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Manipulation of growth in pigs through treatment of the neonate with clenbuterol and somatotropin¹

M. N. Sillence^*,2, K. J. Munn^* and R. G. Campbell^,3

^* School of Agriculture, Charles Sturt University, Wagga Wagga, New South Wales 2768, Australia and and Bunge Meat Industries, Corowa, New South Wales, Australia

² Correspondence:
PO Box 588 (fax [international]: 61 2–69332995; E-mail:
msillence{at}csu.edu.au).

	Abstract

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Neonatal pigs were treated with lipolytic agents to determine whether this would cause a long-term decrease in their ability to deposit fat, with a consequent increase in muscle growth and feed efficiency. Groups of 25 female piglets were given clenbuterol (100 µg/kg BW), porcine somatotropin (pST; 100 µg/kg BW), pST plus clenbuterol, or saline injections from 3 d to 40 d of age. Five piglets from each group were then slaughtered to determine body composition. Clenbuterol and pST both increased ADG up to weaning when given separately (24%, P < 0.05; 20%, P < 0.1 respectively) but did not reduce fat deposition. In contrast, pigs given clenbuterol plus pST showed no increase in ADG and a 41% reduction in carcass fat (P < 0.05). Clenbuterol caused a marked decrease in ß₂-adrenoceptor density in porcine adipose tissue (P < 0.001) and skeletal muscle (P < 0.01). This effect was attenuated by concurrent pST treatment, which helps to explain the synergistic effect of these drugs on fat deposition. Once the drugs were withdrawn at 40 d, the anabolic effect of pST gradually disappeared, so that the live weight of pST-treated and control pigs was identical at 168 d. Clenbuterol withdrawal caused the rapid loss of extra weight gained, plus an additional 4 to 5 kg live weight that was never recovered. During the 4-wk finishing period there was an increase in feed intake in pigs that had previously undergone treatment with pST (23%, P < 0.1), with no increase in ADG, and so feed efficiency was impaired (P < 0.05). Pigs that were treated with pST plus clenbuterol showed no marked increase in feed intake during this period. Carcasses from clenbuterol-treated pigs tended to be leaner at 168 d, but there was no long-term effect of pST or the combined treatment on carcass composition. Overall, the treatment of neonatal pigs with repartitioning agents was counter-productive, due to the withdrawal effects of the ß-adrenergic agonist and the delayed long-term effect of pST on feed intake.

Key Words: ß-Adrenergic Agonists • Growth • Lipolysis • Newborn Animals • Pigs • Somatotropin

	Introduction

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When McCutcheon et al. (1994) administered somatotropin (ST) to neonatal lambs for 3 mo, then stopped the injections, the treated lambs were still leaner than controls 5 mo later. This treatment may have caused a long-term effect on body composition by interfering with fat cell differentiation in the young animal. If a long-term reduction in body fat could be achieved through the short-term treatment of neonatal pigs with a repartitioning agent, this could have several commercial advantages. Reduced fat deposition can lead indirectly to improvements in muscle growth and feed efficiency, as shown in experiments in which older rats and pigs were given a vaccine that destroys fat cells (Panton et al., 1990; Kestin et al., 1993). An antilipogenic effect could be achieved using porcine somatotropin (pST) (Dunshea, 1993), perhaps with fewer daily injections than are needed to cause repartitioning in finishing pigs. Alternatively, a ß-adrenergic agonist could be used, without the risk of tissue residues being found at the time of slaughter.

To evaluate this concept, the present study was designed to measure the long-term consequences of treating neonatal pigs with clenbuterol and(or) pST for 6 wk. The combination of pST with a ß-adrenergic agonist can have a powerful effect on fat deposition in finishing pigs (Hansen et al., 1997). In the present experiment we also tested the hypothesis that the effect on adipose tissue involves a common mechanism through the regulation of ß-adrenoceptors.

	Materials and Methods

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Effect of Clenbuterol and pST Treatment on Neonatal Pigs
Pigs and Treatments.
One hundred twenty neonatal piglets (Large White x Landrace) born over a 72-h period were identified from a commercial herd at Bunge Meat Industries (Corowa, NSW, Australia). Only female piglets were used in this experiment because the scope for reducing fat deposition and improving feed efficiency was believed to be greater in females than in males. Each sow was housed in an individual pen together with her litter. The piglets were fostered where necessary so that each piglet shared a sow with eight others. Within each litter the piglets were allocated at random to one of four treatment groups: controls, clenbuterol-treated, pST-treated, or clenbuterol plus pST-treated. Each treatment group contained 30 piglets initially, but this was reduced to 25 per group over the course of the experiment due to mortalities and other failures to thrive believed to be unrelated to the treatments imposed. Color-coded ear tags were used to identify individual animals and their respective treatment groups.

Commencing at 3 d of age (mean BW 1.77 ± 0.1 kg) each piglet was given a daily injection of saline, pST, clenbuterol, or clenbuterol plus pST. The pST was given at a dose of 100 µg/kg BW dissolved in 2 mL of a buffered solution (pH 9.5) supplied with the product (Reporcin, Southern Cross Biotech Pty. Ltd., Toorak, Victoria, Australia). Clenbuterol was supplied by Nanjing Agricultural University (Nanjing, China). When the ß-adrenergic agonist was given alone it was dissolved in 2 mL isotonic saline. The drug was introduced gradually, using a dose of 25 µg/kg BW for the first 3 d, 50 µg/kg BW for the next 3 d, and 100 µg/kg BW thereafter. The combined treatment was given as a single 2-mL injection, with clenbuterol dissolved in the pST buffer. Doses were based on previous dose-response experiments in grower pigs using pST (Etherton et al., 1987) or clenbuterol (M. N. Sillence, unpublished observations), and were designed to maximize the loss of fat while minimizing the risk of adverse effects.

The pigs were weighed every 7 d. At 28 d of age the pigs were weaned and were moved into a different shed, where they were housed together with members of the same treatment group in pens containing 25 to 30 animals each. The pigs were allowed free access to water and to a stage 1 weaner diet (Table 1). The injections were continued until the pigs were between 40 and 41 d old, when the saline and pST treatments were stopped abruptly. However, in an attempt to avoid possible withdrawal effects of clenbuterol (Sillence, 1996), the dose of ß-adrenergic agonist was decreased gradually during the last 6 d of this period by following the reverse of the dose regimen used when the drug was introduced (50 µg/kg BW for 3 d, then 25 µg/kg BW for 3 d).

A rectangle of skin (approximately 10 x 20 cm) was quickly removed from the back, extending from the base of the neck to the rump, and the subcutaneous adipose tissue (50 to 70 g) was dissected free. The adipose tissue was collected as soon as possible after each pig was killed and was processed immediately into cell membrane fragments as described below. A sample from the center of the longissimus muscle (approximately 30 g) was removed also and frozen in liquid nitrogen, then stored at -80°C until it was processed into membrane fragments at a later date.

After the removal of muscle and fat samples, the carcasses were eviscerated and weighed hot. The weights of the heart, kidneys, and liver were recorded. The carcasses were chilled for 24 h at 4°C, and the weights of the cold carcasses were recorded before they were analyzed for water, fat, and protein content. Carcasses were initially sectioned using a band-saw and then ground without deboning through a 9-mm mesh, followed by a 4-mm and 2-mm mesh. Subsamples of carcass mince were analyzed for water content by freeze-drying to a constant weight. Nitrogen content was measured on the freeze-dried sample following Kjeldahl digestion (Williams and Twine, 1967), and lipid content was determined on another portion of the freeze-dried sample by Soxhlet extraction using ether as the solvent. Carcass water, fat, and protein content (nitrogen x 6.25) were extrapolated from these values, taking into account the weight of the tissue samples removed at slaughter.

Cell Membrane Preparation.
Fat cell membranes were prepared from each pig on the day of killing, to allow the measurement of ß-adrenoceptor density and enzyme activity after long-term treatment with pST or clenbuterol, using methods described previously (Sillence and Matthews, 1994). The backfat removed from each pig was homogenized in ice-cold buffer (50 mM Tris 7.0; 5 mM MgCl₂; 1 mM EGTA; pH 7.4 at 4°C) then centrifuged at 1,000 x g for 10 min at 4°C. The supernatant was filtered through a triple layer of surgical gauze then centrifuged for a further 15 min at 10,000 x g. Next, the supernate was collected and centrifuged at 100,000 x g for 30 min. The supernate was discarded and the pellet resuspended in 2 mL buffer (50 mM Tris 7.7; 10 mM MgCl₂; 0.15 M NaCl; pH 7.4 at 37°C). A fraction of the cell membrane preparation (50 µL) was used for the measurement of protein concentration using the Bradford Protein Assay (Bio-Rad, Hercules, CA), and the remainder was stored at -80°C until assayed for cAMP production or ß-adrenoceptor density.

Cell membranes were also prepared from samples of longissimus muscle that had been collected and frozen on the day of slaughter. The procedure followed was the same as that described for fat-cell membranes. The muscle preparation could be used to measure ß-adrenoceptor density, because ß-adrenoceptors retain their ligand-binding activity whether prepared from fresh or frozen tissue (M. N. Sillence and M. L. Matthews, unpublished observation). Measurements of second-messenger coupling were not made in muscle membranes, however, because adenylyl cyclase activity can only be measured reliably if the tissue is from a freshly killed animal and is processed prior to freezing (Sillence and Matthews, unpublished observation). In the present study it was not possible to process both the muscle and adipose tissue membranes simultaneously.

Measurement of ß-Adrenoceptor Density.
ß-Adrenoceptor density was measured to determine whether receptor down-regulation could account for the tolerance that pigs develop when treated chronically with ß-adrenergic agonists (Spurlock et al., 1994), or whether up-regulation could be a means by which pST causes its lipolytic effects (Dunshea, 1993).

Fat-cell membranes were thawed then incubated for 1 h at 37°C in a shaking water bath with a saturating concentration of the radioligand ³H-CGP12177 (NEN, Boston, MA). The reaction was stopped by the addition of 3 mL of ice-cold buffer (50 mM Tris 7.7; 10 mM MgCl₂; 0.15 M NaCl), and the contents of each tube were filtered over glass-fiber filter papers (Whatman GF/C Crown Scientific, Moorebank, NSW, Australia) to remove any unbound radioligand. This was done using a Brandel 48-well cell harvester modified for this purpose (Beckman Instruments, Sydney, NSW, Australia). Radioligand that was bound to the cell membrane fragments was retained on the filter papers, and this was measured by soaking each filter overnight in 4 mL of scintillation fluid (Emulsifier-Safe, Canberra-Packard, Five Dock, NSW, Australia), before shaking thoroughly and counting each tube in a liquid scintillation beta-counter (LKB-Wallac, Turku, Finland).

All assay tubes were prepared in triplicate and were balanced using an appropriate amount of incubation buffer (50 mM Tris 7.7; 10 mM MgCl₂; 0.15 M NaCl; pH 7.4 at 37°C) so that each tube contained a volume of 250 µL. All the tubes contained cell membrane (100 µL) and ³H-CGP12177 (50 µL), which allowed us to measure total binding of the radioligand. One set of tubes also contained a nonselective ß-adrenoceptor antagonist (l-propranolol; 2 µM; Sigma Chemical, St Louis, MO) and a third set of tubes contained a highly-selective ß₁-adrenoceptor antagonist (CGP20712A; 10 µM; CIBA-Geigy, Basel, Switzerland), which enabled us to determine, by difference, the quantity of radioligand that was bound to ß₁-adrenoceptors, the quantity bound to ß₂-adrenoceptors, and the quantity bound to nonspecific (nonreceptor) binding sites (Sillence and Matthews, 1994).

ß-Adrenoceptor density was measured in muscle cell membranes following the same incubation, filtering, and counting procedure as that used for adipose tissue. However, because the muscle cells had no detectable ß₁-adrenoceptors and yielded a much higher concentration of membrane protein than fat cells, the measurement of ß₂-adrenoceptor density could be made with greater precision. This was achieved by incubating the membranes with a range of concentrations of radioloigand and performing a saturation analysis as described by Sillence and Matthews (1994).

Measurement of Second Messenger Production.
This experiment determined whether chronic treatment with clenbuterol or pST caused any change in the efficiency with which ß-adrenoceptors are coupled to the second-messenger enzyme adenylyl cyclase. Because this enzyme catalyzes the conversion of ATP to cAMP, its activity and receptor coupling efficiency were estimated by incubating cell membrane fragments for a fixed period of time with a potent ß-adrenergic agonist (l-isoproterenol; Sigma Chemical, St. Louis, MO), then measuring the amount of cAMP produced using the procedure of Sillence and Matthews (1994).

The fat-cell membranes were incubated for 10 min in a shaking water bath at 37°C with a high concentration of l-isoproterenol (100 µM) designed to cause near-maximum activation of adenylyl cyclase. The reaction was stopped by placing the tubes in a bath of boiling water for 4 min. After the tubes had been cooled on ice, the cAMP was extracted using a solution of 65% ethanol, followed by centrifuging at 2,500 x g to pellet all proteins. The ethanol was allowed to evaporate overnight in a vacuum oven at 60°C before the cAMP was resuspended in buffer (50 mM Tris 7.0; 4 mM EDTA; pH 7.4 at 37°C) and assayed using a commercially available radioimmunoassay kit (Amersham, North Ryde, NSW, Australia).

Long-Term Effect of Treating Neonatal Pigs with Clenbuterol and(or) pST
Animal Housing and Diet.
After the drug treatments were stopped at 40 to 41 d, the 80 pigs that were not slaughtered remained in the "weaner shed" with free access to food and water. The stage 1 weaner diet was provided until the pigs were 7 wk old, followed by a stage 2 weaner diet for the next 3 wk. At 10 wk of age the pigs were moved to a "grower shed," where they were housed again in four pens, each containing 20 animals of the same treatment group. On entering the grower shed the pigs’ diet was changed to a male grower diet, which was fed until the pigs were 22 wk old. This was replaced by a male finisher diet for the last 2 wk of the experiment. Although the pigs were female, the higher-quality male-type diet was used to facilitate any anabolic response to the treatments. The composition of each diet is shown in Table 1.

From 12 wk of age, feed was provided to the animals via electronic feeding devices that were activated by a transponder located in the pigs’ ear tags (Bunge Meat Industries, Corowa, NSW). This enabled the recording of individual meals for each pig and the estimation of individual feed intake. Live weight was also recorded each week throughout the experiment, with the exception of wk 18 and 21.

Slaughter.
The pigs were killed at 24 wk of age (mean BW 97 ± 3.5 kg). Hot carcass weight and the dressing percentage were recorded. Fat depth was measured at the following locations: on the shoulder (at the deepest point over the saw cut), at P2 (60 mm from the midline adjacent to the last rib), on the middle of the back (on the midline adjacent to the last rib), and on the hind leg (at the deepest point over the leg at the saw cut).

Animal Ethics.
All experimental procedures were conducted with the approval of the Animal Care and Ethics Committees of Bunge Meat Industries and Charles Sturt University, and in accordance with the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes (NHMRC).

Statistical Analysis.
The data were analyzed by two-way ANOVA using the GLM procedure of SAS (SAS Inst. Inc., Cary, NC). No covariates or blocking factors were used in the analysis, and results for carcass components and organ weights are expressed in absolute terms, rather than as a proportion of carcass or body weight. Values expressed as a proportion of body weight can be misleading when two treatments are used that are capable of causing multiple and differential changes in the mass of major body components such as protein and fat. Main effects were sought for the classification variables clenbuterol and pST treatment, as well as clenbuterol x pST interactions. Where significant interactions were observed by ANOVA (P < 0.05) means for the three treatment groups were compared to the mean for the control group using Dunnett’s test, run at two alpha levels (0.05 and 0.1). Pairwise multiple comparisons between all treatment groups were not made because the treatment groups were related (i.e., arranged factorially). Furthermore, by testing clenbuterol and pST at a single dose-rate, any comparison between the two drugs was intended to be qualitative only.

	Results

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Part 1: Effect of Clenbuterol and pST Treatment on Neonatal Pigs
Growth Rate.
Figure 1 illustrates the growth rate of pigs treated with clenbuterol or pST. When these drugs were given together there was a significant negative interaction (P = 0.003) and weight gain was not increased. When they were given separately, clenbuterol and pST both increased ADG up to weaning. By d 28 the control pigs had gained 5.1 ± 0.29 kg, whereas the clenbuterol-treated pigs had gained 24% more weight (6.3 ± 0.39 kg, P < 0.05) and the pST-treated pigs had gained 20% more weight than controls (6.1 ± 0.34 kg, P < 0.1). Although no further increase in ADG occurred, the difference in live weight was maintained until the pigs were killed between d 41 and 43.

View larger version (19K): [in this window] [in a new window]   Figure 1. Cumulative weight gain in a subgroup of female piglets treated daily with clenbuterol (25 µg/kg BW at 3 d of age increasing to 100 µg/kg BW by 9 d of age) and(or) porcine somatotropin (pST; 100 µg/kg BW from 3 d of age). Standard errors are omitted for the sake of clarity but were always < 7% of the mean. Relative to control pigs, cumulative gain was higher at weaning (d 28) in pigs treated with clenbuterol (P < 0.05) or pST alone (P < 0.1), but not in pigs given the combined treatment. n = 5.

ß-Adrenoceptor Density.
Clenbuterol caused a marked reduction in the total number of ß-adrenoceptors in skeletal muscle (P = 0.003) and adipose tissue (P < 0.001, Table 3). Porcine skeletal muscle contains a homogeneous population of ß₂-adrenoceptors (M. N. Sillence, unpublished data), and it is evident that the change in receptor density in adipose tissue was accounted for entirely by down-regulation of the ß₂-subtype (P < 0.001), with no effect on ß₁-adrenoceptors. Contrary to our hypothesis, pST did not increase the expression of ß₂-adrenoceptors, although the hormone might have attenuated the down-regulation caused by clenbuterol. Those pigs treated with pST plus clenbuterol had 40% more ß₂-adrenoceptors in muscle, and twice the density of ß₂-adrenoceptors in adipose tissue, than pigs given clenbuterol alone. The ability of pST to attenuate clenbuterol-induced down-regulation of ß₂-adrenoceptors in adipose tissue might explain why the two drugs produced an interactive effect on fat mass (Table 2).

Second-Messenger Activity.
There was an interaction between clenbuterol and pST with respect to their effects on l-isoproterenol-stimulated cAMP output (P = 0 0.03). Whereas treatment with clenbuterol alone tended to decrease the mean cAMP response, and pST alone had no effect, the combined treatment caused an increase in cAMP output relative to that observed in adipose tissue from untreated pigs (Table 3).

Long-Term Effect of Treating Neonatal Pigs with Clenbuterol and(or) pST
Growth Performance.
Live weight gain is shown in Figure 2. There was no evidence that pST treatment of neonatal pigs caused any long-term effect on weight gain. Indeed, at the end of the experiment the mean live weight of control and pST-treated animals was identical (99.6 kg).

View larger version (21K): [in this window] [in a new window]   Figure 2. Live weight of female pigs treated daily with clenbuterol (25 µg/kg BW at 3 d of age increasing to 100 µg/kg BW by 9 d of age) and(or) porcine somatotropin (pST; 100 µg/kg BW from 3 d of age) from 3 d of age to 40 d of age. Standard errors are omitted for the sake of clarity but were always < 5% of the mean. Relative to controls, pigs that underwent withdrawal from clenbuterol, or clenbuterol plus pST, gained less weight during the grower phase (d 42 to d 70; P < 0.05), but total weight gain was not significantly different by the end of the trial. n = 20.

Feed Intake and Feed Conversion Efficiency.
Figure 3a shows average daily intake at weekly intervals for the last 12 wk of the experiment. Figure 3b shows mean daily feed intake averaged over the entire 12-wk period. Relative to control pigs, pigs that had been treated with pST during the neonatal period showed an increase in feed intake during the grower-finisher period. This increase averaged 10% during the last 12 wk of the experiment (P < 0.05 for clenbuterol x pST interaction; P < 0.1 for pST vs control) but was particularly noticeable during the last 4 wk, averaging 23% until slaughter. Because there was no concurrent increase in weight gain, the increased intake of pST-treated pigs was reflected by a decrease in feed efficiency over the 12-wk period (P = 0.1 for main effect of pST; P < 0.05 for clenbuterol x pST interaction; P < 0.05 for pST vs control, Figures 4a and b). Compared to pST, clenbuterol caused a similar effect on feed intake in the grower-finisher period, with an increase above control values of 19% during the last 4 wk of the experiment. There was a negative interaction between clenbuterol and pST over the last 4 wk (P = 0.06); the combined treatment did not increase feed intake markedly during the grower-finisher period.

View larger version (15K): [in this window] [in a new window]   Figure 3. Feed intake during the growing and finishing period for pigs that were treated from 3 d of age to 40 d of age with porcine somatotropin (pST; 100 µg/kg BW from 3 d of age) and(or) clenbuterol (25 µg/kg BW at 3 d of age increasing to 100 µg/kg BW by 9 d of age). In Figure 3a each time point represents the average daily consumption over the preceding 7 d. Standard errors are omitted for the sake of clarity but were approximately 10% of the mean. Figure 3b shows the daily consumption for each treatment group averaged over the 12-wk period. Relative to controls, pigs that had been treated with pST alone during the neonatal period consumed more feed over the 12-wk growing-finishing period (P < 0.1). n = 20.

View larger version (16K): [in this window] [in a new window]   Figure 4. Feed efficiency (gain/intake) during the growing and finishing period for pigs that were treated from 3 d of age to 40 d of age with porcine somatotropin (pST; 100 µg/kg BW from 3 d of age) and(or) clenbuterol (25 µg/kg BW at 3 d of age increasing to 100 µg/kg BW by 9 d of age). In Figure 4a each time point represents the average feed to gain ratio over the preceding 7 d. Standard errors are omitted for the sake of clarity but were approximately 10% of the mean. Figure 4b shows the feed to gain ratio averaged over the 12-wk period. Relative to controls, pigs that had been treated with pST alone during the neonatal period were less efficient at converting feed to gain over the 12-wk growing-finishing period (P < 0.05). n = 20.

For all the carcass variables measured, the values for pigs treated with pST plus clenbuterol were intermediate between those for pigs treated with either compound alone. This implies that the long-term effect of the two drugs is not synergistic.

	Discussion

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This study tested two hypotheses: that interfering with adipose tissue metabolism early in life can cause a long-term impairment in an animal’s ability to deposit fat, and that energy and protein deposition are linked, so that a reduction in fat deposition would result indirectly in an increase in muscle growth and feed efficiency. The latter hypothesis came from a detailed analysis of the energetics of growth, which revealed a consistent and inverse correlation between the deposition of fat and protein, when different types of anabolic agents were tested in several species, different sexes, and under varying nutritional conditions (Lindsay et al., 1993). Experimental support for the proposed link between energy and protein is also found in studies of a vaccine designed specifically to destroy fat cells but that also causes a compensatory increase in muscle protein deposition in rats and pigs (Panton et al., 1990; Kestin et al., 1993).

Another important finding from vaccine studies is that antibodies to the fat cell can be administered passively to young rats for only 4 d and cause a decrease in adipocyte mass that is still evident 2 mo later, with feed efficiency and protein deposition also increasing during the period 3 to 8 wk after treatment (Panton et al., 1990). Furthermore, the passive immunization of 6-wk-old piglets results in a 25% reduction in backfat that is still evident 14 wk after treatment, with even greater effects seen when the antiserum is administered at 3 wk of age (Kestin et al., 1993). Unfortunately, although this work demonstrates the potential advantages of manipulating growth in the neonate, the antibody approach per se is hampered because of unacceptable side-effects and the difficulty of timing the treatment precisely. Animals must be young enough to show an irreversible response, but old enough to have a well-developed complement system to allow the antibodies to work (Flint, 1994). Thus, an alternative strategy to manipulate adipocyte metabolism in the neonate was sought.

It is known that ST has a physiological role in adipocyte metabolism during the perinatal period. Hypophysectomy of fetal pigs caused increased adipocyte size (Hausman et al., 1987) and hypophysectomy of fetal lambs also caused an increase in fat deposition that was reversed by ST (Stevens and Alexander, 1986). Similar effects were seen in neonatal rats following relatively short-term GH deficiency (Flint and Gardner, 1993). In older pigs, the effects of pST on fat deposition became attenuated following pST withdrawal (Campbell et al., 1989; Smith and Kasson, 1990). However, there is evidence that when ST is used early in life, this can place a life-long restriction on the capacity of an animal to deposit fat. McCutcheon et al. (1994) showed that short-term treatment (11 wk) of neonatal lambs with ST caused a long-term reduction in backfat that was still evident 5 mo later. The mechanism of this effect is not known, but the results imply that neonatal treatment with ST either inhibits adipocyte formation or limits the capacity of adipocytes to accumulate fat. One aim of the present study was to determine whether the treatment of neonatal pigs with pST would cause the same lasting benefits in terms of reduced fat deposition, increased muscle growth, and improved feed efficiency that are seen in ST-treated lambs and antibody-treated pigs.

There is evidence that the lipolytic effects of pST may be achieved more directly through the use of a ß-adrenergic agonist. When porcine adipose tissue was incubated with pST in vitro, there was no acute lipolytic effect (Etherton and Walton, 1986). This was also seen in ovine adipocytes, but after 48 h of incubation with ST the lipolytic response of ovine adipocytes to a ß-adrenergic agonist was increased (Watt et al., 1991). There was also an increase in the number of ß-adrenoceptors in the tissue, consistent with the theory that at least part of the lipolytic action of ST in vivo is mediated indirectly, by increasing the sensitivity of the tissue to the endogenous ß-adrenergic agonist epinephrine (Watt et al., 1991). Therefore, in the present study we determined whether adipocyte metabolism in the neonate could be manipulated using the synthetic ß-adrenergic agonist clenbuterol.

Finally, marked effects on fat deposition have been observed in finishing pigs treated with pST plus the ß-adrenergic agonist salbutamol (Hansen et al., 1997). This is consistent with the hypothesis outlined above, that pST increases the responsiveness of adipose tissue to adrenergic agents. A third aim of the present study was to determine whether pST and clenbuterol would cause a similar synergistic effect in neonatal pigs.

In the present study our results show that treating neonatal pigs with a pST-clenbuterol combination was an effective strategy to reduce fat deposition, even though neither treatment was effective on its own. This indicates a positive interaction of the two drugs, and this was seen at the receptor and post-receptor level, where pST attenuated the down-regulation of ß₂-adrenreceptors and the decrease in maximal cAMP output that occur in response to chronic clenbuterol treatment. From these results it may be inferred that the effects of pST on porcine adipose tissue metabolism are similar to those of ST in sheep, as described by Watt et al. (1991). This involves an indirect action on lipolysis through sensitization to endogenous epinephrine. The antilipogenic effects of pST have been discussed extensively elsewhere (Etherton and Walton, 1986; Dunshea, 1993).

Clenbuterol and pST did not cause an additive increase in muscle growth in young pigs, in contrast to other studies in which an ST plus ß-adrenergic agonist combination was given to older pigs (Hansen et al., 1997), cattle (Maltin et al., 1990), or mice (Bates and Pell, 1991). The present results might have reflected a limitation in feed intake, or dietary protein, even though the protein levels fed were higher than those used in commercial production.

Our strategy of decreasing the daily dose of clenbuterol to avoid withdrawal effects of the drug was not effective. Nevertheless, our results do allow a comparison between the effects of clenbuterol and pST withdrawal. The small anabolic effect of pST disappeared gradually after the young pigs stopped receiving their injections, consistent with the withdrawal effect seen in older pigs (Smith and Kasson, 1990). In Australia, pST withdrawal is not a practical concern, because pST is licensed with no withholding period. By contrast, clenbuterol withdrawal caused a loss of the additional weight gained, plus an extra 4 to 5 kg BW that the pigs never recovered. This is consistent with the withdrawal effects of other ß-adrenergic agonists in pigs and cattle (Jones et al., 1985; Barash et al., 1994). Because clenbuterol leaves tissue residues that take several days or weeks to decline in concentration (Meyer and Rinke, 1991), an inability to withdraw the drug long before slaughter without losing its beneficial effect is a considerable drawback. Reducing the withdrawal effects, perhaps by attenuating the receptor down-regulation caused by clenbuterol, would be a strategy to make ß-adrenergic agonists more useful. We have tried to do this previously without success using a glucocorticoid drug that up-regulates ß-adrenoceptors in smooth muscle (Huang et al., 2000). We showed that the glucocorticoid did not prevent down-regulation of ß-adrenoceptors in skeletal muscle, but our present results suggest that pST may have some efficacy in this regard. This deserves further investigation.

Although the growth rate of clenbuterol-treated pigs decreased when the drug was withdrawn, all pigs were growing at a similar rate by d 70. Several weeks later an increase in feed intake was apparent in pST-treated pigs, and over the last 4 wk of the experiment this effect was quite marked (23%). The treatment of older pigs with pST can sometimes decrease feed intake (Campbell et al., 1990), and so the effect seen in the present experiment could represent a rebound effect of some sort. However, there was no compensatory increase in feed intake after pST withdrawal in the studies by Campbell et al. (1989) or Smith and Kasson (1990). We have no data to suggest a possible mechanism for the increased intake or to explain why the rebound should be delayed in this way. Nevertheless, the hormone leptin is known to be associated with both fat cells and appetite in many species (Houseknecht et al., 1998), and investigations of the long-term effects of pST treatment or withdrawal on leptin secretion may provide some useful information.

Data for the growth, feed efficiency, and carcass composition of the growing-finishing pigs showed that, contrary to our hypothesis, long-term production benefits were not achieved through the treatment of neonatal animals with clenbuterol and(or) pST. In fact, because of withdrawal effects of clenbuterol and the delayed effects of both compounds on feed intake, the long-term effects of neonatal treatment were counterproductive. This study has highlighted several areas for further investigation, however. These include the interactive effects of pST and the adrenergic system in the control of lipolysis, the potential of using pST to counteract the withdrawal effects of clenbuterol, the influence that neonatal stress may have on long-term production efficiency, and the mechanism behind increased feed intake in finishing pigs that have undergone neonatal treatment with pST.

	Implications

Top Abstract Introduction Materials and Methods Results Discussion Implications Literature Cited

 
Fat deposition can be reduced in newborn pigs through treatment for 6 wk with a combination of clenbuterol and porcine somatotropin. However, in this experiment this strategy proved to be unproductive in the long term, with no benefits in terms of growth rate, feed efficiency, or carcass composition observed during the growing or finishing stages. Further research is needed into the mechanism by which somatotropin affects fat deposition and feed intake, the withdrawal effects of clenbuterol, and the long-term consequences of stress in young pigs.

	Footnotes

 
¹ This research was funded by the Pig Research and Development Corp. We thank Avril Ferraro for help in preparing this manuscript.

³ Present address: United Feeds, PO Box 68, Gridley, IL 61744.

Received for publication July 2, 2001. Accepted for publication January 29, 2002.

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Top Abstract Introduction Materials and Methods Results Discussion Implications Literature Cited

 


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