ࡱ> 5@͡bjbj22 !XX'd`7`7`787L7@t8t8"888888h9j9j9j9K9<?$:ARC?]88888?88>@999888h998h99,9,9,98h8 3)`78,9L9T@0@,9RD8RD,9RD,9 88988888??5`78`7 ACUTE AND SUBACUTE METABOLIC AND ENDOCRINE EFFECTS OF CLENBUTEROL IN FEMALE PIGS T.Gojmerac1 * , B. Mandi2 , M. Lojki1 and N. Biland~i1 1 Croatian Veterinary Institute, Zagreb, Croatia; 2 Vuk Vrhovac Institute for Diabetes, Endocrinology and Metabolic Diseases, School of Medicine, Zagreb, Croatia * Correspondence: Croatian Veterinary Institute, 10000 Zagreb, Savska cesta 143, Croatia ABSTRACT Gojmerac,T., Mandi, B., Lojki, M., Biland~i, N., 1999. Acute and subacute metabolic and endocrine effects of clenbuterol in female pigs.Veterinary Research Communications. The aim of the study was to assess the relationship between acute and subacute metabolic and endocrine effects after intravenous administration of (2-adrenergic agonist clenbuterol in a growth-promoting dose in female pigs. Acute metabolic and endocrine effects were investigated by measuring blood glucose, serum insulin and NEFA concentrations during 300 min after single dose administration of clenbuterol. Significantly higher serum insulin and NEFA concentrations (19.90 ( 2.50 (U/mL; p< 0.01; and 0.69 ( 0.04 mmol/L; p<0.001; respectively) were measured 30 min after the preprandial administration of clenbuterol in female pigs. In the same period, the levels of blood glucose (4.42 ( 0.30 mmol/L) showed no difference from control pigs. The postprandial serum NEFA concentration moderately decreased during 210 min after feeding. Postprandial blood glucose and insulin concentrations increased and reached maximal levels at 120 min after clenbuterol administration (10.91 ( 0.60 mmol/L and 85.22 ( 7.24 (U/mL, respectively), and returned to the basal levels at 300 min (4.20 ( 0.21 mmol/L and 7.75 ( 1.60 (U/mL, respectively) after the administration of clenbuterol. Subacute metabolic and endocrine effects were assessed by measuring the blood glucose, serum insulin and NEFA concentrations for 21 days after the repeated dose of clenbuterol. Furthermore, the influence of clenbuterol administration on the endocrine regulation of the onset of the next expected oestrus in female pigs was assessed by mesuring serum 17(-E and P concentrations. Blood glucose, serum insulin and NEFA concentrations after the last administration of clenbuterol did not significantly differ from those in control animals.The onset of the next expected oestrus occurred regularly ("riding test"), without any significant difference in serum 17(-E and P concentrations between the treated (9.83 ( 2.60 pg/mL and 0.15 ( 0.03 ng/mL) and control pigs (8.52 ( 2.70 pg/mL and 0.25 ( 0.06 ng/mL).Study results suggested the duration of intravenous administration of clenbuterol in a growth-promoting dose to influence the metabolic and endocrine activities in female pigs. Key words: (2-adrenergic agonist, clenbuterol, growth-promotor, metabolism, pigs Abbreviations: BAA, (2-adrenergic agonist; NEFA, non-esterified fatty acid; EDTA, etylenediaminetetraacetic acid; NaF, sodium fluoride; 17(-E, 17(-estradiol; P, progesterone INTRODUCTION Clenbuterol (benzenemethanol,4-amino-3,5-dichloro-( (( 1,1-dimethylenethyl) amino( methyl( monohydrochloride) is a synthetic selective BAA widely used as a bronchodilator in veterinary medicine (Sasse, 1987). When administered in 5 - to 10 - fold therapeutic dosages, clenbuterol has marked growth-promoting effects in slaughter animals, reduces body lipids, and promotes muscle growth (Meersmann,1989; Peters,1990). Many countries have imposed total ban on the use of clenbuterol for fattening purposes, since its abuse may have adverse effects on animal welfare and consumer health (Meyer and Karg, 1989; Martinez-Navarro, 1991; Pulce et al. 1991; Sporano et al.,1998). Several experiments have been carried out to study the effects of various BAA such as cimaterol and clenbuterol on growth performance in pigs (Moser et al., 1986; Ricks et al., 1984), however, little is known about its effect on changes in the metabolic and endocrine activity. Previous reports have shown that the administration of clenbuterol influences the metabolic and endocrine status of pigs, which is accompanied by changes in some plasma and serum biochemical parameters, and by morphologic alterations of the ovary and uterus (Baldi et al. 1994; Biolatti et al. 1994). The intensity of these changes in calves depends on the BAA type, animal species and especially on the duration of BAA administration, i. a. acute or long-term (Blum and Fluckiger, 1988; Zimmerli and Blum,1990). The aim of this study was to assess the relationship between acute and subacute metabolic and endocrine effects after intravenous administration of clenbuterol in a growth-promoting dose in female pigs. Acute and subacute effects were assessed by measuring the blood glucose and serum insulin and NEFA concentrations in the animals after single and repeated administration of the drug. Also, the effect of repeated administration of clenbuterol on endocrine regulation of the onset of the next expected oestrus in female pigs was determined by measuring serum 17(-E and P concentrations. MATERIAL AND METHODS Chemicals Clenbuterol was obtained from Sigma-Aldrich-Chemie, Steinheim, Germany. Reagents for quantitative determination of insulin in serum was purchased from Pharmacia ( Upjohn Diagnostics AB, Uppsala, Sweden. Enzymatic glucose reagent was obtained from Trace Scientific, Melbourne, Australia. For quantitative determination of NEFA in serum,the Randox NEFA (Crumlin, UK) reagent was purchased. Delfia 17 (-E and P kits for time-resolved fluoroimmunoassay were purchased from Wallac Oy, Turku, Finland. Animals and blood samples Experiments were carried out in 12 female pigs (6 treated and 6 control) of a known breed (F1 generation of Swedish Landrace X Large Yorkshire) aged 6-7 months, body mass 80-100 kg, farm-bred and kept in the same zoohygienic conditions. The concentration of the required nutrients in complete feed was determined according to recommendation of the National Research Council (NRC, 1988). Thus, 1 kg feed mix fed to pigs weighing ( 50 and 60-100 kg contained 13.43 % MJ-ME and 16.30% CP, and 13.67% MJ-ME and 15.03% CP, respectively. Pigs had access to feed and water ad libitum. From the onset of oestrus manifested by certain behavioural signs (" riding test"), in the acute metabolic and endocrine effect studies (day 1), the pigs were given 10 (g/kg body mass of clenbuterol as an intravenous injection into the ear vein 30 min before the first feeding of the day. Blood samples were collected by jugular venipuncture before 0 min and at 30, 60, 90, 120, 180, 240 and 300 min after the treatment. Blood samples were kept at room temperature for 60 min to allow clot retraction and serum separation by centrifugation. In serum, insulin and NEFA concentrations were determined. Blood samples were collected into tubes containing EDTA and NaF to prevent glycolysis, and immediately analyzed for glucose determination. In the subacute metabolic and endocrine effect studies, the same pigs were given 10 (g/kg body mass of clenbuterol as an intravenous injection into the ear vein twice daily (at about 8.00 a.m. and 3.00 p.m.) from day 2 to day 20. On day 21 (the last day of administration), the pigs were given 10 (g/kg body mass of clenbuterol 30 min before the first feeding of the day. Before 0 and at 30, 60, 90, 120, 180, 240, and 300 min after the last drug administration, blood samples were collected by jugular venipuncture and analyzed for serum insulin, NEFA and blood glucose concentrations. Serum 17(-E and P concentrations were measured in the blood samples collected at 30 ,120 and 300 min after the last administration. Blood samples were collected on days 22 and 23 (at about 8.00 a.m.,11.00 a.m. and 1.00 p.m.) after the last drug administration, encompassing the onset of the next expected oestrus.In the samples, serum 17(-E and P concentrations were determined. The experimental protocol was designed according to the Act on Animal Welfare (Official Gazette of the Republic of Croatia, No.19/1999). Assays Serum 17(-E and P were determined by time-resolved fluoroimmunoassay (FIA). The protocol for each kit of 17(-E and P followed the manufacturer's instructions. The values for both gonadal hormones are means of duplicate determination, and the mean of 3 daily concentrations was used in statistical calculations.Serum insulin concentration was determined by radioimmunoassay (RIA). Characteristics of these FIA and RIA have been described elsewhere (Lovgren et al.1984; Hemmila et al. 1984; Livesey et al.,1980). Serum NEFA concentration was determined by the enzymatic method (Okabe et al., 1980). The glucose oxidase reaction in conjunction with an auxiliary reaction was used for glucose determinatin (Pennock et al.,1973). Data analysis Results are presented as mean ( SE. Student's t-test was used to evaluate the significance of difference between the means (p<0.05 was considered significant). RESULTS Acute metabolic and endocrine effects on serum insulin, NEFA and blood glucose concentrations after a single dose of 10 (g/kg body mass of clenbuterol in female pigs are shown in Figs. 1, 2 and 3, respectively. Significantly higher serum insulin and NEFA concentrations (19.90 ( 2.50 (U/mL p<0.01; and 0.69 ( 0.04 mmol/L, p< 0.001; respectively) were measured 30 min after the preprandial administration of clenbuterol. In the same period, the levels of blood glucose (4.42 ( 0.30 mmol/L) showed no difference from the control animals (5.10 ( 0.20 mmol/L). The higher postprandial serum NEFA levels moderately decreased during 210 min after feeding. The postprandial increase in blood glucose and serum insulin concentrations reached maximal levels at 120 min (10.91 ( 0.60 mmol/L and 85.22 ( 7.24 (U/mL, respectively), and returned to the basal levels at 300 min (4.20( 0.21 mmol/L and 7.75 ( 1.60 (U/mL, respectively) after the administration of clenbuterol. Subacute metabolic and endocrine effects on serum insulin, NEFA and blood glucose concentrations after the repeated dose of clenbuterol, determined for 21 days, are shown in Figs. 1, 2 and 3. Serum insulin, NEFA and blood glucose concentrations in pigs after the last administration of clenbuterol were not significantly different from those in control animals. Serum NEFA concentration decreased to a similar extent in the treated and control animals for the first 120 min after the last administration, whereafter it slowly increased. Serum insulin concentration reached maximal levels at 60 min after the last administration of clenbuterol (10.21 ( 1.3 (U/mL), and all measured values were lower,but not significantly, as compared with control animals. The effects of the repeated administration of clenbuterol on serum 17(-E and P concentrations on days 21, 22 and 23 after the last administration are shown in Fig.4. Serum 17(-E concentration in the treated pigs reached a higher value of 58.50 ( 6.90 pg/mL at about 24 h (day 21) before the onset of the next expected oestrus (day 22 and day 0, respectively). A lower serum 17(-E concentration was measured on day 22 ( 9.83 ( 2.60 pg/mL), corresponding to the onset of the next expected oestrus.Serum P concentration in the treated pigs on days 21, 22 (day 0) and 23 (0.13 ( 0.03 ng/mL; 0.15 ( 0.03 ng/ml; and 1.11 ( 0.08 ng/ml, respectively) did not significantly differ from those in control animals (0.22 ( 0.09 ng/ml; 0.25 ( 0.06 ng/ml; and 1.34 ( 0.14 ng/ml, respectively).The onset of the next expected estrus occurred regularly ("riding test") in the treated pigs ( day 22 and day 0, respectively) DISCUSSION Previous reports have indicated that acute treatment with therapeutic and growth-promoting doses of clenbuterol indirectly mediated effects on several metabolic and endocrine systems, which are manifested as significant changes in the levels of serum insulin, NEFA and blood glucose in calves (Blum and Fluckiger, 1988; Zimmerli and Blum,1990; Luthman and Jacobsson, 1993). A significant preprandial increase in serum NEFA concentration suggested a lipolytic effect of clenbuterol in calves.On the other hand, previous data indicate that the depression in lipogenesis is the mechanism by which clenbuterol decreases subcutaneous fat accretion in cattle (Miller et al. 1988). In our study, the significant increase in preprandial serum NEFA concentration within 30 min after a single growth-promoting dose of clenbuterol in pigs (Fig.2) suggested a notable lipolytic effect of this BAA. Previous reports have also shown that (2-adrenergic agonists induce greater lipolytic effects in fasted cattle than in fed cattle (Blum et al.1982). Furthermore, the significant increase in preprandial serum insulin concentration within 30 min after single dose administration of clenbuterol and at the same time no blood glucose response (Figs 1, and 3) may possibly be explained by specific interaction of this BAA on the beta cells of the islets of Langerhans. It is a well known fact that insulin secretion of the pancreas is increased by the action of (2-adrenergic agonists (Porte, 1967; Loubatieres et al.1971). The significant postprandial increase in the levels of serum insulin and blood glucose as well as a higher value of serum NEFA after single dose administration of clenbuterol in female pigs , were only transient and reversible, showing fast normalization, similar to that found in an earlier study in calves (Luthman and Jacobsson,1993). In addition, decrease in the postprandial levels of serum insulin and blood glucose after 120 min of clenbuterol administration were delayed as compared to controls animals, especially in relation to the levels of blood glucose. This suggested that clenbuterol caused a prolonged hyperglycemia. The repeated dose administratiom of clenbuterol in pigs indicated that changes in the levels of the measured biochemical parameters were similar to those in control animals. Serum insulin concentration was lower than that recorded in controls,but the difference was not statistically significant. This is in agreement with the results reported elsewhere (Baldi et al. 1994). The results of serum 17(-E and P concentration measurements on days encompassing the onset of the next expected oestrus (days 21, 22 and 23) after the last administration of clenbuterol were consistent with the plasma and serum results in intact pigs published elsewhere (Guthrie et al.,1972; Van de Wiel et al. 1981; Gojmerac et al. 1996). Although a lower serum P concentration was obtained, it did not significantly differ from the controls and remained within the physiological range, resulting in regular occurrence of oestrus in the pigs. The levels of biochemical parameters determined showed only subtle changes indicating normal function of the lipoprotein and carbohydrate metabolism and reproduction after the repeated dosage of clenbuterol. The marked difference between the acute and subacute metabolic effects of single and repeated dosage of clenbuterol in pigs could be explained by the possibility that prolonged administration of clenbuterol led to a decrease in the affinity of specific (2-adrenergic receptors for the binding of clenbuterol in target tissues. REFERENCES Act on Animal Welfare,1999. Official Gazette of the Republic of Croatia, No. 19, 1999. Baldi, A., Bontempo, V., Cheli, F., Corino, C. and Polidori, F., 1994. Hormonal and metabolic responses to the stress of transport and slaughterhouse procedures in clenbuterol-fed pigs. Journal of Veterinary Medicine A, 41, 189-196 Biolatti, B., Castagnaro, M., Bollo, E., Appino, S. and Re,G., 1994. Genital lesions following long-term administration of clenbuterol in female pigs. Veterinary Patology, 31, 82-92 Blum, J.W., Froehli,J.D., and Kunz, P.,1982. Effects of catecholamines on plasma free fatty acids in fed and fasted cattle. Endocrinology, 110, 452-459 Blum, J. and Fluckiger, N., 1988. Early metabolic effects of perorally administered (-adrenoceptor agonists in calves. European Journal of Pharmacology, 51, 177-187 Gojmerac, T., Kartal, B., uri, S., }uri, S., Kuaevi, S. and Cvetni, }., 1996. Serum biochemical changes associated with cystic ovarian degeneration in pigs after atrazine treatment. Toxicology Letters, 85, 9-15 Guthrie, H.D., Henricks, D.M. and Handlin, D.L., 1972. Plasma estrogen, progesterone and luteinizing hormone prior to estrus and during early pregnancy in pigs. Endocrinology, 91, 675-679 Livesey,J.H., Hodgkinson, S.C., Roud,H.R. and Donald, R.A.,1980. Effect of time, temperature and freezing on stability of immunoreactive LH, FSH, TSH, growth hormone, prolactin and insulin in plasma.Clinical Biochemistry, 13, 151-155. Loubatieres, A., Mariani, M.M., Sorel, G. and Savi, L., 1971. The action of beta-adrenergic blocking and stimulating agents on insulin secretion. Characterization of the type of beta-receptor. Diabetologia, 7, 127-132 Luthman J. and Jacobsson, S.O., 1993. Acute metabolic effects of clenbuterol in calves. Acta Veterinaria Scandinavica, 34, 169-174 Martinez-Navarro, J.F.,1991. Food poisoning related to consumption of illicit (- agonist in liver. The Lancet, 336, 1311 Meersmann, H.J.,1989. Potential mechanisms for repartitioning of growth by beta-adrenergic agonists. In: Campion, D.R., Hausman, G.J., Martin, R.J. (eds), Animal Growth Regulation. Plenum Press. New York and London, 337-357 Meyer, H.H.D.,and Karg, H., 1989. Growth stimulators for farm animals: Mode of action, effects on meat quality and potential risk originating from residues. In: Proceedings of the FAO/CAAS Workshop on Biotechnology in Animal Production, 49-58 Miller, M.F., Garcia, D.K., Coleman, M.E.,Ekeren, P.A., Lunt, D.K., Wagner, K.A., Procknor, M., Welsh, Jr.T.H. and Smith, S.B., 1988. Adipose tissue, longissimus muscle and anterior pituitary growth and function in clenbuterol- fed heifers. Journal of Animal Science, 66, 12-20 Moser, R.J.,Dalrymple, R.H., Cornelius, S.G.,Pettigrew, E. and Allen, C.E., 1986. Effects of cimaterol (CL 263,780) as a repartitioning agent in the diet for finishing pigs. Journal of Animal Science, 62, 21-26 National Research Council,1988. Nutrient requirements of swine. Washington, D.C. Okabe,H., Uji,Y.,Nagashima, K., and Noma, A.,1980. Enzymic determination of free fatty acids in serum. Clinical Chemistry, 26, 1540-1543. Pennock,C.A., Murphy,D.,Sellers,J. and Longdon, K.J. 1973. A comparison of autoanalyser methods for the estimation of glucose in blood. Clinica Chimica Acta, 48,193-201. Peters, A.R. 1990. Beta-agonists and pig production. Pig News and Information, 11, 519-522 Porte, D., 1967. Beta adrenergic stimulation of insulin release in man. Diabetes 16, 150-155 Pulce, C., Lamaison, D.,Keck, G., Bostvironnois, C., Nicolas, J. and Descotes, J., 1991. Collective human food poisoning by clenbuterol residues in veal liver. Veterinary and Human Toxicology, 33, 480-481 Ricks, C.A., Backer, P.K., Dalryamle, R.H., Doschner, M.E., Ingle, D.L. and Pankhavich,J., 1984. Use of clenbuterol to alter muscle and fat accretion in swine. Federation Proceedings, 43, 857 Sasse, H.H.L.,1987. Clinical aspects of current (-agonist use in veterinary medicine. In: Hanrahan, J.P. (ed), (-Agonists and their effects on animal growth and carcass quality. Elsevier Applied Scence. London, New York, 60-71 Sporano, V., Grasso, L., Esposito, M., Oliviero, G., Brambilla, G. and Loizzo, A., 1998. Clenbuterol residues in non-liver containing meat as a cause of collective food poisoning. Veterinary and Human Toxicology, 40, 141-143 Van de Wiel, D.F.M., Erkens,J., Koops, W., Vos, E. and Van Landeghem, A.A.J., 1981. Periestrous and midluteal time courses of circulating LH, FSH, prolactin, estradiol -17( and progesterone in the domestic pig. Biology of Reproduction, 24, 223-233 Zimmerli, U.V., and Blum, J.W.,1990. Acute and longterm metabolic, endocrine, respiratory, cardiac and skeletal muscle activity changes in response to perorally administered (-adrenoceptor agonists in calves. Journal of Animal Physiology and Animal Nutrition, 63, 157-172 Fig.1. Mean (( SE) serum insulin concentration ((U/mL) in pigsa after singleb and repeatedc dose of clenbuterol a Female pigs (6 crossbred between Swedish Landrace and Large Yorkshire) aged 6-7 months, body mass 80-100 kg b Female pigs were given 10 (g/kg body mass of clenbuterol intravenously 30 min before feeding (day 1) c Female pigs were given clenbuterol intravenously for 21 days; day 1: 10 (g/kg body mass 30 min before feeding; day 2-20: 10 (g/kg body mass twice daily; day 21: 10 (g/kg body mass 30 min before feeding * p< 0.01; ** p< 0.001 vs control; statistical evaluation by Student's t-test Fig.2. Mean ((SE) serum non-esterified fatty acids concentration (mmol/L) in pigsa after singleb and repeatedc dose of clenbuterol a Female pigs (6 crossbred between Swedish Landrace and Large Yorkshire) aged 6-7 months, body mass 80-100 kg b Female pigs were given 10 (g/kg body mass of clenbuterol intravenously 30 min before feeding (day 1) c Female pigs were given clenbuterol intravenously for 21 days; day 1: 10 (g/kg body mass 30 min before feeding; day 2-20: 10 (g/kg body mass twice daily; day 21: 10 (g/kg body mass 30 min before feeding * p< 0.01; ** p< 0.001 vs control; statistical evaluation by Student's t-test Fig.3. Mean (( SE) blood glucose concentration ( mmol/L) in pigsa after singleb and repeatedc dose of clenbuterol a Female pigs (6 crossbred between Swedish Landrace and Large Yorkshire) aged 6-7 months, body mass 80-100 kg b Female pigs were given 10 (g/kg body mass of clenbuterol intravenously 30 min before feeding (day 1) c Female pigs were given clenbuterol intravenously for 21 days; day 1: 10 (g/kg body mass 30 min before feeding; day 2-20: 10 (g/kg body mass twice daily; day 21: 10 (g/kg body mass 30 min before feeding *p< 0.01; ** p<0.001 vs control ; statistical evaluation by Student's t-test Fig.4. 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