CON = Animals that received non-immune yolk power. c Percentage of right carcass weight. d Percentage of carcass weight.

Number of pigs 12 12 Glucose, mg/dl 81.95 ± 16.70 63.73 ± 12.99 TG, mmol/L 0.78 ± 0.07 0.68 ± 0.05 NEFA, μmol/L 436.54 ± 85.07⁎⁎ 317.92 ± 75.96 Urea-N, mmol/L 5.54 ± 1.55⁎ 8.29 ± 2.37 Insulin, μIU/ml 4.50 ± 0.47⁎ 6.10 ± 0.55 Leptin, ng/ml 0.36 ± 0.03⁎ 0.49 ± 0.06 ME (μmol/mg protein·min) 0.51 ± 0.09 0.50 ± 0.06 aLPL(μmol oleic acid/h/g tissue) c 25.00 ± 1.84⁎ 26.78 ± 1.54 mLPL (μmol oleic acid/h/g 13.30 ± 2.04 12.24 ± 2.63 tissue) d aLPL/ cmLPL d ratio 1.97 ± 0.31⁎ 2.32 ± 0.36 ⁎ P b 0.05 differs from control. ⁎⁎ P b 0.01 differs from control. a TRT = Animals that received yolk power containing antibody against pig adipose tissue plasma membrane. b CON = Animals that received non-immune yolk power. c aLPL:LPL activity in adipose tissue. d mLPL: LPL activity in the longissimus muscle.

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Fig. 2. Morphological alterations of white adipose tissue. (A) Representative photos of adipose tissue sections stained with HE. Magnification 200x. (B) Statistical analysis of the average diameters of adipocytes from three fat pads (⁎ P b 0.05; ⁎⁎ P b 0.01). Results are expressed as the mean ± SD (n = 10–12/group). SF = Subcutaneous fat, MF = Mesenteric fat, PF = Perirenal fat.

3.2. Growth performance, carcass and meat quality As shown in Table 2, dietary supplementation of AIgYenhanced average daily gain and feed efficiency by 13.03% (P b 0.01) and 7.49%, respectively, with no influence on feed consumption. AIgY increased the lean mass by 10.3% (P b 0.01) without affecting the dressing percentage. Backfat thickness at 6th–7th rib was reduced by 24.14% (P b 0.01), the perirenal, mesenteric and

subcutaneous fat depots relative to carcass weight were reduced by 27.27% (P b 0.05), 20.42% (P b 0.01) and 29.21% (P b 0.01), respectively. The meat color was improved (P b 0.05) whereas the marbling score, the intramuscular fat content, and pH45 of the longissimus muscle remained unaffected (Table 3). No significant changes were observed for the relative weight of liver and kidney, yet the spleen weight was increased (P b 0.05) (Table 2).

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3.3. Hormones, metabolites and enzymes The serum concentrations of insulin and leptin were significantly reduced in AIgY-treated group. Meanwhile, serum NEFA concentration was elevated (P b 0.05) and urea-N content was reduced (P b 0.05). However, no alterations were found for serum concentrations of glucose and triglyceride. AIgY reduced LPL activity in subcutaneous fat by 6.65% (P b 0.05), whereas muscle LPL activity was slightly higher in AIgY-treated pigs, resulting in significantly lower fat to muscle ratio of LPL activity (P b 0.05) in AIgY-treated group. In contrast, ME activity in subcutaneous fat was unaffected (Table 4). 3.4. Adipose tissue morphology As shown in Fig. 2, dietary supplementation of AIgY reduced the size of adipocytes in all the three fat pads (P b 0.05). There were no histopathological evidences observed in heart, liver, kidney, and spleen in AIgYtreated pigs (data not shown). 4. Discussion We have demonstrated the consistently effective production of high-titer yolk antibody against pig adipose tissue plasma membranes in laying hens. The hens immunized with pig adipose tissue plasma membranes were found continuously to lay eggs without any change in the egg laying rate and the yolk of the eggs laid over 6 months showed a high titer after the 4th booster immunization (results not shown). Compared with mammalian antibodies, IgY possesses several biochemical advantages. Chicken housing is inexpensive, egg collection is noninvasive, and IgY isolation is fast and simple. Another advantage is that very small quantity of antigen is required to obtain high titer and long-lasting production of IgY in the yolk from immunized hens (Carlander et al., 1999). Despite slight cross-reactivity of AIgY to plasma membranes of various tissues detected by ELISA, we did not observe loss of appetite or other noticeable abnormalities of internal organs in treated pigs at slaughter. In addition, no histopathological evidences in various other tissues (i.e. heart, liver, kidney, and spleen) were observed, except an increase in spleen weight. It indicated that the cross-reactivity of AIgY detected by ELISA was not sufficiently high to result in a cytotoxic effect to nonadipose tissue. Spleen enlargement could be a sign of an immune response triggered by treatment or might reflect the presence of common antigens on the adipocyte and splenocyte plasma membranes. In all species examined to date, antibodies to adipose tissue showed a relatively high

degree of reactivity towards the adipocyte plasma membrane but reactivity towards other tissues was also observed (Dulor et al., 1990; Flint et al., 1986; Kestin et al., 1993). However, much of this cross-reactivity could be eliminated by adsorption of antibodies with non-adipose tissue plasma membranes prior to immunization without impairment of the efficacy of the remaining antibodies (Futter et al., 1992). It has been documented that yolk antibodies can act effectively in the gastrointestinal tract to prevent and treat a variety of GI infections, such as enterotoxigenic Escherichia coli, Salmonella spp., etc. (Mine and Kovacs-Nolan, 2002). This function is reasonable since the action takes place within the GI tract. However, there are some reports describing the effect of IgY on other tissues, such as the feed efficiency-promoting effect of CCK yolk antibody (Danny, 1998) which was protected by US Patents 1998, 5 827 517 (Cook et al., 1998) and 2000, 6 086 878 (Adalsteinsson et al., 2000). Nevertheless, it was still a puzzle whether yolk antibody could cross the intestinal barrier of adult animals. In order to resolve this question, we conducted an absorption experiment on rats, and detected with Western blot analysis the full-length (≈ 200 kD) CCK yolk antibody in duodenal venous plasma 2–3 h after gastric infusion of CCK yolk antibody. The immune reactivity of this absorbed yolk antibody was further confirmed by ELISA with the titer of 1:128 (Zhang et al., 2004). The present study provided the first evidence that dietary supplementation of AIgY improves the average daily gain and feed conversion efficiency. The increase in lean mass percentage is accompanied with the decrease in fat deposition. Similar results were reported in pigs (Kestin et al., 1993) and rats (Panton et al., 1990) where using polyclonal antisera against fat tissue decreased fat tissue mass and increased percentage of lean tissue. It was reported that administration of antibodies against chicken adipocytes plasma membranes was more sensitive to reduce the abdominal fat pad mass in male chickens than female chicken (Wu et al., 2000). In our present experiment, however, we did not observe any gender differences in any observed indices responding to AIgY treatment. This discrepancy may be induced by different species, doses, types of antibodies, as well as the routes of administration in different experiment. The mechanism by which the antisera against adipocytes decrease adiposity remains to be elucidated. Some studies have demonstrated the cytotoxic potential of antisera to adipocytes in vivo (Futter et al., 1992; Kestin et al., 1993) and in vitro (De clercq et al., 1997). There have been also some evidences that antibodies against various protein components of adipocyte plasma membranes could block the binding of various hormones to their receptors on

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the plasma membrane to interrupt the signal transduction, and/or to disturb the assembly of various transporters to the membrane to influence the substance transport across the membrane (Kaise et al., 1988; Oka et al., 1988; Ros et al., 1988; Suzuki et al., 1988). It was reported (De clercq et al., 1997) that the sizes of adipocytes became smaller when treated with antibody against porcine adipocyte plasma membrane. We observed the same phenomenon that adipocytes from all the three fat depots of AIgY-treated pigs are smaller in size. It has been of great concern whether a decrease in fat deposition would compromise meat quality. Recombinant growth hormone and beta-adrenergic agonist have been shown to improve carcass composition (Crome et al., 1996; Mikel et al., 1993; See et al., 2004; White et al., 1993), but also reported to reduce intramuscular lipid content in pigs (Bidanel et al., 1991; Lafaucheur et al., 1992; Stoller et al., 2003). We did not observe any alteration in marbling score or the intramuscular fat content of the longissimus muscle in AIgY-treated pigs, except improvement of meat color. This finding is in agreement with previous report (Monoley et al., 2002) in which donkey antisera against sheep adipose tissue membrane protein did not produce any detrimental effect on meat quality. Fat deposition is determined by a complex balance between lipogenic and lipolytic enzymes. Lipoprotein lipase (LPL) is a key enzyme regulating the disposal of lipid fuels in the body. LPL is abundant in adipose and muscle tissues, and its gene expression correlates highly with the uptake of lipid fuels by the tissue. Therefore, LPL is considered as the key factor determining the dietary lipid disposition between tissues, and described as the ‘metabolic gatekeeper’ (Farese et al., 1991; Greenwood, 1985; Zechner, 1997). Higher muscle/fat ratio of LPL activity could indicate partition fatty acids away from adipose tissue towards muscle tissue (Ellis et al., 1994). NADPH is essential for fatty acid synthesis (Ratledge, 2004) while malic enzyme catalyzes the reaction which provides NADPH for de novo lipid biosynthesis of fatty acids. The activity of ME is correlated positively with the rate of fatty acid synthesis (Vidal et al., 2006). It was reported that rabbits treated with antibodies to adipocytes exhibited decreased glycerophosphate dehydrogenase and lipoprotein lipase activities in adipose tissue (Dulor et al., 1990). In the present study, the muscle/fat ratio of LPL activity was significantly higher (P b 0.05) whereas ME activity in subcutaneous fat was unaffected in AIgYtreated group. These changes indicated impaired ability to store triglyceride in adipocytes, and NEFA might have provided the muscle with extra energy, contributing to the increased protein accretion in AIgY-treated pigs.

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Insulin and leptin are known to regulate lipid metabolism and affect LPL activity (Farese et al., 1991). Insulin could increase both activity and mRNA expression of LPL in adipose tissue (Ong et al., 1988) and induce changes in LPL mRNA stability (Raynolds et al., 1990). Leptin plays an important role in regulating food intake and energy expenditure. Leptin is known to affect insulin sensitivity (Margetic et al., 2002) and to determine LPL activity in adipose tissue. In accordance with the previous finding in rats treated with antibodies against adipocytes (Flint, 1990), dietary supplementation of AIgY significantly reduced serum levels of insulin and leptin, which was accompanied by a significant decrease in LPL activity in adipose tissue. In conclusion, dietary supplementation of AIgY can effectively improve growth and carcass composition of pigs and induce changes of tissue LPL activity that favor a redistribution of fatty acids from fat to muscle thereby inhibiting fat deposition while improving lean mass accretion. Insulin and leptin may regulate this repartitioning process through suppression of LPL activity in adipose tissue. Acknowledgements This work was supported by National Basic Research Program of China (2004CB117505).

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