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Body composition and adipose tissue accretion in lambs passively immunised against adipose tissue
Livestock Production Science 74 (2002) 165–174 www.elsevier.com / locate / livprodsci
Body composition and adipose tissue accretion in lambs passively immunised against adipose tissue a, b a ,1 A.P. Moloney *, P. Allen , W.J. Enright b
a Teagasc, Grange Research Centre, Dunsany, Co. Meath, Ireland Teagasc, The National Food Centre, Dunsinea, Castleknock, Co. Dublin, Ireland
Received 8 March 2001; received in revised form 26 July 2001; accepted 26 July 2001
Abstract Thirty-eight cross-bred wether lambs, initial liveweight 35.2160.75 kg were used to evaluate the effects on growth and carcass composition of passive immunisation with anti-ovine adipose plasma membrane immunoglobulin G (IgG). Antibodies were raised in donkeys by active immunisation with sheep adipocyte membrane protein in Freund’s complete adjuvant followed by boosting at 3, 5 and 15 weeks and subsequently at 4-month intervals with membrane protein in Freund’s incomplete adjuvant. An IgG fraction was prepared from pooled serum by ammonium sulphate precipitation and dialysis. Ten lambs were slaughtered and the remaining lambs (n 5 14 / group) were injected subcutaneously with 80 ml of IgG from immunised (treated) or non-immunised donkeys (control) on 4 consecutive days at the beginning of the study. Following a 91-day test period the animals were slaughtered, the weights of internal organs and fat depots recorded and carcass composition determined by dissection. Treatment did not affect preslaughter liveweight (Control 5 47.8 kg, Treated 5 47.4 kg) or carcass weight (Control 5 25.9 kg, Treated 5 24.7 kg). Liver weight tended to be increased (P 5 0.07) by treatment (from 751 to 905 g), while the weights of other organs were not affected. The weights of the omentum and kidney / channel fat depots were reduced (P , 0.05) by 18.1 and 27.7%, respectively, by treatment. Treatment decreased the weight of dissectable subcutaneous fat (from 2.7 to 2.4 kg, P , 0.1), dissectable intermuscular fat (from 2.9 to 2.3 kg, P , 0.05) and total dissectable fat (from 5.6 to 4.7 kg, P , 0.05) without a decrease in dissectable meat. Daily adipose tissue accretion was decreased by 25% approximately for omentum, intermuscular, subcutaneous and total carcass fat and by 37% for kidney / channel fat by treatment. Treatment resulted in an acute decrease in plasma glucose and insulin concentrations, an acute increase in non-esterified fatty acid and b-hydroxybutyrate concentrations and a chronic increase in insulin concentration. It is concluded that passive immunisation of lambs with anti-ovine adipose plasma membrane IgG can reduce body fatness without decreasing lean meat accretion. 2002 Elsevier Science B.V. All rights reserved. Keywords: Immunisation; Adipose tissue; Sheep; Carcass; Growth
*Corresponding author. Tel.: 1 353-046-26700; fax: 1 35346-26154. E-mail address: [email protected] (A.P. Moloney). 1 Present address: Intervet International BCV, Wim de Korverstraat 35, P.O. Box 31, 5830AA Boxmeer, The Netherlands.
1. Introduction Consumer interest in the fat content of meat has been heightened by medical advice to decrease their intake of total and saturated fat (e.g., National
0301-6226 / 02 / $ – see front matter 2002 Elsevier Science B.V. All rights reserved. PII: S0301-6226( 01 )00299-8
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Research Council, 1988). Moreover, much of the fat stored by domestic animals is discarded following slaughter and, as such, is a source of inefficiency in (lean) meat production. One approach to decreasing the fat content of domestic livestock is to manipulate the immune system to destroy existing fat cells (or adipocytes) with cytotoxic antibodies. The capacity of the animal to store fat would thereby be decreased, resulting in leaner meat. Flint et al. (1986) first demonstrated that antibodies raised in sheep against rat adipocyte plasma membranes were cytotoxic in vitro and caused a decrease in rat body fat in vivo. In pigs, Kestin et al. (1993) reported a 25% decrease in dissectable fat in the fore- and hind-limb joints due to passive immunisation with antibodies to pig adipocyte plasma membranes. In sheep, Thornton and Tume (1987) reported no effect on body fat content, and while Nassar and Hu (1991a) observed a 46% reduction in perirenal adipose tissue weight, there was no reduction in total carcass fatness due to passive immunisation with anti-sheep adipocyte plasma membrane sera. Recently, Moloney et al. (1998) reported that passive immunisation of lambs and ewes with antiadipocyte plasma membrane sera decreased the weight of selected internal fat depots and that the subcutaneous (SC) route of antiserum administration was superior to the intravenous route. However, while dissectable fat was decreased close to the site of SC immunisation, overall carcass fat accretion was not affected. The objective of this experiment was to progress this approach to decreasing adiposity by examining the effect of administration of a higher dose of antiserum with a more dispersed pattern of administration than that used in our previous study.
2. Materials and methods
2.1. Antibody preparation Preparation of adipose plasma membranes and antibody production were as described by Moloney et al. (1998). In brief, plasma membranes were prepared from a pool of subcutaneous, perirenal and omentum adipose tissue by a combination of homogenisation, density gradient and differential centrifugation. Enriched plasma membranes, emulsified with
Freund’s complete adjuvant were used to immunise donkeys by SC injection at four sites in the neck ventral to the jugular groove. Booster injections using Freund’s incomplete adjuvant were administered after 3, 5 and 15 weeks, and subsequently at 4-month intervals. Additional donkeys were similarly immunised, but using buffer instead of adipocyte plasma membrane protein to provide control serum. Blood was collected at intervals throughout the antibody production phase of the study, allowed to clot overnight at 48C and centrifuged at 2000 g for 30 min. Serum was stored at 2 208C. For immunisation, serum was pooled and the immunoglobulin G (IgG) fraction precipitated using ammonium sulphate (Hudson and Hay, 1980). Thus, to 4 l of pooled serum from control donkeys or 4 l pooled serum from membrane-immunised donkeys, 1.164 kg ammonium sulphate was slowly added (over 4 h) while stirring at 48C. The serum was stirred at 48C overnight and then centrifuged at 2000 g for 90 min. The precipitate was dissolved in 70 ml phosphate-buffered saline (PBS) and dialysed against 9 l PBS with two changes of buffer. After dialysis, no residual ammonia was detected using Nessler’s reagent and the IgG solution was adjusted to 5 l with PBS.
2.2. Passive immunisation of sheep Thirty-eight Finnish–Landrace wether lambs, approximately 8 months old were penned together and offered straw in decreasing quantities (ad libitum at the beginning) and increasing quantities of whole barley until the ad libitum level of intake of unsupplemented barley was achieved. This phase lasted approximately 3 weeks. The animals were then offered ad libitum a pelleted concentrate diet consisting of (g / kg) 700 barley, 100 soyabean meal, 150 grain screenings, 30 molasses and 20 vitamin / mineral premix. The concentrate contained 155 g crude protein (CP), 91 g crude fibre and 56 g ash / kg dry matter (DM). Animals were weighed on 2 consecutive days, and 10 were chosen at random for slaughter at the beginning of the study. The remainder were blocked in pairs on a descending bodyweight basis (mean of both weights) and within block assigned at random to receive control IgG (Control) or anti-ovine adipocyte plasma membrane
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IgG (Treated). The animals were penned individually and offered fresh feed daily at 10:30 h. Feed intake was measured daily until slaughter 91 days after first immunisation. Animals were injected SC with 10 ml IgG at each of four sites on both sides of the midline. The immunisation procedure was repeated on 4 consecutive days. Upon completion of immunisation on day 1 and for 4 subsequent days, lambs received 3 ml penicillin and 3 ml streptomycin. Subcutaneous fat depth was measured bilaterally over the 12th rib in vivo using an A-mode ultrasound scanner (Delphi Instruments, New Zealand) on days 33, 61 and 85 after the completion of immunisation. The mean of the bilateral readings for each day was used in the statistical analyses. Animals were weighed at weekly intervals (at 10:00 h) and on 2 consecutive days prior to slaughter. Blood samples (10 ml) were taken immediately before the first immunisation and then periodically (at 13:30 h) by jugular venipuncture using vacutainers containing either sodium EDTA or potassium oxalate / sodium fluoride as anticoagulants. Blood was centrifuged at 2000 g and the plasma stored at 2 208C. On the day of slaughter, animals were stunned prior to exsanguination and carcass weight, fleece weight and the weights of individual offal components were recorded. The carcasses were chilled at 08C for 48 h, split medially and carcass composition determined by cutting the left side into joints based on the method of Carroll and O’Carroll (1964). The cross-sectional area of the longissimus thoracis et lumborum muscle (LTL) at the 12–13th rib interface and SC fat depth over the sixth and 12th rib, the leg and the loin were measured. Each joint was dissected into subcutaneous and intermuscular fat, lean and bone and the weights of each tissue recorded. All lean tissue was pooled, minced, subsampled and stored at 2 208C for subsequent chemical analysis. A sample of LTL was stored at 2 208C for subsequent chemical analysis.
2.3. Chemical and biological analyses Feed samples were analysed for DM, CP, crude fibre and ash, and tissue samples for CP (Kjeldahl), ash, moisture and total fat by standard procedures (AOAC, 1990). Antibodies were quantified using the modification of the assay of Flint et al. (1986) as
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described by Moloney et al. (1998). Plasma samples were analysed for insulin and metabolites as previously described (Moloney et al., 1998).
2.4. Statistical analyses The relationship between bodyweight or carcass weight and body components of the animals slaughtered at the beginning of the experiment was examined using linear regression. These relationships were then used to calculate accretion rates of various tissues for each animal over the course of the study. All animal data were subjected to analysis of variance using a model that had block and treatment as sources as variation. Weekly feed intake, periodic bodyweight and blood data were analysed at each measurement time using the above model. A onesided t-test was used for all measurements of fatness, since the hypothesis tested was that treatment would have no effect or cause a decrease (but never increase) in fatness.
3. Results The descriptive statistics of the lambs slaughtered at the beginning of the study are shown in Table 1. The relationships used to estimate the initial composition of the remaining animals are shown in Table 2. Table 1 Characteristics of lambs (n 5 10) slaughtered at the beginning of the study Variable Whole body (kg) Cold carcass (kg) Omentum fat (g) Kidney / channel fat (g) Carcass composition (g) Fat Subcutaneous Intermuscular Total Meat Bone Lipid in pooled carcass meat (g / kg) Lipid in LTL a (g / kg) a
Longissimus thoracis et lumborum.
Mean 34.4 16.2 560 387
1164 968 2132 11 159 2908 73 29
S.D. 4.41 2.53 233.7 77.7
459.2 389.3 800.3 178.5 355.7 8.6 6.2
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Table 2 Relationships between bodyweight (BW) and carcass weight (CAR) or internal adipose tissue depots, and between CAR and composition in lambs slaughtered at the beginning of the study
CAR weight (kg) Kidney / channel fat (g) Omentum fat (g) CAR fat (g) Subcutaneous Intermuscular Total Meat (g) Lipid in pooled carcass lean (g) a
R.S.D.
R2
Significance a
0.55 (BW) 2 2.8 11.5 (BW) 2 7.5 43.1 (BW) 2 922
0.74 62.6 144.4
0.92 0.42 0.66
*** * **
158 (CAR) 2 1398 138 (CAR) 2 1263 296 (CAR) 2 2661 563 (CAR) 1 2032 61.5 (CAR) 2 186
274.1 211.4 377.2 373.5 192.6
0.71 0.75 0.82 0.94 0.76
** ** *** ** **
* 5 P , 0.05; ** 5 P , 0.01; *** 5 P , 0.001.
3.1. Antiserum
3.3. Live animal variables
The binding pattern of the anti-adipose tissue IgG preparation to immobilised plasma membranes is shown in Fig. 1. For comparison, the binding pattern of the pooled unfractionated antiserum used by Moloney et al. (1998), measured in the same assay, is also shown in Fig. 1.
Bodyweight was greater (P , 0.05) in control than treated animals up to day 37 after first immunisation (Fig. 2). Treatment decreased (P , 0.001) daily DM intake during the week of immunisation (1.29 vs. 0.65 kg / animal) and during the first week after immunisation (1.40 and 0.97 kg / animal). Feed conversion efficiency data over the whole experiment are summarised in Table 3. There was no effect of treatment on any efficiency variables. Ultrasoundestimated backfat was lower (P , 0.05) at all measurement times in treated animals (Table 3). Mean packed cell volume (PCV) before immunisation was 35.563.21 (S.D.). From the day after completion of immunisation (day 4) until approximately 5 weeks later, PCV was lower (P , 0.05) in the treated group (Fig. 3). Treatment resulted in an immediate decrease in plasma glucose and insulin concentrations, an immediate increase in non-esterified fatty acid (NEFA) and b-hydroxybutyrate
3.2. Immediate effects of immunisation There were no obvious immediate effects of injection with control or immune sera on any of the 4 days of immunisation. During the 4 days of immunisation, the anti-adipocyte plasma membrane sera had an apparent sedating effect on most sheep. However, this effect was absent from day 6 of the experiment onwards.
Fig. 1. Binding of I 125 -protein A to an immobilised donkey immunoglobulin G-adipocyte plasma membrane complex. (j) Immunoglobulin G from donkeys immunised against adipose tissue plasma membranes. (m) Sera used by Moloney et al. (1998). (d) Immunoglobulin G from donkeys immunised against buffer.
Fig. 2. Bodyweight of control sheep (d) and of sheep immunised with antibodies to adipocyte plasma membranes (j).
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Table 3 Growth, efficiency and backfat depth in lambs passively immunised against adipose tissue a Trait
CON
TRT
S.E.
Significance
Initial weight (kg) Dry matter intake (DMI; g / day) Growth (g / day) Whole body Carcass Lean tissue Efficiency (g / kg DMI) Whole body Carcass Lean tissue Backfat depth (mm) Day 33 Day 61 Day 85
35.5 1294
35.1 1201
0.48 36.1
NS NS
132 99 52
138 91 50
11.0 7.0 3.3
NS NS NS
101 77 40
115 75 41
8.0 5.0 2.6
NS NS NS
0.10 0.12 0.12
** * *
2.0 2.1 2.2
1.5 1.7 1.8
a CON 5 Animals that received non-immune serum; TRT 5 animals that received antiserum to adipose tissue; NS 5 not significant; * 5 P , 0.05; ** 5 P , 0.01.
Fig. 3. Packed cell volume (PCV) in control sheep (d) and in sheep immunised with antibodies to adipocyte plasma membranes (j).
(BHB) concentrations and a chronic increase in insulin concentration (Table 4).
3.4. Body composition Treatment decreased (P , 0.05) the weights of the kidney / pelvic and omentum fat depots and increased (P , 0.10) the weight of the liver when expressed either in absolute terms or as a proportion of preslaughter BW (Table 5). Treatment did not affect carcass weight in absolute terms but decreased (P , 0.01) carcass weight when expressed as a proportion of pre-slaughter BW (Table 5). Treatment decreased (P , 0.05) the depth of fat covering the 12th rib and the leg and loin joints, without an effect on fat score,
decreased (P , 0.05) carcass conformation (Table 6), did not affect the composition of the shoulder joint (data not shown), decreased (P , 0.05) the total fat proportion of the leg, rib and loin joints, and increased (P , 0.05) the lean proportion of the loin joint (Table 7). The weights and proportions of intermuscular and total dissectable fat were decreased (P , 0.05) and the proportion of bone in the carcass increased (P , 0.01) by treatment. Treatment tended (P , 0.10) to increase the proportion of moisture and protein [717 and 729 g / kg (S.E. 3.7), and 220 and 226 g / kg (S.E. 2.0) for control and treated lambs, respectively] and decreased (P , 0.05) the proportion of fat (50 vs. 37 g / kg) in the LTL. The rate of fat accretion in internal depots and in the carcass was decreased (P , 0.05) due to treatment (Table 8) but deposition of lipid in carcass lean meat was not affected.
4. Discussion The wether lambs used were typical of those in an Irish lamb production system (Connolly, 1996) and their mean carcass weight was similar to that (23.8 kg) of the wethers used by Moloney et al. (1998). An immunological procedure may be a more acceptable approach to satisfy consumer and producer demands for decreased fat in meat animals than the
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Table 4 Temporal changes in plasma metabolite and insulin concentrations Variable
Treatment a
Day (post immunisation) 0
1
6
12
27
55
67
88
Glucose (mmol / l)
CON TRT S.E.D. Significance
5.21 5.32 0.178 NS
5.33 3.99 0.201 *
5.44 5.01 0.218 NS
5.15 4.90 0.148 NS
4.91 4.87 0.175 NS
3.51 3.72 0.429 NS
4.66 4.83 0.249 NS
4.99 4.63 0.187 NS
TAG b (mequiv. / l)
CON TRT S.E.D. Significance
86 101 10.3 NS
66 98 9.4 *
74 73 7.6 NS
82 83 5.4 NS
65 64 7.0 NS
135 94 28.8 NS
119 138 24.7 NS
155 142 18.8 NS
NEFAc (mequiv. / l)
CON TRT S.E.D. Significance
79 86 18.2 NS
74 388 54.0 *
111 251 34.1 *
38 55 33.3 NS
72 19 24.1 *
205 114 45.6 NS
105 113 28.5 NS
173 254 84.2 NS
BHB d (mmol / l)
CON TRT S.E.D. Significance
395 446 32.0 NS
294 478 61.2 *
294 320 48.9 NS
329 324 58.5 NS
281 224 31.7 NS
290 310 58.4 NS
244 324 55.0 NS
144 186 29.9 NS
Insulin (mU / ml)
CON TRT S.E.D. Significance
30.1 26.7 6.64 NS
27.2 14.1 2.46 *
28.0 32.2 4.32 NS
21.9 43.7 6.19 *
30.8 46.6 6.71 *
24.8 30.2 4.47 NS
– – –
15.1 17.5 25.1 NS
a
CON 5 Animals that received non-immune serum; TRT 5 animals that received antiserum to adipose tissue, NS 5 not significant; * 5 P , 0.05. b TAG 5 Triacylglycerol. c NEFA 5 Non-esterified fatty acid. d BHB 5 b-Hydroxybutyrate.
use of exogenous hormones or antibiotics. Moloney et al. (1998) demonstrated the potential of adipocyte destruction by treatment with antibodies to proteins on the adipocyte plasma membrane as a means of decreasing fat accretion in sheep. The objective of this study was to develop this concept further by using a higher concentration of antibodies (and a larger number of animals) and a more dispersed pattern of administration than used previously. A larger quantity (320 ml vs. 150 ml) of a higher titre (24 vs. 17% bound at 1:10 000 dilution) was administered at eight injection sites / 4 days compared to six injection sites / 3 days by Moloney et al. (1998). The apparent sedative effect of passive immunisation against adipocyte plasma membranes has been observed in rats (Futter et al., 1992, Hu et al., 1992),
pigs (Kestin et al., 1993) and sheep (Moloney et al., 1998), and may reflect the production of immune complexes (sub-fatal anaphylaxis) in these animals. The decrease in feed consumption in the week after immunisation (also observed by the above authors) may in part be a reflection of this lethargy in the treated animals; however, it may also be due to endogenous energy supply from lysed adipocytes as evidenced by the acute increase in plasma NEFA concentrations in treated animals. A short-term increase in plasma non-esterified fatty acid concentrations has also been observed in rats (Futter et al., 1992, Hu et al., 1992) and rabbits (Dulor et al., 1990) immunised with antibodies to adipose plasma membranes. The acute increase in plasma triacyglycerol and BHB concentrations reflect re-esterification and metabolism of the released NEFA,
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Table 5 Whole body and component weights in lambs passively immunised against adipose tissue a Trait
CON
TRT
S.E.
Significance
Pre-slaughter weight (kg) Lungs (g) Liver (g) Kidney (g) Heart (g) Kidney / pelvic fat (g) Omentum fat (g) Cold carcass (kg)
47.8 955 751 126 208 1549 1985 25.9
47.4 998 905 132 216 1120 1625 24.7
0.98 27.8 56.2 8.0 7.9 104.7 108.5 0.62
NS NS 0.06 NS NS * * NS
19.9 15.9 2.7 4.4 31.9 41.1 543
21.0 19.1 2.8 4.5 23.7 34.3 520
0.63 1.07 0.16 0.13 1.75 1.82 5.0
g / kg BW Lungs Liver Kidney Heart Kidney / pelvic fat Omentum fat Cold carcass
NS * NS NS ** * **
a
CON 5 Animals that received non-immune serum; TRT 5 animals that received antiserum to adipose tissue, NS 5 not significant; * 5 P , 0.05; ** 5 P , 0.01.
Table 6 Carcass characteristics of lambs passively immunised against adipose tissue a Trait Fat depth (mm) 6th rib 12th rib Leg Loin Area of longissimus thoracis et lumborum (cm 2 ) Conformation Fat score
CON
TRT
S.E.
Significance
1.5 3.9 13.6 4.1
1.3 2.8 10.5 3.0
0.23 0.40 0.87 0.34
NS * * *
20.4 3.2 3.6
18.9 2.6 3.5
0.50 0.15 0.21
1 * NS
a
CON 5 Animals that received non-immune serum; TRT 5 animals that received antiserum to adipose tissue, NS 5 not significant; 1 5 P , 0.1; * 5 P , 0.05.
respectively. No long-term differences in plasma glucose, insulin or insulin-like growth factor-1 concentrations were observed in rats (Futter and Flint, 1987; Panton et al., 1990), while Futter et al. (1992) observed no differences in plasma glucose or insulin concentrations in rats sampled up to 48 h after immunisation. Data in the present study support those of Moloney et al. (1998) which suggested that insulin resistance may develop in the earlier stages of immunisation (i.e., higher insulin concentrations in treated animals compared with control animals). This
may reflect an adaptation to a decreased capacity to remove glucose from the circulation and to store triacyglycerol. The decrease in PCV caused by immunisation is due to lysis in vivo or during blood sample collection (increased erythrocyte fragility) and may reflect the presence of common antigens on the adipocyte and erythrocyte plasma membranes. In all species examined to date, antisera to adipose tissue showed a relatively high degree of reactivity towards the adipocyte plasma membrane but reactivity towards
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Table 7 Composition of individual joints and total carcass from lambs passively immunised against adipose tissue a CON
TRT
S.E.
Leg (g / kg) Subcutaneous fat Intermuscular fat Total fat Lean Bone
Significance
83 35 118 726 156
76 31 107 730 163
4.3 2.5 4.6 4.5 2.3
NS NS * NS *
Rib (g / kg) Subcutaneous fat Intermuscular fat Total fat Lean Bone
116 189 305 533 162
122 146 268 551 181
8.2 11.7 11.8 10.3 3.9
NS * * NS **
Loin (g / kg) Subcutaneous fat Intermuscular fat Total fat Lean Bone
124 169 293 582 125
97 137 234 623 143
11.9 13.8 9.0 9.1 5.5
0.06 0.06 *** ** *
Carcass (g / kg) Fat Intermuscular Subcutaneous Total Meat Bone
114 104 218 623 149
97 100 197 639 159
6.1 4.2 7.3 5.8 2.1
* NS * NS **
a CON 5 Animals that received non-immune serum; TRT 5 animals that received antiserum to adipose tissue, NS 5 not significant, * 5 P , 0.05, ** 5 P , 0.01; *** 5 P , 0.01.
Table 8 Adipose tissue accretion (g / day) in lambs passively immunised against adipose tissue a Depot
CON
TRT
S.E.
Significance
Omentum Kidney / pelvic Intermuscular Subcutaneous Total carcass Lipid in pooled meat
15.1 12.8 21.2 16.2 37.4 8.34
11.6 8.1 15.3 12.5 27.9 8.96
1.14 1.30 1.75 1.35 2.72 0.58
* * * * * NS
a
CON 5 Animals that received non-immune serum; TRT 5 animals that received antiserum to adipose tissue, NS 5 not significant, * 5 P , 0.05.
other tissues was also observed (Flint et al., 1986; Dulor et al., 1990; Nasser and Hu, 1991b). However, much of this cross-reactivity could be removed by
adsorption to plasma membranes from non-adipose tissue preparations. The lack of a decrease in bodyweight gain contrasts with that observed by Nasser and Hu (1991a) and Moloney et al. (1998) and since estimated lean tissue accretion was not impaired by immunisation, the reductions in adiposity observed do not reflect a general impairment of growth. Moloney et al. (1998) examined the effect of the interval between immunisation and slaughter on fat deposition and noted a trend for the effect on internal adipose tissue depots to increase with time. In the present study, the SC fat depot, measured in vivo, appeared to recover with time after immunisation. In rabbits (Dulor et al., 1990), adipose tissue growth appeared similar in control and treated animals for up to 9 weeks after immunisation, i.e., the initial decrease in adipose
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tissue was maintained. In contrast, Panton et al. (1990) reported that differences in the weights of the perirenal and omentum depots in rats 3 weeks after immunisation had disappeared by 7 weeks. For most animals, correct timing of slaughter relative to immunisation is critical to achieve the maximum effect of treatment. The decrease of 18% in the weight of the omentum fat depot compared with corresponding value of 9% for the wether lambs in the study of Moloney et al. (1998), indicates that there is a dose–response pattern to immunisation. For commercial application, the optimum dose would need to be determined. A 46% reduction in perirenal adipose tissue weight with no reduction in carcass fat content due to immunisation of lambs was reported by Nasser and Hu (1991a). In contrast, Thornton and Tume (1987) failed to detect a reduction in adipose tissue deposition in sheep. Since the antibodies used were subsequently shown to be cytotoxic in vitro (Tume, 1991), insufficient antibodies may have been administered to cause a detectable reduction in fat deposition in vivo. Kestin et al. (1993) demonstrated that SC immunisation of pigs with antibodies to pig adipose tissue resulted in localised reductions in backfat thickness at the site of injection without alteration of the fat content of the foreloin joint. Similar results were obtained by Moloney et al. (1998). Immunisation via the intraperitoneal route in the studies of Kestin et al. (1993), resulted in a 25% reduction in the fat content of the foreloin joint, suggesting that this route rather than localised administration of antibodies is the more effective strategy. That the fat concentration of three of the four joints examined was decreased by immunisation in the present study indicates that our strategy of antiserum dispersal was successful. However, the visual effect of this strategy was that carcasses from immunised animals did not have the uniform SC fatness appearance of control carcasses. This appears to have resulted from a loss of integrity of SC adipose tissue which contributed to variable removal of adipose tissue during fleece removal. Such an effect could decrease the market value of the carcasses from animals immunised by this method. In some studies (Flint et al., 1986; Moloney et al., 1998), the effects of immunisation appeared to be adipose tissue depot specific with SC adipocytes
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more resistant than those from internal depots. Since equal weights of tissue from SC, perirenal and omentum depots were used to prepare plasma membranes in the present study and since tissue from the SC depot is more collagenous than that from internal depots (Jeffrey, 1983), the pool of plasma membranes may therefore contain relatively more membranes from, and consequently more antibodies to, the SC depot. However, differences in the scale of the decrease in size of the internal and carcass adipose tissue depots were not as pronounced (28, 18 and 16% for kidney / pelvic fat, omentum fat and carcass fat depots, respectively) in the present study. Adipose tissue accretion rates were calculated by reference to animals slaughtered at the beginning of the study, to estimate the true impact of immunisation. The marked inhibition of the rate of adipose tissue accretion (25% approximately for omentum, intermuscular, SC and total carcass fat and 37% for kidney / channel fat), with a 4% decrease in lean tissue growth illustrate the potential of this approach to decrease adiposity and increase carcass leanness in sheep. A decrease in intramuscular fat concentration accompanied the decreases in other fat depots due to immunisation. This decrease is in line with some consumer demands and was not of such a magnitude as to compromise the eating quality of the meat. In conclusion, the data from the present study confirm the potential of passive immunisation against adipose tissue plasma membranes as a means of decreasing adiposity in farm animals. However, to be commercially viable, this approach must be further developed so that decreases in the fat content of the carcass can be achieved without deterioration in the appearance of the carcass.
Acknowledgements The technical assistance of V. McHugh and M. Murray, and the secretarial assistance of M. Smith is gratefully acknowledged.
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Carroll, M.A., O’Carroll, F.M., 1964. Differences between left and right sides of lamb carcasses. Irish J. Agric. Res. 3, 223–237. Connolly, L., 1996. Competitiveness of Irish sheep production. Irish Grassland Anim. Prod. Assoc. J. 30, 168–177. Dulor, J.P., Reyne, Y., Nougues, J., 1990. In vivo effects of treatment with antibodies to adipocyte plasma membranes in the rabbit. Reprod. Nutr. Dev. 30, 49–58. Flint, D.J., Cograve, H., Futter, C.E., Gardner, J., Clarke, T.J., 1986. Stimulatory and cytotoxic effects of an antiserum to adipocyte plasma membranes on adipose tissue metabolism in vitro and in vivo. Int. J. Obesity 10, 69–77. Futter, C.E., Flint, D.J., 1987. Long-term reduction of adiposity in rats after passive immunisation with antibodies to rat fat cell plasma membranes. In: Berry, E.M., Blondheim, S.H., Eliahou, H.E., Shafrir, E. (Eds.). Recent Advances in Obesity Research, Vol. 5. Bibby, London, pp. 181–185. Futter, C.E., Panton, D., Kestin, S., Flint, D.J., 1992. Mechanism of action of cytotoxic antibodies to adipocytes on adipose tissue, liver and food intake in the rat. Int. J. Obesity 16, 615–622. Hu, C.Y., Suryawan, A., Killefer, J., Wehr, N.B., 1992. Effects of antiserum to rat adipocytes on growth and body composition of the rat. Comp. Biochem. Physiol. 101A, 669–673. Hudson, L., Hay, F.C., 1980. In: Practical Immunology. Blackwell, London, p. 1. Jeffrey, A.B., 1983. Studies On Beefburgers. 1. Effects of the Use of Different Fats On the Costs and Performance of Pure Beef Beefburgers. Research Report No. 406. Leatherhead Food, Leatherhead. Kestin, S., Kennedy, R., Tonner, E., Kiernan, M., Cryer, A.,
Griffin, H., Butterwith, S., Rhind, S., Flint, D., 1993. Decreased fat content and increased lean in pigs treated with antibodies to adipocyte plasma membranes. J. Anim. Sci. 71, 1486–1494. Moloney, A.P., Allen, P., Enright, W.J., 1998. Passive immunisation of sheep against adipose tissue: effects on metabolism, growth and body composition. Livest. Prod. Sci. 56, 233–244. Nassar, A.H., Hu, C.Y., 1991a. Growth and carcass characteristics of lambs passively immunised with antibodies developed against ovine adipocyte plasma membranes. J. Anim. Sci. 69, 578–586. Nasser, A.H., Hu, C.Y., 1991b. Antibodies to ovine adipocyte plasma membranes recognise tissue and species specific plasma membrane components. Comp. Biochem. Physiol. 98B, 361– 367. National Research Council, 1988. In: Designing Foods: Animal Product Options in the Marketplace. Committee on Technological Options to Improve the Nutritional Attributes of Animal Products. National Academy Press, Washington, DC, pp. 45–62. Panton, D., Futter, C., Kestin, S., Flint, D., 1990. Increased growth and protein deposition in rats treated with antibodies to adipocytes. Am. J. Physiol. 258, E985–E989. Thornton, R.D., Tume, R.K., 1987. Factors controlling fat deposition in ruminants. In: Farrell, D.J. (Ed.), Recent Advances in Animal Nutrition in Australia. University of New England Printery, pp. 28–49. Tume, R.K., 1991. Characterisation of polyclonal antibodies to cell-surface antigens from ovine adipose tissue. Biochem. Int. 25, 221–232.
Body composition and adipose tissue accretion in lambs passively immunised against adipose tissue a, b a ,1 A.P. Moloney *, P. Allen , W.J. Enright b
a Teagasc, Grange Research Centre, Dunsany, Co. Meath, Ireland Teagasc, The National Food Centre, Dunsinea, Castleknock, Co. Dublin, Ireland
Received 8 March 2001; received in revised form 26 July 2001; accepted 26 July 2001
Abstract Thirty-eight cross-bred wether lambs, initial liveweight 35.2160.75 kg were used to evaluate the effects on growth and carcass composition of passive immunisation with anti-ovine adipose plasma membrane immunoglobulin G (IgG). Antibodies were raised in donkeys by active immunisation with sheep adipocyte membrane protein in Freund’s complete adjuvant followed by boosting at 3, 5 and 15 weeks and subsequently at 4-month intervals with membrane protein in Freund’s incomplete adjuvant. An IgG fraction was prepared from pooled serum by ammonium sulphate precipitation and dialysis. Ten lambs were slaughtered and the remaining lambs (n 5 14 / group) were injected subcutaneously with 80 ml of IgG from immunised (treated) or non-immunised donkeys (control) on 4 consecutive days at the beginning of the study. Following a 91-day test period the animals were slaughtered, the weights of internal organs and fat depots recorded and carcass composition determined by dissection. Treatment did not affect preslaughter liveweight (Control 5 47.8 kg, Treated 5 47.4 kg) or carcass weight (Control 5 25.9 kg, Treated 5 24.7 kg). Liver weight tended to be increased (P 5 0.07) by treatment (from 751 to 905 g), while the weights of other organs were not affected. The weights of the omentum and kidney / channel fat depots were reduced (P , 0.05) by 18.1 and 27.7%, respectively, by treatment. Treatment decreased the weight of dissectable subcutaneous fat (from 2.7 to 2.4 kg, P , 0.1), dissectable intermuscular fat (from 2.9 to 2.3 kg, P , 0.05) and total dissectable fat (from 5.6 to 4.7 kg, P , 0.05) without a decrease in dissectable meat. Daily adipose tissue accretion was decreased by 25% approximately for omentum, intermuscular, subcutaneous and total carcass fat and by 37% for kidney / channel fat by treatment. Treatment resulted in an acute decrease in plasma glucose and insulin concentrations, an acute increase in non-esterified fatty acid and b-hydroxybutyrate concentrations and a chronic increase in insulin concentration. It is concluded that passive immunisation of lambs with anti-ovine adipose plasma membrane IgG can reduce body fatness without decreasing lean meat accretion. 2002 Elsevier Science B.V. All rights reserved. Keywords: Immunisation; Adipose tissue; Sheep; Carcass; Growth
*Corresponding author. Tel.: 1 353-046-26700; fax: 1 35346-26154. E-mail address: [email protected] (A.P. Moloney). 1 Present address: Intervet International BCV, Wim de Korverstraat 35, P.O. Box 31, 5830AA Boxmeer, The Netherlands.
1. Introduction Consumer interest in the fat content of meat has been heightened by medical advice to decrease their intake of total and saturated fat (e.g., National
0301-6226 / 02 / $ – see front matter 2002 Elsevier Science B.V. All rights reserved. PII: S0301-6226( 01 )00299-8
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Research Council, 1988). Moreover, much of the fat stored by domestic animals is discarded following slaughter and, as such, is a source of inefficiency in (lean) meat production. One approach to decreasing the fat content of domestic livestock is to manipulate the immune system to destroy existing fat cells (or adipocytes) with cytotoxic antibodies. The capacity of the animal to store fat would thereby be decreased, resulting in leaner meat. Flint et al. (1986) first demonstrated that antibodies raised in sheep against rat adipocyte plasma membranes were cytotoxic in vitro and caused a decrease in rat body fat in vivo. In pigs, Kestin et al. (1993) reported a 25% decrease in dissectable fat in the fore- and hind-limb joints due to passive immunisation with antibodies to pig adipocyte plasma membranes. In sheep, Thornton and Tume (1987) reported no effect on body fat content, and while Nassar and Hu (1991a) observed a 46% reduction in perirenal adipose tissue weight, there was no reduction in total carcass fatness due to passive immunisation with anti-sheep adipocyte plasma membrane sera. Recently, Moloney et al. (1998) reported that passive immunisation of lambs and ewes with antiadipocyte plasma membrane sera decreased the weight of selected internal fat depots and that the subcutaneous (SC) route of antiserum administration was superior to the intravenous route. However, while dissectable fat was decreased close to the site of SC immunisation, overall carcass fat accretion was not affected. The objective of this experiment was to progress this approach to decreasing adiposity by examining the effect of administration of a higher dose of antiserum with a more dispersed pattern of administration than that used in our previous study.
2. Materials and methods
2.1. Antibody preparation Preparation of adipose plasma membranes and antibody production were as described by Moloney et al. (1998). In brief, plasma membranes were prepared from a pool of subcutaneous, perirenal and omentum adipose tissue by a combination of homogenisation, density gradient and differential centrifugation. Enriched plasma membranes, emulsified with
Freund’s complete adjuvant were used to immunise donkeys by SC injection at four sites in the neck ventral to the jugular groove. Booster injections using Freund’s incomplete adjuvant were administered after 3, 5 and 15 weeks, and subsequently at 4-month intervals. Additional donkeys were similarly immunised, but using buffer instead of adipocyte plasma membrane protein to provide control serum. Blood was collected at intervals throughout the antibody production phase of the study, allowed to clot overnight at 48C and centrifuged at 2000 g for 30 min. Serum was stored at 2 208C. For immunisation, serum was pooled and the immunoglobulin G (IgG) fraction precipitated using ammonium sulphate (Hudson and Hay, 1980). Thus, to 4 l of pooled serum from control donkeys or 4 l pooled serum from membrane-immunised donkeys, 1.164 kg ammonium sulphate was slowly added (over 4 h) while stirring at 48C. The serum was stirred at 48C overnight and then centrifuged at 2000 g for 90 min. The precipitate was dissolved in 70 ml phosphate-buffered saline (PBS) and dialysed against 9 l PBS with two changes of buffer. After dialysis, no residual ammonia was detected using Nessler’s reagent and the IgG solution was adjusted to 5 l with PBS.
2.2. Passive immunisation of sheep Thirty-eight Finnish–Landrace wether lambs, approximately 8 months old were penned together and offered straw in decreasing quantities (ad libitum at the beginning) and increasing quantities of whole barley until the ad libitum level of intake of unsupplemented barley was achieved. This phase lasted approximately 3 weeks. The animals were then offered ad libitum a pelleted concentrate diet consisting of (g / kg) 700 barley, 100 soyabean meal, 150 grain screenings, 30 molasses and 20 vitamin / mineral premix. The concentrate contained 155 g crude protein (CP), 91 g crude fibre and 56 g ash / kg dry matter (DM). Animals were weighed on 2 consecutive days, and 10 were chosen at random for slaughter at the beginning of the study. The remainder were blocked in pairs on a descending bodyweight basis (mean of both weights) and within block assigned at random to receive control IgG (Control) or anti-ovine adipocyte plasma membrane
A.P. Moloney et al. / Livestock Production Science 74 (2002) 165 – 174
IgG (Treated). The animals were penned individually and offered fresh feed daily at 10:30 h. Feed intake was measured daily until slaughter 91 days after first immunisation. Animals were injected SC with 10 ml IgG at each of four sites on both sides of the midline. The immunisation procedure was repeated on 4 consecutive days. Upon completion of immunisation on day 1 and for 4 subsequent days, lambs received 3 ml penicillin and 3 ml streptomycin. Subcutaneous fat depth was measured bilaterally over the 12th rib in vivo using an A-mode ultrasound scanner (Delphi Instruments, New Zealand) on days 33, 61 and 85 after the completion of immunisation. The mean of the bilateral readings for each day was used in the statistical analyses. Animals were weighed at weekly intervals (at 10:00 h) and on 2 consecutive days prior to slaughter. Blood samples (10 ml) were taken immediately before the first immunisation and then periodically (at 13:30 h) by jugular venipuncture using vacutainers containing either sodium EDTA or potassium oxalate / sodium fluoride as anticoagulants. Blood was centrifuged at 2000 g and the plasma stored at 2 208C. On the day of slaughter, animals were stunned prior to exsanguination and carcass weight, fleece weight and the weights of individual offal components were recorded. The carcasses were chilled at 08C for 48 h, split medially and carcass composition determined by cutting the left side into joints based on the method of Carroll and O’Carroll (1964). The cross-sectional area of the longissimus thoracis et lumborum muscle (LTL) at the 12–13th rib interface and SC fat depth over the sixth and 12th rib, the leg and the loin were measured. Each joint was dissected into subcutaneous and intermuscular fat, lean and bone and the weights of each tissue recorded. All lean tissue was pooled, minced, subsampled and stored at 2 208C for subsequent chemical analysis. A sample of LTL was stored at 2 208C for subsequent chemical analysis.
2.3. Chemical and biological analyses Feed samples were analysed for DM, CP, crude fibre and ash, and tissue samples for CP (Kjeldahl), ash, moisture and total fat by standard procedures (AOAC, 1990). Antibodies were quantified using the modification of the assay of Flint et al. (1986) as
167
described by Moloney et al. (1998). Plasma samples were analysed for insulin and metabolites as previously described (Moloney et al., 1998).
2.4. Statistical analyses The relationship between bodyweight or carcass weight and body components of the animals slaughtered at the beginning of the experiment was examined using linear regression. These relationships were then used to calculate accretion rates of various tissues for each animal over the course of the study. All animal data were subjected to analysis of variance using a model that had block and treatment as sources as variation. Weekly feed intake, periodic bodyweight and blood data were analysed at each measurement time using the above model. A onesided t-test was used for all measurements of fatness, since the hypothesis tested was that treatment would have no effect or cause a decrease (but never increase) in fatness.
3. Results The descriptive statistics of the lambs slaughtered at the beginning of the study are shown in Table 1. The relationships used to estimate the initial composition of the remaining animals are shown in Table 2. Table 1 Characteristics of lambs (n 5 10) slaughtered at the beginning of the study Variable Whole body (kg) Cold carcass (kg) Omentum fat (g) Kidney / channel fat (g) Carcass composition (g) Fat Subcutaneous Intermuscular Total Meat Bone Lipid in pooled carcass meat (g / kg) Lipid in LTL a (g / kg) a
Longissimus thoracis et lumborum.
Mean 34.4 16.2 560 387
1164 968 2132 11 159 2908 73 29
S.D. 4.41 2.53 233.7 77.7
459.2 389.3 800.3 178.5 355.7 8.6 6.2
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Table 2 Relationships between bodyweight (BW) and carcass weight (CAR) or internal adipose tissue depots, and between CAR and composition in lambs slaughtered at the beginning of the study
CAR weight (kg) Kidney / channel fat (g) Omentum fat (g) CAR fat (g) Subcutaneous Intermuscular Total Meat (g) Lipid in pooled carcass lean (g) a
R.S.D.
R2
Significance a
0.55 (BW) 2 2.8 11.5 (BW) 2 7.5 43.1 (BW) 2 922
0.74 62.6 144.4
0.92 0.42 0.66
*** * **
158 (CAR) 2 1398 138 (CAR) 2 1263 296 (CAR) 2 2661 563 (CAR) 1 2032 61.5 (CAR) 2 186
274.1 211.4 377.2 373.5 192.6
0.71 0.75 0.82 0.94 0.76
** ** *** ** **
* 5 P , 0.05; ** 5 P , 0.01; *** 5 P , 0.001.
3.1. Antiserum
3.3. Live animal variables
The binding pattern of the anti-adipose tissue IgG preparation to immobilised plasma membranes is shown in Fig. 1. For comparison, the binding pattern of the pooled unfractionated antiserum used by Moloney et al. (1998), measured in the same assay, is also shown in Fig. 1.
Bodyweight was greater (P , 0.05) in control than treated animals up to day 37 after first immunisation (Fig. 2). Treatment decreased (P , 0.001) daily DM intake during the week of immunisation (1.29 vs. 0.65 kg / animal) and during the first week after immunisation (1.40 and 0.97 kg / animal). Feed conversion efficiency data over the whole experiment are summarised in Table 3. There was no effect of treatment on any efficiency variables. Ultrasoundestimated backfat was lower (P , 0.05) at all measurement times in treated animals (Table 3). Mean packed cell volume (PCV) before immunisation was 35.563.21 (S.D.). From the day after completion of immunisation (day 4) until approximately 5 weeks later, PCV was lower (P , 0.05) in the treated group (Fig. 3). Treatment resulted in an immediate decrease in plasma glucose and insulin concentrations, an immediate increase in non-esterified fatty acid (NEFA) and b-hydroxybutyrate
3.2. Immediate effects of immunisation There were no obvious immediate effects of injection with control or immune sera on any of the 4 days of immunisation. During the 4 days of immunisation, the anti-adipocyte plasma membrane sera had an apparent sedating effect on most sheep. However, this effect was absent from day 6 of the experiment onwards.
Fig. 1. Binding of I 125 -protein A to an immobilised donkey immunoglobulin G-adipocyte plasma membrane complex. (j) Immunoglobulin G from donkeys immunised against adipose tissue plasma membranes. (m) Sera used by Moloney et al. (1998). (d) Immunoglobulin G from donkeys immunised against buffer.
Fig. 2. Bodyweight of control sheep (d) and of sheep immunised with antibodies to adipocyte plasma membranes (j).
A.P. Moloney et al. / Livestock Production Science 74 (2002) 165 – 174
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Table 3 Growth, efficiency and backfat depth in lambs passively immunised against adipose tissue a Trait
CON
TRT
S.E.
Significance
Initial weight (kg) Dry matter intake (DMI; g / day) Growth (g / day) Whole body Carcass Lean tissue Efficiency (g / kg DMI) Whole body Carcass Lean tissue Backfat depth (mm) Day 33 Day 61 Day 85
35.5 1294
35.1 1201
0.48 36.1
NS NS
132 99 52
138 91 50
11.0 7.0 3.3
NS NS NS
101 77 40
115 75 41
8.0 5.0 2.6
NS NS NS
0.10 0.12 0.12
** * *
2.0 2.1 2.2
1.5 1.7 1.8
a CON 5 Animals that received non-immune serum; TRT 5 animals that received antiserum to adipose tissue; NS 5 not significant; * 5 P , 0.05; ** 5 P , 0.01.
Fig. 3. Packed cell volume (PCV) in control sheep (d) and in sheep immunised with antibodies to adipocyte plasma membranes (j).
(BHB) concentrations and a chronic increase in insulin concentration (Table 4).
3.4. Body composition Treatment decreased (P , 0.05) the weights of the kidney / pelvic and omentum fat depots and increased (P , 0.10) the weight of the liver when expressed either in absolute terms or as a proportion of preslaughter BW (Table 5). Treatment did not affect carcass weight in absolute terms but decreased (P , 0.01) carcass weight when expressed as a proportion of pre-slaughter BW (Table 5). Treatment decreased (P , 0.05) the depth of fat covering the 12th rib and the leg and loin joints, without an effect on fat score,
decreased (P , 0.05) carcass conformation (Table 6), did not affect the composition of the shoulder joint (data not shown), decreased (P , 0.05) the total fat proportion of the leg, rib and loin joints, and increased (P , 0.05) the lean proportion of the loin joint (Table 7). The weights and proportions of intermuscular and total dissectable fat were decreased (P , 0.05) and the proportion of bone in the carcass increased (P , 0.01) by treatment. Treatment tended (P , 0.10) to increase the proportion of moisture and protein [717 and 729 g / kg (S.E. 3.7), and 220 and 226 g / kg (S.E. 2.0) for control and treated lambs, respectively] and decreased (P , 0.05) the proportion of fat (50 vs. 37 g / kg) in the LTL. The rate of fat accretion in internal depots and in the carcass was decreased (P , 0.05) due to treatment (Table 8) but deposition of lipid in carcass lean meat was not affected.
4. Discussion The wether lambs used were typical of those in an Irish lamb production system (Connolly, 1996) and their mean carcass weight was similar to that (23.8 kg) of the wethers used by Moloney et al. (1998). An immunological procedure may be a more acceptable approach to satisfy consumer and producer demands for decreased fat in meat animals than the
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170
Table 4 Temporal changes in plasma metabolite and insulin concentrations Variable
Treatment a
Day (post immunisation) 0
1
6
12
27
55
67
88
Glucose (mmol / l)
CON TRT S.E.D. Significance
5.21 5.32 0.178 NS
5.33 3.99 0.201 *
5.44 5.01 0.218 NS
5.15 4.90 0.148 NS
4.91 4.87 0.175 NS
3.51 3.72 0.429 NS
4.66 4.83 0.249 NS
4.99 4.63 0.187 NS
TAG b (mequiv. / l)
CON TRT S.E.D. Significance
86 101 10.3 NS
66 98 9.4 *
74 73 7.6 NS
82 83 5.4 NS
65 64 7.0 NS
135 94 28.8 NS
119 138 24.7 NS
155 142 18.8 NS
NEFAc (mequiv. / l)
CON TRT S.E.D. Significance
79 86 18.2 NS
74 388 54.0 *
111 251 34.1 *
38 55 33.3 NS
72 19 24.1 *
205 114 45.6 NS
105 113 28.5 NS
173 254 84.2 NS
BHB d (mmol / l)
CON TRT S.E.D. Significance
395 446 32.0 NS
294 478 61.2 *
294 320 48.9 NS
329 324 58.5 NS
281 224 31.7 NS
290 310 58.4 NS
244 324 55.0 NS
144 186 29.9 NS
Insulin (mU / ml)
CON TRT S.E.D. Significance
30.1 26.7 6.64 NS
27.2 14.1 2.46 *
28.0 32.2 4.32 NS
21.9 43.7 6.19 *
30.8 46.6 6.71 *
24.8 30.2 4.47 NS
– – –
15.1 17.5 25.1 NS
a
CON 5 Animals that received non-immune serum; TRT 5 animals that received antiserum to adipose tissue, NS 5 not significant; * 5 P , 0.05. b TAG 5 Triacylglycerol. c NEFA 5 Non-esterified fatty acid. d BHB 5 b-Hydroxybutyrate.
use of exogenous hormones or antibiotics. Moloney et al. (1998) demonstrated the potential of adipocyte destruction by treatment with antibodies to proteins on the adipocyte plasma membrane as a means of decreasing fat accretion in sheep. The objective of this study was to develop this concept further by using a higher concentration of antibodies (and a larger number of animals) and a more dispersed pattern of administration than used previously. A larger quantity (320 ml vs. 150 ml) of a higher titre (24 vs. 17% bound at 1:10 000 dilution) was administered at eight injection sites / 4 days compared to six injection sites / 3 days by Moloney et al. (1998). The apparent sedative effect of passive immunisation against adipocyte plasma membranes has been observed in rats (Futter et al., 1992, Hu et al., 1992),
pigs (Kestin et al., 1993) and sheep (Moloney et al., 1998), and may reflect the production of immune complexes (sub-fatal anaphylaxis) in these animals. The decrease in feed consumption in the week after immunisation (also observed by the above authors) may in part be a reflection of this lethargy in the treated animals; however, it may also be due to endogenous energy supply from lysed adipocytes as evidenced by the acute increase in plasma NEFA concentrations in treated animals. A short-term increase in plasma non-esterified fatty acid concentrations has also been observed in rats (Futter et al., 1992, Hu et al., 1992) and rabbits (Dulor et al., 1990) immunised with antibodies to adipose plasma membranes. The acute increase in plasma triacyglycerol and BHB concentrations reflect re-esterification and metabolism of the released NEFA,
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Table 5 Whole body and component weights in lambs passively immunised against adipose tissue a Trait
CON
TRT
S.E.
Significance
Pre-slaughter weight (kg) Lungs (g) Liver (g) Kidney (g) Heart (g) Kidney / pelvic fat (g) Omentum fat (g) Cold carcass (kg)
47.8 955 751 126 208 1549 1985 25.9
47.4 998 905 132 216 1120 1625 24.7
0.98 27.8 56.2 8.0 7.9 104.7 108.5 0.62
NS NS 0.06 NS NS * * NS
19.9 15.9 2.7 4.4 31.9 41.1 543
21.0 19.1 2.8 4.5 23.7 34.3 520
0.63 1.07 0.16 0.13 1.75 1.82 5.0
g / kg BW Lungs Liver Kidney Heart Kidney / pelvic fat Omentum fat Cold carcass
NS * NS NS ** * **
a
CON 5 Animals that received non-immune serum; TRT 5 animals that received antiserum to adipose tissue, NS 5 not significant; * 5 P , 0.05; ** 5 P , 0.01.
Table 6 Carcass characteristics of lambs passively immunised against adipose tissue a Trait Fat depth (mm) 6th rib 12th rib Leg Loin Area of longissimus thoracis et lumborum (cm 2 ) Conformation Fat score
CON
TRT
S.E.
Significance
1.5 3.9 13.6 4.1
1.3 2.8 10.5 3.0
0.23 0.40 0.87 0.34
NS * * *
20.4 3.2 3.6
18.9 2.6 3.5
0.50 0.15 0.21
1 * NS
a
CON 5 Animals that received non-immune serum; TRT 5 animals that received antiserum to adipose tissue, NS 5 not significant; 1 5 P , 0.1; * 5 P , 0.05.
respectively. No long-term differences in plasma glucose, insulin or insulin-like growth factor-1 concentrations were observed in rats (Futter and Flint, 1987; Panton et al., 1990), while Futter et al. (1992) observed no differences in plasma glucose or insulin concentrations in rats sampled up to 48 h after immunisation. Data in the present study support those of Moloney et al. (1998) which suggested that insulin resistance may develop in the earlier stages of immunisation (i.e., higher insulin concentrations in treated animals compared with control animals). This
may reflect an adaptation to a decreased capacity to remove glucose from the circulation and to store triacyglycerol. The decrease in PCV caused by immunisation is due to lysis in vivo or during blood sample collection (increased erythrocyte fragility) and may reflect the presence of common antigens on the adipocyte and erythrocyte plasma membranes. In all species examined to date, antisera to adipose tissue showed a relatively high degree of reactivity towards the adipocyte plasma membrane but reactivity towards
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172
Table 7 Composition of individual joints and total carcass from lambs passively immunised against adipose tissue a CON
TRT
S.E.
Leg (g / kg) Subcutaneous fat Intermuscular fat Total fat Lean Bone
Significance
83 35 118 726 156
76 31 107 730 163
4.3 2.5 4.6 4.5 2.3
NS NS * NS *
Rib (g / kg) Subcutaneous fat Intermuscular fat Total fat Lean Bone
116 189 305 533 162
122 146 268 551 181
8.2 11.7 11.8 10.3 3.9
NS * * NS **
Loin (g / kg) Subcutaneous fat Intermuscular fat Total fat Lean Bone
124 169 293 582 125
97 137 234 623 143
11.9 13.8 9.0 9.1 5.5
0.06 0.06 *** ** *
Carcass (g / kg) Fat Intermuscular Subcutaneous Total Meat Bone
114 104 218 623 149
97 100 197 639 159
6.1 4.2 7.3 5.8 2.1
* NS * NS **
a CON 5 Animals that received non-immune serum; TRT 5 animals that received antiserum to adipose tissue, NS 5 not significant, * 5 P , 0.05, ** 5 P , 0.01; *** 5 P , 0.01.
Table 8 Adipose tissue accretion (g / day) in lambs passively immunised against adipose tissue a Depot
CON
TRT
S.E.
Significance
Omentum Kidney / pelvic Intermuscular Subcutaneous Total carcass Lipid in pooled meat
15.1 12.8 21.2 16.2 37.4 8.34
11.6 8.1 15.3 12.5 27.9 8.96
1.14 1.30 1.75 1.35 2.72 0.58
* * * * * NS
a
CON 5 Animals that received non-immune serum; TRT 5 animals that received antiserum to adipose tissue, NS 5 not significant, * 5 P , 0.05.
other tissues was also observed (Flint et al., 1986; Dulor et al., 1990; Nasser and Hu, 1991b). However, much of this cross-reactivity could be removed by
adsorption to plasma membranes from non-adipose tissue preparations. The lack of a decrease in bodyweight gain contrasts with that observed by Nasser and Hu (1991a) and Moloney et al. (1998) and since estimated lean tissue accretion was not impaired by immunisation, the reductions in adiposity observed do not reflect a general impairment of growth. Moloney et al. (1998) examined the effect of the interval between immunisation and slaughter on fat deposition and noted a trend for the effect on internal adipose tissue depots to increase with time. In the present study, the SC fat depot, measured in vivo, appeared to recover with time after immunisation. In rabbits (Dulor et al., 1990), adipose tissue growth appeared similar in control and treated animals for up to 9 weeks after immunisation, i.e., the initial decrease in adipose
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tissue was maintained. In contrast, Panton et al. (1990) reported that differences in the weights of the perirenal and omentum depots in rats 3 weeks after immunisation had disappeared by 7 weeks. For most animals, correct timing of slaughter relative to immunisation is critical to achieve the maximum effect of treatment. The decrease of 18% in the weight of the omentum fat depot compared with corresponding value of 9% for the wether lambs in the study of Moloney et al. (1998), indicates that there is a dose–response pattern to immunisation. For commercial application, the optimum dose would need to be determined. A 46% reduction in perirenal adipose tissue weight with no reduction in carcass fat content due to immunisation of lambs was reported by Nasser and Hu (1991a). In contrast, Thornton and Tume (1987) failed to detect a reduction in adipose tissue deposition in sheep. Since the antibodies used were subsequently shown to be cytotoxic in vitro (Tume, 1991), insufficient antibodies may have been administered to cause a detectable reduction in fat deposition in vivo. Kestin et al. (1993) demonstrated that SC immunisation of pigs with antibodies to pig adipose tissue resulted in localised reductions in backfat thickness at the site of injection without alteration of the fat content of the foreloin joint. Similar results were obtained by Moloney et al. (1998). Immunisation via the intraperitoneal route in the studies of Kestin et al. (1993), resulted in a 25% reduction in the fat content of the foreloin joint, suggesting that this route rather than localised administration of antibodies is the more effective strategy. That the fat concentration of three of the four joints examined was decreased by immunisation in the present study indicates that our strategy of antiserum dispersal was successful. However, the visual effect of this strategy was that carcasses from immunised animals did not have the uniform SC fatness appearance of control carcasses. This appears to have resulted from a loss of integrity of SC adipose tissue which contributed to variable removal of adipose tissue during fleece removal. Such an effect could decrease the market value of the carcasses from animals immunised by this method. In some studies (Flint et al., 1986; Moloney et al., 1998), the effects of immunisation appeared to be adipose tissue depot specific with SC adipocytes
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more resistant than those from internal depots. Since equal weights of tissue from SC, perirenal and omentum depots were used to prepare plasma membranes in the present study and since tissue from the SC depot is more collagenous than that from internal depots (Jeffrey, 1983), the pool of plasma membranes may therefore contain relatively more membranes from, and consequently more antibodies to, the SC depot. However, differences in the scale of the decrease in size of the internal and carcass adipose tissue depots were not as pronounced (28, 18 and 16% for kidney / pelvic fat, omentum fat and carcass fat depots, respectively) in the present study. Adipose tissue accretion rates were calculated by reference to animals slaughtered at the beginning of the study, to estimate the true impact of immunisation. The marked inhibition of the rate of adipose tissue accretion (25% approximately for omentum, intermuscular, SC and total carcass fat and 37% for kidney / channel fat), with a 4% decrease in lean tissue growth illustrate the potential of this approach to decrease adiposity and increase carcass leanness in sheep. A decrease in intramuscular fat concentration accompanied the decreases in other fat depots due to immunisation. This decrease is in line with some consumer demands and was not of such a magnitude as to compromise the eating quality of the meat. In conclusion, the data from the present study confirm the potential of passive immunisation against adipose tissue plasma membranes as a means of decreasing adiposity in farm animals. However, to be commercially viable, this approach must be further developed so that decreases in the fat content of the carcass can be achieved without deterioration in the appearance of the carcass.
Acknowledgements The technical assistance of V. McHugh and M. Murray, and the secretarial assistance of M. Smith is gratefully acknowledged.
References AOAC, 1990. Official Methods of Analysis, 15th Edition. Association of Official Analytical Chemists, Washington, DC.
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