Effects on health, performance, and tissue residues of the ionophore antibiotic salinomycin in finishing broilers (21 to 38 d)

Effects on health, performance, and tissue residues of the ionophore antibiotic salinomycin in... ABSTRACT A study was conducted to evaluate the effects of feeding salinomycin at the recommended prophylactic level, and at 2 and 3 times this level, to finishing male broilers (d 21 to 38). Four treatment groups were given the experimental diets containing 0, 60, 120, or 180 parts per million (ppm) salinomycin from d 21 to 38. Performance, relative organ weights, selected serum enzymes, and salinomycin residues in liver, muscle, and serum were determined. Salinomycin supplementation had no effect on body weight, feed intake, or feed conversion, and caused no overt signs of toxicity. After a week of being fed the salinomycin diets, the serum activity of aspartate aminotransferase was significantly increased in chickens fed 180 ppm compared with controls. These birds also showed microscopic lesions in breast and thigh muscles, but not in cardiac muscle. Salinomycin residues were not detected by high-performance liquid chromatography coupled to tandem mass spectrometry in liver or muscle samples from the birds fed 0, 60, or 120 ppm salinomycin. However, chickens fed 180 ppm salinomycin had detectable levels in liver and muscle above the maximum residue level of 5 μg/kg established by the European Union. All birds fed salinomycin had salinomycin in their sera with levels ranging from N.D. (not detected) in the controls to 24.4 ± 7.9, 61.4 ± 18.9, and 94.5 ± 9.1 μg/L for salinomycin dietary levels of 60, 120, and 180 ppm, respectively. Serum salinomycin concentration was linearly related with salinomycin content in feed (y = 0.584x − 10, r2 = 0.999). The results showed that even at 3 times the prophylactic level, salinomycin does not induce clinical toxicosis or mortality. No salinomycin residues were found in edible tissues at the recommended dietary level or at 2 times this level. However, salinomycin was detected in serum regardless of the dietary level. A simple method for salinomycin determination in serum is described which can be used as a marker of exposure and/or to predict levels in the diet. INTRODUCTION Ionophore antibiotics are commonly used as feed additives for the prevention of coccidiosis in poultry and as growth promoters in cattle and swine. These compounds are produced by 53 different bacteria of the Streptomycetaceae family and are characterized by multiple tetrahydrofuran rings connected together as spiroketal moieties (Clarke et al., 2014). Among the most commonly used ionophores in poultry are monensin, maduramicin, lasalocid, narasin, and salinomycin. The latter was discovered as a fermentation product of a strain of Streptomyces albus (Kinashi et al., 1973; Miyazaki et al., 1974). Migaki and Babcock (1979) conducted a safety evaluation of salinomycin in broiler chickens by feeding them diets containing 50, 60, 80, 100, and 160 mg/g salinomycin from 1 to 56 d. In this study, the weight gains at 50 and 60 mg/kg salinomycin were not significantly different from the control, at 80 mg/kg the weight gain was slightly below controls, and at 100 and 160 mg/kg salinomycin depressed weight gain. Feed conversion was only affected by 160 mg/kg (Migaki and Babcock, 1979). Currently, salinomycin is commonly added to poultry diets at a concentration of 60 mg/kg and it is compatible with most ingredients, except several antibiotics (tiamulin, erythromycin, sulfachloropyrazine, sulfaquinoxaline, and chloramphenicol) and the antioxidant XAX-M (Dowling, 1992). Generally, ionophores are safe at the recommended inclusion levels; however, toxicosis can result from overdose and misuse situations (Novilla, 1992). The effects of feeding chickens with salinomycin at 1.5, 2, 2.5, and 3 times the recommended levels (Keshavarz and McDougald, 1982) and at 2 and 3 times the recommended levels have been reported (Rizvi and Anjum, 1999). However, in both of these experiments the salinomycin diets were started at d 8 and were given for 3 and 8 weeks, respectively. In Colombia and other countries, salinomycin is used in finishing diets (d 21 to 38), until the market weight is reached. Salinomycin residues in poultry meat are not regulated in Colombia but the European Union has a maximum residue level (MRL) of 5 μg/kg in liver, kidney, and muscle (Commission Regulation (EC) No. 496/2007). Given the widespread use of salinomycin in Colombian poultry diets and also that there have been complaints of growers blaming salinomycin for causing mortality in their flocks, the present study aimed at determining the potential adverse effects of feeding salinomycin to 3-week-old chickens at the recommended prophylactic level (60 ppm) and at 2 and 3 times this level. Further, salinomycin residues in muscle, liver, and serum were determined by high-performance liquid chromatography coupled to tandem mass spectrometry (HPLC-MS/MS). MATERIALS AND METHODS The experiment was approved by the Ethics Committee of the College of Veterinary Medicine of the National University of Colombia under approval No. CB-FMVZ-UN-008–17. A total of 400 male day-old chicks (Ross 308) were received in the experimental premises (Poultry Research Building, College of Veterinary Medicine, located in Bogota, Colombia, at 2,600 m above sea level) and individually wing banded. For the first 20 d, all chicks received the same starter ration, which contained the mixture of anticoccidials narasin, and nicarbazin at dietary concentrations of 40 ppm each (Maxiban, Elanco, IN). At d 21, all birds were weighed and the heaviest (24) and lightest (24) were removed from the experiment. The remaining 352 birds were distributed at random into 4 dietary treatments with 4 replicates of 22 birds each, for a total of 88 chickens per treatment. The experiment consisted of a completely randomized dietary arrangement of treatments with a control diet (without salinomycin), a diet with the recommended salinomycin level (60 mg/kg), and diets with 2 times (120 mg/kg) and 3 times (180 mg/kg) the recommended level. These levels were chosen in order to simulate a formulation mistake or an improper mixing of the diet. The experimental diets were provided ad libitum for 18 d (d 21 to 38). Birds were kept in floor pens at a density of 10 birds/m2 at an initial temperature of 27°C, decreased by 2°C every 7 d until the end of the experiment. Response variables measured or calculated during and at the end of the experiment were as follows: Performance variables: body weight (d 21, 28, 35, and 38), feed intake (measured daily and analyzed weekly), and feed conversion (calculated weekly). For these variables the experimental unit was the replicate pen. Blood chemistry: blood samples from 3 birds per replicate pen (12 birds per treatment) were taken at d 21, 28, and 35 (d 1, 7, and 14 of the experiment) for the determination of the serum activity of creatine kinase (CK), lactate dehydrogenase (LDH), and aspartate aminotransferase (AST). Serum samples were processed using commercial spectrophotometric kits (Spinreact, S.A., Barcelona, Spain), part numbers 1,001,050, 1,001,260, 1,001,161 for CK, LDH, and AST, respectively. Relative organ weights: at the end of the experiment (d 38), 30 birds taken at random from each treatment were sacrificed for the determination of the relative weight of proventriculus, gizzard, liver, heart, pancreas, spleen, and bursa of Fabricius. Salinomycin determination in muscle, liver, and serum: at the end of the experiment, liver and muscle (left deep pectoral) samples were taken from 4 birds per treatment for the determination of salinomycin by HPLC-MS/MS. The analysis was performed on a Shimadzu Prominence HPLC (Shimadzu, Kyoto, Japan) coupled to an ABI 3200 QTrap triple quadrupole mass spectrometer detector (MDS Sciex, Toronto, Canada). Also, blood samples from 4 birds per treatment were taken at d 35 (d 15 of the experiment) for salinomycin determination. Liver and muscle samples were analyzed according to the method described by Matabudul et al. (2002), whereas the serum determination was conducted after serum deproteinization according to Blanchard (1981), as follows: to 400 μL of serum, 1.6 mL of acetonitrile was added in a 2 mL centrifuge vial. The contents were shaken in vortex for 30 seconds and centrifuged at 9,000 × g for 15 minutes. An aliquot of 200 μL of the supernatant was further diluted with 1.8 mL of acetonitrile, and shaken and centrifuged as described previously. An aliquot of approximately 1 mL was then filtered through a 0.22 μm pore size polytetrafluoroethylene membrane into an autosampler vial for the determination of salinomycin by HPLC-MS/MS. Histology: tissue samples of liver, skeletal muscle (left deep pectoral and left cranial iliotibial), and cardiac muscle were taken from 4 birds per treatment at the end of the experiment. Tissue samples were fixed in neutral-buffered formalin, and stained with hematoxylin-eosin before microscopic examination. Quantitative response variables were analyzed using Statistix 9 for Windows (2008) by one-way ANOVA. When the P value of the ANOVA test was <0.05, means were separated using the Tukey's test (Statistix 9 for Windows, 2008, Tallahassee, FL). RESULTS Table 1 shows the mean body weight of the 4 experimental treatments at d 21, 28, 35, and 38 (corresponding to d 1, 8, 15, and 18 of the experiment) and the 18-d body weight gain. Significant differences in body weight were observed at d 28, 35, and 38. At d 28 and 38, the birds receiving 120 and 180 ppm salinomycin showed significantly lower body weights compared with the birds fed 0 or 60 ppm salinomycin. Further, the chickens fed the diet with 180 ppm salinomycin had significantly lower body weights than the birds fed 120 ppm at d 28, 35, and 38. Body weight gain was significantly different among the 4 treatments and followed an inverse dose-response relationship. Chickens without salinomycin had the highest weight gain and chickens receiving 3 times the recommended level, the lowest. The body weight gain in these chickens was only 56% of the control group. Feed intake and feed conversion for the 18 experimental d are shown in Table 2. The first 2 weeks feed intake was significantly lower only for the chickens receiving 3 times the recommended dose of salinomycin; however from d 35 to 38, feed intake was significantly lower for the groups receiving 2 and 3 times the recommended level. For all time periods evaluated only the group receiving 180 ppm salinomycin had a feed conversion significantly higher than the control group. Overall (d 21 to 38), feed intake and feed conversion were significantly different only between the group receiving 180 ppm salinomycin and the other 3 groups. Table 1. Effect of dietary supplementation with salinomycin from d 21 to 38 on body weight and body weight gain of broiler chickens.1 Days of age Total body weight gain (g) and % of control Salinomycin level (ppm) 21 28 35 38 21 to 38 0 781.3 ± 3.8ª 1,639 ± 12ª 2,392 ± 19a 2,610 ± 26a 1,828 ± 24a (100) 60 794.6 ± 3.7a 1,614 ± 6.5ª 2,291 ± 45ab 2,538 ± 17a 1,744 ± 18b (95) 120 790.0 ± 6.6ª 1,557 ± 7.5b 2,210 ± 16b 2,388 ± 4.8b 1,598 ± 6c (87) 180 800.0 ± 9.5ª 1,303 ± 16c 1,708 ± 21c 1,832 ± 24c 1,032 ± 17d (56) P 0.262 0.000 0.000 0.000 0.000 Days of age Total body weight gain (g) and % of control Salinomycin level (ppm) 21 28 35 38 21 to 38 0 781.3 ± 3.8ª 1,639 ± 12ª 2,392 ± 19a 2,610 ± 26a 1,828 ± 24a (100) 60 794.6 ± 3.7a 1,614 ± 6.5ª 2,291 ± 45ab 2,538 ± 17a 1,744 ± 18b (95) 120 790.0 ± 6.6ª 1,557 ± 7.5b 2,210 ± 16b 2,388 ± 4.8b 1,598 ± 6c (87) 180 800.0 ± 9.5ª 1,303 ± 16c 1,708 ± 21c 1,832 ± 24c 1,032 ± 17d (56) P 0.262 0.000 0.000 0.000 0.000 1Values correspond to mean ± SEM of 4 replicates per treatment. Within a column, means with different superscripts differ significantly (P < 0.05). View Large Table 1. Effect of dietary supplementation with salinomycin from d 21 to 38 on body weight and body weight gain of broiler chickens.1 Days of age Total body weight gain (g) and % of control Salinomycin level (ppm) 21 28 35 38 21 to 38 0 781.3 ± 3.8ª 1,639 ± 12ª 2,392 ± 19a 2,610 ± 26a 1,828 ± 24a (100) 60 794.6 ± 3.7a 1,614 ± 6.5ª 2,291 ± 45ab 2,538 ± 17a 1,744 ± 18b (95) 120 790.0 ± 6.6ª 1,557 ± 7.5b 2,210 ± 16b 2,388 ± 4.8b 1,598 ± 6c (87) 180 800.0 ± 9.5ª 1,303 ± 16c 1,708 ± 21c 1,832 ± 24c 1,032 ± 17d (56) P 0.262 0.000 0.000 0.000 0.000 Days of age Total body weight gain (g) and % of control Salinomycin level (ppm) 21 28 35 38 21 to 38 0 781.3 ± 3.8ª 1,639 ± 12ª 2,392 ± 19a 2,610 ± 26a 1,828 ± 24a (100) 60 794.6 ± 3.7a 1,614 ± 6.5ª 2,291 ± 45ab 2,538 ± 17a 1,744 ± 18b (95) 120 790.0 ± 6.6ª 1,557 ± 7.5b 2,210 ± 16b 2,388 ± 4.8b 1,598 ± 6c (87) 180 800.0 ± 9.5ª 1,303 ± 16c 1,708 ± 21c 1,832 ± 24c 1,032 ± 17d (56) P 0.262 0.000 0.000 0.000 0.000 1Values correspond to mean ± SEM of 4 replicates per treatment. Within a column, means with different superscripts differ significantly (P < 0.05). View Large Table 2. Effect of dietary supplementation with salinomycin from d 21 to 38 on feed intake and feed conversion of broiler chickens.1 Days of age—Feed intake (g/chicken) Total feed intake and % of control Days of age—Feed conversion (g/g) Overall feed conversion and % of control Salinomycin level (ppm) 21 to 27 28 to 35 35 to 38 21 to 38 21 to 27 28 to 35 35 to 38 21 to 38 0 988 ± 12ª 1050 ± 27ª 402 ± 24a 2,427 ± 56a (100) 1.24 ± 0.02b 1.38 ± 0.03b 1.52 ± 0.05b 1.33 ± 0.03b (100) 60 987 ± 13a 1017 ± 57ª 386 ± 16a 2,391 ± 65a (98) 1.25 ± 0.02b 1.44 ± 0.04b 1.63 ± 0.04ab 1.37 ± 0.02b (103) 120 945 ± 14ª 1005 ± 24ab 377 ± 9b 2,328 ± 31ab (96) 1.30 ± 0.03b 1.40 ± 0.03b 1.49 ± 0.02b 1.46 ± 0.01b (109) 180 820 ± 14b 956 ± 39b 338 ± 9c 2,115 ± 46b (87) 1.40 ± 0.03a 1.63 ± 0.02a 1.70 ± 0.02a 2.05 ± 0.04a (154) P 0.000 0.002 0.000 0.004 0.003 0.000 0.003 0.000 Days of age—Feed intake (g/chicken) Total feed intake and % of control Days of age—Feed conversion (g/g) Overall feed conversion and % of control Salinomycin level (ppm) 21 to 27 28 to 35 35 to 38 21 to 38 21 to 27 28 to 35 35 to 38 21 to 38 0 988 ± 12ª 1050 ± 27ª 402 ± 24a 2,427 ± 56a (100) 1.24 ± 0.02b 1.38 ± 0.03b 1.52 ± 0.05b 1.33 ± 0.03b (100) 60 987 ± 13a 1017 ± 57ª 386 ± 16a 2,391 ± 65a (98) 1.25 ± 0.02b 1.44 ± 0.04b 1.63 ± 0.04ab 1.37 ± 0.02b (103) 120 945 ± 14ª 1005 ± 24ab 377 ± 9b 2,328 ± 31ab (96) 1.30 ± 0.03b 1.40 ± 0.03b 1.49 ± 0.02b 1.46 ± 0.01b (109) 180 820 ± 14b 956 ± 39b 338 ± 9c 2,115 ± 46b (87) 1.40 ± 0.03a 1.63 ± 0.02a 1.70 ± 0.02a 2.05 ± 0.04a (154) P 0.000 0.002 0.000 0.004 0.003 0.000 0.003 0.000 1Values correspond to mean ± SEM of 4 replicates per treatment. Within a column, means with different superscripts differ significantly (P < 0.05). View Large Table 2. Effect of dietary supplementation with salinomycin from d 21 to 38 on feed intake and feed conversion of broiler chickens.1 Days of age—Feed intake (g/chicken) Total feed intake and % of control Days of age—Feed conversion (g/g) Overall feed conversion and % of control Salinomycin level (ppm) 21 to 27 28 to 35 35 to 38 21 to 38 21 to 27 28 to 35 35 to 38 21 to 38 0 988 ± 12ª 1050 ± 27ª 402 ± 24a 2,427 ± 56a (100) 1.24 ± 0.02b 1.38 ± 0.03b 1.52 ± 0.05b 1.33 ± 0.03b (100) 60 987 ± 13a 1017 ± 57ª 386 ± 16a 2,391 ± 65a (98) 1.25 ± 0.02b 1.44 ± 0.04b 1.63 ± 0.04ab 1.37 ± 0.02b (103) 120 945 ± 14ª 1005 ± 24ab 377 ± 9b 2,328 ± 31ab (96) 1.30 ± 0.03b 1.40 ± 0.03b 1.49 ± 0.02b 1.46 ± 0.01b (109) 180 820 ± 14b 956 ± 39b 338 ± 9c 2,115 ± 46b (87) 1.40 ± 0.03a 1.63 ± 0.02a 1.70 ± 0.02a 2.05 ± 0.04a (154) P 0.000 0.002 0.000 0.004 0.003 0.000 0.003 0.000 Days of age—Feed intake (g/chicken) Total feed intake and % of control Days of age—Feed conversion (g/g) Overall feed conversion and % of control Salinomycin level (ppm) 21 to 27 28 to 35 35 to 38 21 to 38 21 to 27 28 to 35 35 to 38 21 to 38 0 988 ± 12ª 1050 ± 27ª 402 ± 24a 2,427 ± 56a (100) 1.24 ± 0.02b 1.38 ± 0.03b 1.52 ± 0.05b 1.33 ± 0.03b (100) 60 987 ± 13a 1017 ± 57ª 386 ± 16a 2,391 ± 65a (98) 1.25 ± 0.02b 1.44 ± 0.04b 1.63 ± 0.04ab 1.37 ± 0.02b (103) 120 945 ± 14ª 1005 ± 24ab 377 ± 9b 2,328 ± 31ab (96) 1.30 ± 0.03b 1.40 ± 0.03b 1.49 ± 0.02b 1.46 ± 0.01b (109) 180 820 ± 14b 956 ± 39b 338 ± 9c 2,115 ± 46b (87) 1.40 ± 0.03a 1.63 ± 0.02a 1.70 ± 0.02a 2.05 ± 0.04a (154) P 0.000 0.002 0.000 0.004 0.003 0.000 0.003 0.000 1Values correspond to mean ± SEM of 4 replicates per treatment. Within a column, means with different superscripts differ significantly (P < 0.05). View Large Table 3 shows the serum activities of AST and CK measured at d 21, 28, and 35. As expected, no significant differences in enzyme activities were seen at d 21 (d 1 of the experiment). After 7 d of salinomycin supplementation AST activity was significantly higher in the chickens receiving 180 ppm salinomycin compared with the other 3 groups and reached 1.74 times that of the control group; however, after 14 d, no significant differences were observed. CK levels were not significantly different after 7 d of salinomycin supplementation but after 14 d CK activity was significantly lower in the chickens fed 180 ppm salinomycin compared with the other 2 groups receiving salinomycin. No significant differences among the experimental groups were observed in LDH activity at any sampling time (data not shown). Table 3. Effect of dietary supplementation with salinomycin from d 21 to 38 on serum activity of aspartate aminotransferasa (AST) and creatine kinase (CK) of broiler chickens.1 AST serum activity (U/L) CK serum activity (U/L) Days of age Salinomycin level (ppm) 21 28 35 21 28 35 0 89 ± 3a 98 ± 7b 139 ± 10a 1,286 ± 178a 2,438 ± 302a 5,997 ± 1,183a,b 60 81 ± 2a 101 ± 5b 146 ± 15a 1,032 ± 47a 2,640 ± 339a 7,003 ± 956a 120 83 ± 2a 113 ± 13b 140 ± 12a 1,062 ± 96a 2,335 ± 334a 7,145 ± 962a 180 86 ± 3a 170 ± 17a 156 ± 13a 1,068 ± 69a 3,191 ± 413a 3,501 ± 580b P 0.209 0.000 0.771 0.347 0.341 0.033 AST serum activity (U/L) CK serum activity (U/L) Days of age Salinomycin level (ppm) 21 28 35 21 28 35 0 89 ± 3a 98 ± 7b 139 ± 10a 1,286 ± 178a 2,438 ± 302a 5,997 ± 1,183a,b 60 81 ± 2a 101 ± 5b 146 ± 15a 1,032 ± 47a 2,640 ± 339a 7,003 ± 956a 120 83 ± 2a 113 ± 13b 140 ± 12a 1,062 ± 96a 2,335 ± 334a 7,145 ± 962a 180 86 ± 3a 170 ± 17a 156 ± 13a 1,068 ± 69a 3,191 ± 413a 3,501 ± 580b P 0.209 0.000 0.771 0.347 0.341 0.033 1Values correspond to mean ± SEM of 12 observations per treatment. Within a column, means with different superscripts differ significantly (P < 0.05). View Large Table 3. Effect of dietary supplementation with salinomycin from d 21 to 38 on serum activity of aspartate aminotransferasa (AST) and creatine kinase (CK) of broiler chickens.1 AST serum activity (U/L) CK serum activity (U/L) Days of age Salinomycin level (ppm) 21 28 35 21 28 35 0 89 ± 3a 98 ± 7b 139 ± 10a 1,286 ± 178a 2,438 ± 302a 5,997 ± 1,183a,b 60 81 ± 2a 101 ± 5b 146 ± 15a 1,032 ± 47a 2,640 ± 339a 7,003 ± 956a 120 83 ± 2a 113 ± 13b 140 ± 12a 1,062 ± 96a 2,335 ± 334a 7,145 ± 962a 180 86 ± 3a 170 ± 17a 156 ± 13a 1,068 ± 69a 3,191 ± 413a 3,501 ± 580b P 0.209 0.000 0.771 0.347 0.341 0.033 AST serum activity (U/L) CK serum activity (U/L) Days of age Salinomycin level (ppm) 21 28 35 21 28 35 0 89 ± 3a 98 ± 7b 139 ± 10a 1,286 ± 178a 2,438 ± 302a 5,997 ± 1,183a,b 60 81 ± 2a 101 ± 5b 146 ± 15a 1,032 ± 47a 2,640 ± 339a 7,003 ± 956a 120 83 ± 2a 113 ± 13b 140 ± 12a 1,062 ± 96a 2,335 ± 334a 7,145 ± 962a 180 86 ± 3a 170 ± 17a 156 ± 13a 1,068 ± 69a 3,191 ± 413a 3,501 ± 580b P 0.209 0.000 0.771 0.347 0.341 0.033 1Values correspond to mean ± SEM of 12 observations per treatment. Within a column, means with different superscripts differ significantly (P < 0.05). View Large Table 4 shows the relative organ weights measured at the end of the experiment. Proventriculus, gizzard, and pancreas showed significantly higher mean values in the birds receiving 180 ppm salinomycin compared with the other 3 groups. Table 4. Effect of dietary supplementation with salinomycin from d 21 to 38 on the relative weights of liver, heart, spleen, proventriculus, gizzard, pancreas, and bursa of Fabricius of broiler chickens.1 Relative organ weight (% of body weight) Salinomycin level (ppm) Liver Heart Spleen Proventriculus Gizzard Pancreas Bursa of Fabricius 0 1.83 ± 0.06a 0.54 ± 0.03a 0.101 ± 0.006ab 0.32 ± 0.01b 1.00 ± 0.04b 0.164 ± 0.007b 0.056 ± 0.004a 60 1.81 ± 0.05a 0.59 ± 0.03a 0.094 ± 0.006b 0.31 ± 0.01b 1.01 ± 0.04b 0.182 ± 0.006b 0.063 ± 0.004a 120 1.87 ± 0.05a 0.61 ± 0.05a 0.084 ± 0.006b 0.33 ± 0.01b 0.97 ± 0.04b 0.174 ± 0.006b 0.056 ± 0.004a 180 1.96 ± 0.05a 0.62 ± 0.03a 0.118 ± 0.006a 0.43 ± 0.01a 1.26 ± 0.04a 0.246 ± 0.006a 0.067 ± 0.004a P 0.174 0.274 0.001 0.000 0.000 0.000 0.064 Relative organ weight (% of body weight) Salinomycin level (ppm) Liver Heart Spleen Proventriculus Gizzard Pancreas Bursa of Fabricius 0 1.83 ± 0.06a 0.54 ± 0.03a 0.101 ± 0.006ab 0.32 ± 0.01b 1.00 ± 0.04b 0.164 ± 0.007b 0.056 ± 0.004a 60 1.81 ± 0.05a 0.59 ± 0.03a 0.094 ± 0.006b 0.31 ± 0.01b 1.01 ± 0.04b 0.182 ± 0.006b 0.063 ± 0.004a 120 1.87 ± 0.05a 0.61 ± 0.05a 0.084 ± 0.006b 0.33 ± 0.01b 0.97 ± 0.04b 0.174 ± 0.006b 0.056 ± 0.004a 180 1.96 ± 0.05a 0.62 ± 0.03a 0.118 ± 0.006a 0.43 ± 0.01a 1.26 ± 0.04a 0.246 ± 0.006a 0.067 ± 0.004a P 0.174 0.274 0.001 0.000 0.000 0.000 0.064 1Values are mean ± SEM of 4 replicate pens per treatment. Within a column, means with different superscripts differ significantly (P < 0.05). View Large Table 4. Effect of dietary supplementation with salinomycin from d 21 to 38 on the relative weights of liver, heart, spleen, proventriculus, gizzard, pancreas, and bursa of Fabricius of broiler chickens.1 Relative organ weight (% of body weight) Salinomycin level (ppm) Liver Heart Spleen Proventriculus Gizzard Pancreas Bursa of Fabricius 0 1.83 ± 0.06a 0.54 ± 0.03a 0.101 ± 0.006ab 0.32 ± 0.01b 1.00 ± 0.04b 0.164 ± 0.007b 0.056 ± 0.004a 60 1.81 ± 0.05a 0.59 ± 0.03a 0.094 ± 0.006b 0.31 ± 0.01b 1.01 ± 0.04b 0.182 ± 0.006b 0.063 ± 0.004a 120 1.87 ± 0.05a 0.61 ± 0.05a 0.084 ± 0.006b 0.33 ± 0.01b 0.97 ± 0.04b 0.174 ± 0.006b 0.056 ± 0.004a 180 1.96 ± 0.05a 0.62 ± 0.03a 0.118 ± 0.006a 0.43 ± 0.01a 1.26 ± 0.04a 0.246 ± 0.006a 0.067 ± 0.004a P 0.174 0.274 0.001 0.000 0.000 0.000 0.064 Relative organ weight (% of body weight) Salinomycin level (ppm) Liver Heart Spleen Proventriculus Gizzard Pancreas Bursa of Fabricius 0 1.83 ± 0.06a 0.54 ± 0.03a 0.101 ± 0.006ab 0.32 ± 0.01b 1.00 ± 0.04b 0.164 ± 0.007b 0.056 ± 0.004a 60 1.81 ± 0.05a 0.59 ± 0.03a 0.094 ± 0.006b 0.31 ± 0.01b 1.01 ± 0.04b 0.182 ± 0.006b 0.063 ± 0.004a 120 1.87 ± 0.05a 0.61 ± 0.05a 0.084 ± 0.006b 0.33 ± 0.01b 0.97 ± 0.04b 0.174 ± 0.006b 0.056 ± 0.004a 180 1.96 ± 0.05a 0.62 ± 0.03a 0.118 ± 0.006a 0.43 ± 0.01a 1.26 ± 0.04a 0.246 ± 0.006a 0.067 ± 0.004a P 0.174 0.274 0.001 0.000 0.000 0.000 0.064 1Values are mean ± SEM of 4 replicate pens per treatment. Within a column, means with different superscripts differ significantly (P < 0.05). View Large The analysis of the liver, breast muscle, and serum samples for salinomycin showed no detectable levels in muscle or liver except for the samples from the birds receiving 180 ppm salinomycin. The mean ± SD values of salinomycin in these samples were 8.9 ± 1.2 and 13.3 ± 6.5 μg/kg for breast muscle and liver, respectively. In contrast with the tissue samples, salinomycin was detected in the serum samples of all birds receiving salinomycin in the diet. The mean ± SD of salinomycin in serum samples were N.D. (not detected), 24.4 ± 7.9, 61.4 ± 18.9, and 94.5 ± 9.1 μg/L for salinomycin levels in the diet of 0, 60, 120, and 180 ppm, respectively. Serum salinomycin content followed a linear dose relationship with salinomycin content in feed (r2 = 0.999, regression equation: y = 0.584x – 10) as shown in Figure 1. Chromatograms of the salinomycin standard and of serum samples from birds receiving 0 and 180 ppm salinomycin are shown in Figure 2. Figure 1. View largeDownload slide Salinomycin concentration in serum vs. salinomycin content of feed. Figure 1. View largeDownload slide Salinomycin concentration in serum vs. salinomycin content of feed. Figure 2. View largeDownload slide HPLC-MS/MS chromatograms: (A) salinomycin standard equivalent to 50 μg/L in sample; (B) serum from a control chicken with no detectable levels of salinomycin; (C) serum from a chicken fed 180 ppm salinomycin containing 77.5 μg/L of salinomycin. The retention time of salinomycin was 11.5 min. Figure 2. View largeDownload slide HPLC-MS/MS chromatograms: (A) salinomycin standard equivalent to 50 μg/L in sample; (B) serum from a control chicken with no detectable levels of salinomycin; (C) serum from a chicken fed 180 ppm salinomycin containing 77.5 μg/L of salinomycin. The retention time of salinomycin was 11.5 min. The mortality registered during the 18 experimental d was 14, 22, 7, and 0 birds, from the total of 88, for salinomycin levels of 0, 60, 120, and 180 ppm, respectively. Post-mortem examination established that in all cases, the chickens died from right ventricular hypertrophy, with or without hydropericardium or ascites. Histological examination of the tissues collected at the end of the experiment showed cellular necrosis in pectoral and cranial iliotibial muscles as the major findings in chickens exposed to 180 ppm salinomycin (Figure 3). No alterations were seen in the other groups. Figure 3. View largeDownload slide Cellular necrosis in breast muscle (A) and cranial iliotibial muscle (B) in a chicken fed 180 ppm salinomycin in the diet from d 21 to 38. Bar = 50 μm. Figure 3. View largeDownload slide Cellular necrosis in breast muscle (A) and cranial iliotibial muscle (B) in a chicken fed 180 ppm salinomycin in the diet from d 21 to 38. Bar = 50 μm. DISCUSSION Although the signs and lesions of ionophore toxicosis are not pathognomonic, the toxicosis is clinically characterized by anorexia, diarrhea, dyspnea, ataxia, depression, recumbence, and death, and pathologically by focal degenerative cardiomyopathy, skeletal muscle necrosis, and congestive heart failure (Novilla, 1992). With the exception of anorexia (shown as decreased feed intake) none of the clinical signs of ionophore toxicosis were observed in the present experiment; however, histological examination revealed skeletal muscle necrosis. Our findings indicate that exposure to salinomycin at 3 times the recommended prophylactic level for 18 d does not cause overt toxicity in chickens. However, all salinomycin levels tested, even at the recommended dosage, caused at least one adverse effect on performance. At the recommended level of 60 ppm no differences in body weight, feed intake, or feed conversion were recorded on d 1, 15, or 18 of the experiment. However, the 18-d body weight gain was significantly lower in all chickens receiving salinomycin, although this difference was only 5% compared to the chickens receiving the recommended level and in contrast with the findings of Keshavarz and McDougald (1982), who reported no effects on performance at the recommended salinomycin level. In regard to the decrease in body weight and increase in feed conversion caused by 2 and 3 times the recommended salinomycin level, our results agree with those of Keshavarz and McDougald (1982) who reported decreased body weight in one-week-old chickens fed salinomycin levels 1.5, 2, 2.5, and 3 times the recommended level for 3 weeks; at 2.5 and 3 times the recommended levels the feed conversion was affected as well. Our results also agree with those of Migaki and Babcock (1979) who reported decreased growth with dietary salinomycin levels >80 ppm and partially agree with the results of Rizvi and Anjum (1999), who reported decreased body weight and increased feed conversion with 120 and 180 ppm salinomycin in the diet. In the present study, feed conversion was affected by 180 ppm but not by 120 ppm. Further, in our experiment chickens fed 180 ppm salinomycin had a feed conversion rate of 1.7 compared to 1.52 in the control group. This difference was much lower than the one reported by Rizvi and Anjum (1999) who observed feed conversion values of 2.21, 3.25, 3.85, and 4.16 with salinomycin levels of 0, 60, 120, and 180 ppm, respectively. These differences can be the result of the different exposure times (2.5 weeks in the present study vs. 8 weeks in the one from Rizvi and Anjum) and/or the difference in broiler performance between the current strains of chickens and those from 1999. In almost 3 decades, genetic selection have reduced significantly the time required to reach the target body weight with better feed conversion rates. The clinical enzymology results were contradictory. AST is a cytosolic enzyme present in many tissues and it is considered to be a non-specific but highly sensitive marker of muscle damage; even though CK is more specific for muscle damage, AST is often used to confirm changes in CK (Kramer and Hoffmann, 1997). In the present study, a significant increase in AST activity was seen after 7 d of exposure to 180 ppm salinomycin, confirming the sensitivity of this enzyme to muscular damage; however, no difference was observed after 14 d of exposure. A possible explanation for this finding could be the high prevalence of pulmonary hypertension syndrome (which affects soft tissues like the heart and the liver) that affected the birds growing faster (0 and 60 ppm salinomycin). CK increases are usually associated with damage to cardiac and/or skeletal muscle, the target tissues of ionophore antibiotics. In contrast to expectations, the group receiving the highest salinomycin level had the lowest CK serum levels after 14 d of exposure. This finding could also be related to the high incidence of right ventricular hypertrophy found in the birds receiving 0, 60, and 120 ppm salinomycin, all of which showed high mortality due to ascites syndrome. The CK kit used in the present study determines the isoenzyme CK-MB, which has traditionally been used as a marker protein for acute myocardial infarction (Adams et al., 1994); the damage to the myocardium from the pulmonary hypertension could have caused the increase in serum CK in the present study, not necessarily the exposure to salinomycin. The enzyme LDH has 2 main monomers found in heart (LDH-1) and liver and skeletal muscle (LDH-5); however, it is also found in kidneys and red blood cells (Kramer and Hoffmann, 1997). In broiler chickens, the ionophore monensin caused an increase in LDH activity, even at the prophylactic dose of 100 ppm (Dowling, 1992). In the present experiment, no difference in LDH activity among experimental groups was observed at any sampling time. It is possible that LDH is not a sensitive enough muscle damage marker or that the serum enzyme activity had also been influenced by the high morbidity of pulmonary hypertension and ascites as previously mentioned. Measurement of relative organ weights is common in toxico-pathological studies. In the present study a significant increase in the relative weights of proventriculus, gizzard, spleen, and pancreas were observed only in the birds fed 180 ppm salinomycin. Our results are in contrast with those of Rizvi and Anjum (1999), who found a significant decrease in the relative weight of the gizzard and kidneys and a significant increase in the relative heart weight in chickens that received 180 ppm salinomycin for 8 weeks. These differences could be due to different exposure times, since the effects of anticoccidials depend not only on the dose but also on the exposure time. No detectable levels of salinomycin were found in muscle or liver tissue from the birds fed 0, 60, or 120 ppm. Only the chickens receiving 3 times the prophylactic level showed residues in these tissues and in both cases they were above the MRL of 5 μg/kg recommended by the European Union (8.9 μg/kg in breast muscle and 13.3 μg/kg in liver). The results obtained confirm the current regulations regarding withdrawal times for salinomycin. In the United States there is no withdrawal time required, whereas the European Union requires withdrawal of salinomycin from the chicken diets at least 24 hours before slaughter. In the present study the feed was suspended 12 hours before the chickens were sacrificed and even at double the recommended prophylactic dose there were no detectable residues of salinomycin in muscle or liver. These results are in contrast with those from the serum samples taken 14 d after the beginning of the experiment, at which time the chickens were still being fed. All chickens receiving salinomycin had detectable levels in serum and the salinomycin concentration was linearly related to the dietary levels. This finding suggests that it is possible to measure salinomycin in serum as a marker of exposure and even estimate the concentration in feed by using the regression equation y = 0.584x – 10. No mortality associated with salinomycin was observed in any of the treatments. Mortality was found to be due only to ascitic syndrome. Interestingly, the growth decrease caused by 180 ppm salinomycin prevented the presentation of ascitic syndrome since no mortality was observed in this group. Our results agree with those of Rizvi and Anjum (1999), who reported no mortality in chickens fed 60, 120, or 180 ppm salinomycin for 8 weeks. In summary, the results of the present study showed that no mortality or clinical signs of toxicosis occur in broiler chickens fed 2 or 3 times the recommended prophylactic salinomycin dose. Even though chickens receiving 60 ppm salinomycin had a 5% decrease in 18-d body weight gain compared with controls, it is important to note that the birds were not exposed to coccidia as occurs under field conditions. Birds without a coccidiostat in their diet would experience severe adverse effects on their health and performance in field conditions. Clinical biochemistry showed AST as the most sensitive enzyme for muscle damage. It may be possible that the other enzymes measured (CK and LDH) might also be used as markers of muscular damage in situations where the confounding effect of ascites syndrome is not present (chickens are not raised at high altitude). The chemical analysis results confirm the fast elimination of salinomycin and the lack of the requirement for a withdrawal period. Determination of salinomycin in serum could be used as a marker of exposure and to predict the salinomycin level in the diet. FUNDING Partial funding was provided by Alimentos Balanceados Tequendama, Albateq, S.A. Bogotá, Colombia. DECLARATION OF CONFLICTING INTERESTS The authors declare no potential conflicts of interest with respect to the research, authorship, and/or publication of this article. REFERENCES Adams J. E. , Schechtman K. B. , Landt Y. , Ladenson J. H. , Jaffe A. S. . 1994 . Comparable detection of acute myocardial infarction by creatine kinase MB isoenzyme and cardiac troponin I . Clin. Chem. 40 : 1291 – 1295 . Google Scholar PubMed Blanchard J. 1981 . Evaluation of the relative efficacy of various techniques for deproteinizing plasma samples prior to high-performance liquid chromatographic analysis . J. of Chromatogr. B: Biomed. Sci. and Appl. 226 : 455 – 460 . Google Scholar CrossRef Search ADS Clarke L. , Fodey T. L. , Crooks S. R. H. , Moloney M. , O’Mahony J. , Delahaut P. , O’Kennedy R. , Danaher M. . 2014 . A review of coccidiostats and the analysis of their residues in meat and other food . Meat Sci. 97 : 358 – 374 . Google Scholar CrossRef Search ADS PubMed Commission Regulation (EC) No. 496/2007 of 4 May 2007 amending Regulation (EC) No. 600/2005 as regards the introduction of a maximum residue limit for the feed additive ‘Salinomax 120G’, belonging to the group of coccidiostats and other medicinal substances. Official Journal of the European Union L117/9, 5.5 . 2007 . Dowling L. 1992 . Ionophore toxicity in chickens: a review of pathology and diagnosis . Avian Pathol. 21 : 355 – 368 . Google Scholar CrossRef Search ADS PubMed Keshavarz K. , McDougald L. R. . 1982 . Anticoccidial Drugs: Growth and Performance Depressing Effects in Young Chickens . Poult. Sci. 61 : 699 – 705 . Google Scholar CrossRef Search ADS PubMed Kinashi H. , Otake N. , Yonehara H. , Sate S. , Saito Y. . 1973 . The structure of salinomycin, a new member of the polyether antibiotics . Tetrahedron Lett. 14 : 4955 – 4958 . Google Scholar CrossRef Search ADS Kramer J. W. , Hoffmann W. E. . 1997 . Clinical Enzymology . Pages 303 – 325 in Clinical Biochemistry of Domestic Animals , 5th Edn . Kaneko J. J. , Harvey J. W. , Bruss M. L. , eds. Academic Press , San Diego . Matabudul D. K. , Lumley I. D. , Points J. S. . 2002 . The determination of 5 anticoccidial drugs (nicarbazin, lasalocid, monensin, salinomycin and narasin) in animal livers and eggs by liquid chromatography linked with tandem mass spectrometry (LC-MS-MS) . Analyst . 127 : 760 – 768 . Google Scholar CrossRef Search ADS PubMed Migaki T. T. , Babcock W. E. . 1979 . Safety evaluation of salinomycin in broiler chickens reared in floor pens . Poult. Sci. 58 : 481 – 482 . Google Scholar CrossRef Search ADS PubMed Miyazaki Y. , Shibuya M. , Sugawara H. , Kawaguchi O. , Hirose C. , Nagatsu J. , Esumi S. . 1974 . Salinomycin, a new polyether antibiotic . J. Antibiot. 27 : 814 – 821 . Google Scholar CrossRef Search ADS PubMed Novilla M. 1992 . The veterinary importance of the toxic syndrome induced by ionophores . Vet. Hum. Toxicol. 34 : 66 – 70 . Google Scholar PubMed Rizvi F. , Anjum A. D. . 1999 . Effect of salinomycin on broiler health . Vet. Arh. 69 : 39 – 47 . Statistix 9 for Windows . 2008 . User's Manual. Tallahassee Analytical Software . © 2018 Poultry Science Association Inc. This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Poultry Science Oxford University Press

Effects on health, performance, and tissue residues of the ionophore antibiotic salinomycin in finishing broilers (21 to 38 d)

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© 2018 Poultry Science Association Inc.
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0032-5791
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Abstract

ABSTRACT A study was conducted to evaluate the effects of feeding salinomycin at the recommended prophylactic level, and at 2 and 3 times this level, to finishing male broilers (d 21 to 38). Four treatment groups were given the experimental diets containing 0, 60, 120, or 180 parts per million (ppm) salinomycin from d 21 to 38. Performance, relative organ weights, selected serum enzymes, and salinomycin residues in liver, muscle, and serum were determined. Salinomycin supplementation had no effect on body weight, feed intake, or feed conversion, and caused no overt signs of toxicity. After a week of being fed the salinomycin diets, the serum activity of aspartate aminotransferase was significantly increased in chickens fed 180 ppm compared with controls. These birds also showed microscopic lesions in breast and thigh muscles, but not in cardiac muscle. Salinomycin residues were not detected by high-performance liquid chromatography coupled to tandem mass spectrometry in liver or muscle samples from the birds fed 0, 60, or 120 ppm salinomycin. However, chickens fed 180 ppm salinomycin had detectable levels in liver and muscle above the maximum residue level of 5 μg/kg established by the European Union. All birds fed salinomycin had salinomycin in their sera with levels ranging from N.D. (not detected) in the controls to 24.4 ± 7.9, 61.4 ± 18.9, and 94.5 ± 9.1 μg/L for salinomycin dietary levels of 60, 120, and 180 ppm, respectively. Serum salinomycin concentration was linearly related with salinomycin content in feed (y = 0.584x − 10, r2 = 0.999). The results showed that even at 3 times the prophylactic level, salinomycin does not induce clinical toxicosis or mortality. No salinomycin residues were found in edible tissues at the recommended dietary level or at 2 times this level. However, salinomycin was detected in serum regardless of the dietary level. A simple method for salinomycin determination in serum is described which can be used as a marker of exposure and/or to predict levels in the diet. INTRODUCTION Ionophore antibiotics are commonly used as feed additives for the prevention of coccidiosis in poultry and as growth promoters in cattle and swine. These compounds are produced by 53 different bacteria of the Streptomycetaceae family and are characterized by multiple tetrahydrofuran rings connected together as spiroketal moieties (Clarke et al., 2014). Among the most commonly used ionophores in poultry are monensin, maduramicin, lasalocid, narasin, and salinomycin. The latter was discovered as a fermentation product of a strain of Streptomyces albus (Kinashi et al., 1973; Miyazaki et al., 1974). Migaki and Babcock (1979) conducted a safety evaluation of salinomycin in broiler chickens by feeding them diets containing 50, 60, 80, 100, and 160 mg/g salinomycin from 1 to 56 d. In this study, the weight gains at 50 and 60 mg/kg salinomycin were not significantly different from the control, at 80 mg/kg the weight gain was slightly below controls, and at 100 and 160 mg/kg salinomycin depressed weight gain. Feed conversion was only affected by 160 mg/kg (Migaki and Babcock, 1979). Currently, salinomycin is commonly added to poultry diets at a concentration of 60 mg/kg and it is compatible with most ingredients, except several antibiotics (tiamulin, erythromycin, sulfachloropyrazine, sulfaquinoxaline, and chloramphenicol) and the antioxidant XAX-M (Dowling, 1992). Generally, ionophores are safe at the recommended inclusion levels; however, toxicosis can result from overdose and misuse situations (Novilla, 1992). The effects of feeding chickens with salinomycin at 1.5, 2, 2.5, and 3 times the recommended levels (Keshavarz and McDougald, 1982) and at 2 and 3 times the recommended levels have been reported (Rizvi and Anjum, 1999). However, in both of these experiments the salinomycin diets were started at d 8 and were given for 3 and 8 weeks, respectively. In Colombia and other countries, salinomycin is used in finishing diets (d 21 to 38), until the market weight is reached. Salinomycin residues in poultry meat are not regulated in Colombia but the European Union has a maximum residue level (MRL) of 5 μg/kg in liver, kidney, and muscle (Commission Regulation (EC) No. 496/2007). Given the widespread use of salinomycin in Colombian poultry diets and also that there have been complaints of growers blaming salinomycin for causing mortality in their flocks, the present study aimed at determining the potential adverse effects of feeding salinomycin to 3-week-old chickens at the recommended prophylactic level (60 ppm) and at 2 and 3 times this level. Further, salinomycin residues in muscle, liver, and serum were determined by high-performance liquid chromatography coupled to tandem mass spectrometry (HPLC-MS/MS). MATERIALS AND METHODS The experiment was approved by the Ethics Committee of the College of Veterinary Medicine of the National University of Colombia under approval No. CB-FMVZ-UN-008–17. A total of 400 male day-old chicks (Ross 308) were received in the experimental premises (Poultry Research Building, College of Veterinary Medicine, located in Bogota, Colombia, at 2,600 m above sea level) and individually wing banded. For the first 20 d, all chicks received the same starter ration, which contained the mixture of anticoccidials narasin, and nicarbazin at dietary concentrations of 40 ppm each (Maxiban, Elanco, IN). At d 21, all birds were weighed and the heaviest (24) and lightest (24) were removed from the experiment. The remaining 352 birds were distributed at random into 4 dietary treatments with 4 replicates of 22 birds each, for a total of 88 chickens per treatment. The experiment consisted of a completely randomized dietary arrangement of treatments with a control diet (without salinomycin), a diet with the recommended salinomycin level (60 mg/kg), and diets with 2 times (120 mg/kg) and 3 times (180 mg/kg) the recommended level. These levels were chosen in order to simulate a formulation mistake or an improper mixing of the diet. The experimental diets were provided ad libitum for 18 d (d 21 to 38). Birds were kept in floor pens at a density of 10 birds/m2 at an initial temperature of 27°C, decreased by 2°C every 7 d until the end of the experiment. Response variables measured or calculated during and at the end of the experiment were as follows: Performance variables: body weight (d 21, 28, 35, and 38), feed intake (measured daily and analyzed weekly), and feed conversion (calculated weekly). For these variables the experimental unit was the replicate pen. Blood chemistry: blood samples from 3 birds per replicate pen (12 birds per treatment) were taken at d 21, 28, and 35 (d 1, 7, and 14 of the experiment) for the determination of the serum activity of creatine kinase (CK), lactate dehydrogenase (LDH), and aspartate aminotransferase (AST). Serum samples were processed using commercial spectrophotometric kits (Spinreact, S.A., Barcelona, Spain), part numbers 1,001,050, 1,001,260, 1,001,161 for CK, LDH, and AST, respectively. Relative organ weights: at the end of the experiment (d 38), 30 birds taken at random from each treatment were sacrificed for the determination of the relative weight of proventriculus, gizzard, liver, heart, pancreas, spleen, and bursa of Fabricius. Salinomycin determination in muscle, liver, and serum: at the end of the experiment, liver and muscle (left deep pectoral) samples were taken from 4 birds per treatment for the determination of salinomycin by HPLC-MS/MS. The analysis was performed on a Shimadzu Prominence HPLC (Shimadzu, Kyoto, Japan) coupled to an ABI 3200 QTrap triple quadrupole mass spectrometer detector (MDS Sciex, Toronto, Canada). Also, blood samples from 4 birds per treatment were taken at d 35 (d 15 of the experiment) for salinomycin determination. Liver and muscle samples were analyzed according to the method described by Matabudul et al. (2002), whereas the serum determination was conducted after serum deproteinization according to Blanchard (1981), as follows: to 400 μL of serum, 1.6 mL of acetonitrile was added in a 2 mL centrifuge vial. The contents were shaken in vortex for 30 seconds and centrifuged at 9,000 × g for 15 minutes. An aliquot of 200 μL of the supernatant was further diluted with 1.8 mL of acetonitrile, and shaken and centrifuged as described previously. An aliquot of approximately 1 mL was then filtered through a 0.22 μm pore size polytetrafluoroethylene membrane into an autosampler vial for the determination of salinomycin by HPLC-MS/MS. Histology: tissue samples of liver, skeletal muscle (left deep pectoral and left cranial iliotibial), and cardiac muscle were taken from 4 birds per treatment at the end of the experiment. Tissue samples were fixed in neutral-buffered formalin, and stained with hematoxylin-eosin before microscopic examination. Quantitative response variables were analyzed using Statistix 9 for Windows (2008) by one-way ANOVA. When the P value of the ANOVA test was <0.05, means were separated using the Tukey's test (Statistix 9 for Windows, 2008, Tallahassee, FL). RESULTS Table 1 shows the mean body weight of the 4 experimental treatments at d 21, 28, 35, and 38 (corresponding to d 1, 8, 15, and 18 of the experiment) and the 18-d body weight gain. Significant differences in body weight were observed at d 28, 35, and 38. At d 28 and 38, the birds receiving 120 and 180 ppm salinomycin showed significantly lower body weights compared with the birds fed 0 or 60 ppm salinomycin. Further, the chickens fed the diet with 180 ppm salinomycin had significantly lower body weights than the birds fed 120 ppm at d 28, 35, and 38. Body weight gain was significantly different among the 4 treatments and followed an inverse dose-response relationship. Chickens without salinomycin had the highest weight gain and chickens receiving 3 times the recommended level, the lowest. The body weight gain in these chickens was only 56% of the control group. Feed intake and feed conversion for the 18 experimental d are shown in Table 2. The first 2 weeks feed intake was significantly lower only for the chickens receiving 3 times the recommended dose of salinomycin; however from d 35 to 38, feed intake was significantly lower for the groups receiving 2 and 3 times the recommended level. For all time periods evaluated only the group receiving 180 ppm salinomycin had a feed conversion significantly higher than the control group. Overall (d 21 to 38), feed intake and feed conversion were significantly different only between the group receiving 180 ppm salinomycin and the other 3 groups. Table 1. Effect of dietary supplementation with salinomycin from d 21 to 38 on body weight and body weight gain of broiler chickens.1 Days of age Total body weight gain (g) and % of control Salinomycin level (ppm) 21 28 35 38 21 to 38 0 781.3 ± 3.8ª 1,639 ± 12ª 2,392 ± 19a 2,610 ± 26a 1,828 ± 24a (100) 60 794.6 ± 3.7a 1,614 ± 6.5ª 2,291 ± 45ab 2,538 ± 17a 1,744 ± 18b (95) 120 790.0 ± 6.6ª 1,557 ± 7.5b 2,210 ± 16b 2,388 ± 4.8b 1,598 ± 6c (87) 180 800.0 ± 9.5ª 1,303 ± 16c 1,708 ± 21c 1,832 ± 24c 1,032 ± 17d (56) P 0.262 0.000 0.000 0.000 0.000 Days of age Total body weight gain (g) and % of control Salinomycin level (ppm) 21 28 35 38 21 to 38 0 781.3 ± 3.8ª 1,639 ± 12ª 2,392 ± 19a 2,610 ± 26a 1,828 ± 24a (100) 60 794.6 ± 3.7a 1,614 ± 6.5ª 2,291 ± 45ab 2,538 ± 17a 1,744 ± 18b (95) 120 790.0 ± 6.6ª 1,557 ± 7.5b 2,210 ± 16b 2,388 ± 4.8b 1,598 ± 6c (87) 180 800.0 ± 9.5ª 1,303 ± 16c 1,708 ± 21c 1,832 ± 24c 1,032 ± 17d (56) P 0.262 0.000 0.000 0.000 0.000 1Values correspond to mean ± SEM of 4 replicates per treatment. Within a column, means with different superscripts differ significantly (P < 0.05). View Large Table 1. Effect of dietary supplementation with salinomycin from d 21 to 38 on body weight and body weight gain of broiler chickens.1 Days of age Total body weight gain (g) and % of control Salinomycin level (ppm) 21 28 35 38 21 to 38 0 781.3 ± 3.8ª 1,639 ± 12ª 2,392 ± 19a 2,610 ± 26a 1,828 ± 24a (100) 60 794.6 ± 3.7a 1,614 ± 6.5ª 2,291 ± 45ab 2,538 ± 17a 1,744 ± 18b (95) 120 790.0 ± 6.6ª 1,557 ± 7.5b 2,210 ± 16b 2,388 ± 4.8b 1,598 ± 6c (87) 180 800.0 ± 9.5ª 1,303 ± 16c 1,708 ± 21c 1,832 ± 24c 1,032 ± 17d (56) P 0.262 0.000 0.000 0.000 0.000 Days of age Total body weight gain (g) and % of control Salinomycin level (ppm) 21 28 35 38 21 to 38 0 781.3 ± 3.8ª 1,639 ± 12ª 2,392 ± 19a 2,610 ± 26a 1,828 ± 24a (100) 60 794.6 ± 3.7a 1,614 ± 6.5ª 2,291 ± 45ab 2,538 ± 17a 1,744 ± 18b (95) 120 790.0 ± 6.6ª 1,557 ± 7.5b 2,210 ± 16b 2,388 ± 4.8b 1,598 ± 6c (87) 180 800.0 ± 9.5ª 1,303 ± 16c 1,708 ± 21c 1,832 ± 24c 1,032 ± 17d (56) P 0.262 0.000 0.000 0.000 0.000 1Values correspond to mean ± SEM of 4 replicates per treatment. Within a column, means with different superscripts differ significantly (P < 0.05). View Large Table 2. Effect of dietary supplementation with salinomycin from d 21 to 38 on feed intake and feed conversion of broiler chickens.1 Days of age—Feed intake (g/chicken) Total feed intake and % of control Days of age—Feed conversion (g/g) Overall feed conversion and % of control Salinomycin level (ppm) 21 to 27 28 to 35 35 to 38 21 to 38 21 to 27 28 to 35 35 to 38 21 to 38 0 988 ± 12ª 1050 ± 27ª 402 ± 24a 2,427 ± 56a (100) 1.24 ± 0.02b 1.38 ± 0.03b 1.52 ± 0.05b 1.33 ± 0.03b (100) 60 987 ± 13a 1017 ± 57ª 386 ± 16a 2,391 ± 65a (98) 1.25 ± 0.02b 1.44 ± 0.04b 1.63 ± 0.04ab 1.37 ± 0.02b (103) 120 945 ± 14ª 1005 ± 24ab 377 ± 9b 2,328 ± 31ab (96) 1.30 ± 0.03b 1.40 ± 0.03b 1.49 ± 0.02b 1.46 ± 0.01b (109) 180 820 ± 14b 956 ± 39b 338 ± 9c 2,115 ± 46b (87) 1.40 ± 0.03a 1.63 ± 0.02a 1.70 ± 0.02a 2.05 ± 0.04a (154) P 0.000 0.002 0.000 0.004 0.003 0.000 0.003 0.000 Days of age—Feed intake (g/chicken) Total feed intake and % of control Days of age—Feed conversion (g/g) Overall feed conversion and % of control Salinomycin level (ppm) 21 to 27 28 to 35 35 to 38 21 to 38 21 to 27 28 to 35 35 to 38 21 to 38 0 988 ± 12ª 1050 ± 27ª 402 ± 24a 2,427 ± 56a (100) 1.24 ± 0.02b 1.38 ± 0.03b 1.52 ± 0.05b 1.33 ± 0.03b (100) 60 987 ± 13a 1017 ± 57ª 386 ± 16a 2,391 ± 65a (98) 1.25 ± 0.02b 1.44 ± 0.04b 1.63 ± 0.04ab 1.37 ± 0.02b (103) 120 945 ± 14ª 1005 ± 24ab 377 ± 9b 2,328 ± 31ab (96) 1.30 ± 0.03b 1.40 ± 0.03b 1.49 ± 0.02b 1.46 ± 0.01b (109) 180 820 ± 14b 956 ± 39b 338 ± 9c 2,115 ± 46b (87) 1.40 ± 0.03a 1.63 ± 0.02a 1.70 ± 0.02a 2.05 ± 0.04a (154) P 0.000 0.002 0.000 0.004 0.003 0.000 0.003 0.000 1Values correspond to mean ± SEM of 4 replicates per treatment. Within a column, means with different superscripts differ significantly (P < 0.05). View Large Table 2. Effect of dietary supplementation with salinomycin from d 21 to 38 on feed intake and feed conversion of broiler chickens.1 Days of age—Feed intake (g/chicken) Total feed intake and % of control Days of age—Feed conversion (g/g) Overall feed conversion and % of control Salinomycin level (ppm) 21 to 27 28 to 35 35 to 38 21 to 38 21 to 27 28 to 35 35 to 38 21 to 38 0 988 ± 12ª 1050 ± 27ª 402 ± 24a 2,427 ± 56a (100) 1.24 ± 0.02b 1.38 ± 0.03b 1.52 ± 0.05b 1.33 ± 0.03b (100) 60 987 ± 13a 1017 ± 57ª 386 ± 16a 2,391 ± 65a (98) 1.25 ± 0.02b 1.44 ± 0.04b 1.63 ± 0.04ab 1.37 ± 0.02b (103) 120 945 ± 14ª 1005 ± 24ab 377 ± 9b 2,328 ± 31ab (96) 1.30 ± 0.03b 1.40 ± 0.03b 1.49 ± 0.02b 1.46 ± 0.01b (109) 180 820 ± 14b 956 ± 39b 338 ± 9c 2,115 ± 46b (87) 1.40 ± 0.03a 1.63 ± 0.02a 1.70 ± 0.02a 2.05 ± 0.04a (154) P 0.000 0.002 0.000 0.004 0.003 0.000 0.003 0.000 Days of age—Feed intake (g/chicken) Total feed intake and % of control Days of age—Feed conversion (g/g) Overall feed conversion and % of control Salinomycin level (ppm) 21 to 27 28 to 35 35 to 38 21 to 38 21 to 27 28 to 35 35 to 38 21 to 38 0 988 ± 12ª 1050 ± 27ª 402 ± 24a 2,427 ± 56a (100) 1.24 ± 0.02b 1.38 ± 0.03b 1.52 ± 0.05b 1.33 ± 0.03b (100) 60 987 ± 13a 1017 ± 57ª 386 ± 16a 2,391 ± 65a (98) 1.25 ± 0.02b 1.44 ± 0.04b 1.63 ± 0.04ab 1.37 ± 0.02b (103) 120 945 ± 14ª 1005 ± 24ab 377 ± 9b 2,328 ± 31ab (96) 1.30 ± 0.03b 1.40 ± 0.03b 1.49 ± 0.02b 1.46 ± 0.01b (109) 180 820 ± 14b 956 ± 39b 338 ± 9c 2,115 ± 46b (87) 1.40 ± 0.03a 1.63 ± 0.02a 1.70 ± 0.02a 2.05 ± 0.04a (154) P 0.000 0.002 0.000 0.004 0.003 0.000 0.003 0.000 1Values correspond to mean ± SEM of 4 replicates per treatment. Within a column, means with different superscripts differ significantly (P < 0.05). View Large Table 3 shows the serum activities of AST and CK measured at d 21, 28, and 35. As expected, no significant differences in enzyme activities were seen at d 21 (d 1 of the experiment). After 7 d of salinomycin supplementation AST activity was significantly higher in the chickens receiving 180 ppm salinomycin compared with the other 3 groups and reached 1.74 times that of the control group; however, after 14 d, no significant differences were observed. CK levels were not significantly different after 7 d of salinomycin supplementation but after 14 d CK activity was significantly lower in the chickens fed 180 ppm salinomycin compared with the other 2 groups receiving salinomycin. No significant differences among the experimental groups were observed in LDH activity at any sampling time (data not shown). Table 3. Effect of dietary supplementation with salinomycin from d 21 to 38 on serum activity of aspartate aminotransferasa (AST) and creatine kinase (CK) of broiler chickens.1 AST serum activity (U/L) CK serum activity (U/L) Days of age Salinomycin level (ppm) 21 28 35 21 28 35 0 89 ± 3a 98 ± 7b 139 ± 10a 1,286 ± 178a 2,438 ± 302a 5,997 ± 1,183a,b 60 81 ± 2a 101 ± 5b 146 ± 15a 1,032 ± 47a 2,640 ± 339a 7,003 ± 956a 120 83 ± 2a 113 ± 13b 140 ± 12a 1,062 ± 96a 2,335 ± 334a 7,145 ± 962a 180 86 ± 3a 170 ± 17a 156 ± 13a 1,068 ± 69a 3,191 ± 413a 3,501 ± 580b P 0.209 0.000 0.771 0.347 0.341 0.033 AST serum activity (U/L) CK serum activity (U/L) Days of age Salinomycin level (ppm) 21 28 35 21 28 35 0 89 ± 3a 98 ± 7b 139 ± 10a 1,286 ± 178a 2,438 ± 302a 5,997 ± 1,183a,b 60 81 ± 2a 101 ± 5b 146 ± 15a 1,032 ± 47a 2,640 ± 339a 7,003 ± 956a 120 83 ± 2a 113 ± 13b 140 ± 12a 1,062 ± 96a 2,335 ± 334a 7,145 ± 962a 180 86 ± 3a 170 ± 17a 156 ± 13a 1,068 ± 69a 3,191 ± 413a 3,501 ± 580b P 0.209 0.000 0.771 0.347 0.341 0.033 1Values correspond to mean ± SEM of 12 observations per treatment. Within a column, means with different superscripts differ significantly (P < 0.05). View Large Table 3. Effect of dietary supplementation with salinomycin from d 21 to 38 on serum activity of aspartate aminotransferasa (AST) and creatine kinase (CK) of broiler chickens.1 AST serum activity (U/L) CK serum activity (U/L) Days of age Salinomycin level (ppm) 21 28 35 21 28 35 0 89 ± 3a 98 ± 7b 139 ± 10a 1,286 ± 178a 2,438 ± 302a 5,997 ± 1,183a,b 60 81 ± 2a 101 ± 5b 146 ± 15a 1,032 ± 47a 2,640 ± 339a 7,003 ± 956a 120 83 ± 2a 113 ± 13b 140 ± 12a 1,062 ± 96a 2,335 ± 334a 7,145 ± 962a 180 86 ± 3a 170 ± 17a 156 ± 13a 1,068 ± 69a 3,191 ± 413a 3,501 ± 580b P 0.209 0.000 0.771 0.347 0.341 0.033 AST serum activity (U/L) CK serum activity (U/L) Days of age Salinomycin level (ppm) 21 28 35 21 28 35 0 89 ± 3a 98 ± 7b 139 ± 10a 1,286 ± 178a 2,438 ± 302a 5,997 ± 1,183a,b 60 81 ± 2a 101 ± 5b 146 ± 15a 1,032 ± 47a 2,640 ± 339a 7,003 ± 956a 120 83 ± 2a 113 ± 13b 140 ± 12a 1,062 ± 96a 2,335 ± 334a 7,145 ± 962a 180 86 ± 3a 170 ± 17a 156 ± 13a 1,068 ± 69a 3,191 ± 413a 3,501 ± 580b P 0.209 0.000 0.771 0.347 0.341 0.033 1Values correspond to mean ± SEM of 12 observations per treatment. Within a column, means with different superscripts differ significantly (P < 0.05). View Large Table 4 shows the relative organ weights measured at the end of the experiment. Proventriculus, gizzard, and pancreas showed significantly higher mean values in the birds receiving 180 ppm salinomycin compared with the other 3 groups. Table 4. Effect of dietary supplementation with salinomycin from d 21 to 38 on the relative weights of liver, heart, spleen, proventriculus, gizzard, pancreas, and bursa of Fabricius of broiler chickens.1 Relative organ weight (% of body weight) Salinomycin level (ppm) Liver Heart Spleen Proventriculus Gizzard Pancreas Bursa of Fabricius 0 1.83 ± 0.06a 0.54 ± 0.03a 0.101 ± 0.006ab 0.32 ± 0.01b 1.00 ± 0.04b 0.164 ± 0.007b 0.056 ± 0.004a 60 1.81 ± 0.05a 0.59 ± 0.03a 0.094 ± 0.006b 0.31 ± 0.01b 1.01 ± 0.04b 0.182 ± 0.006b 0.063 ± 0.004a 120 1.87 ± 0.05a 0.61 ± 0.05a 0.084 ± 0.006b 0.33 ± 0.01b 0.97 ± 0.04b 0.174 ± 0.006b 0.056 ± 0.004a 180 1.96 ± 0.05a 0.62 ± 0.03a 0.118 ± 0.006a 0.43 ± 0.01a 1.26 ± 0.04a 0.246 ± 0.006a 0.067 ± 0.004a P 0.174 0.274 0.001 0.000 0.000 0.000 0.064 Relative organ weight (% of body weight) Salinomycin level (ppm) Liver Heart Spleen Proventriculus Gizzard Pancreas Bursa of Fabricius 0 1.83 ± 0.06a 0.54 ± 0.03a 0.101 ± 0.006ab 0.32 ± 0.01b 1.00 ± 0.04b 0.164 ± 0.007b 0.056 ± 0.004a 60 1.81 ± 0.05a 0.59 ± 0.03a 0.094 ± 0.006b 0.31 ± 0.01b 1.01 ± 0.04b 0.182 ± 0.006b 0.063 ± 0.004a 120 1.87 ± 0.05a 0.61 ± 0.05a 0.084 ± 0.006b 0.33 ± 0.01b 0.97 ± 0.04b 0.174 ± 0.006b 0.056 ± 0.004a 180 1.96 ± 0.05a 0.62 ± 0.03a 0.118 ± 0.006a 0.43 ± 0.01a 1.26 ± 0.04a 0.246 ± 0.006a 0.067 ± 0.004a P 0.174 0.274 0.001 0.000 0.000 0.000 0.064 1Values are mean ± SEM of 4 replicate pens per treatment. Within a column, means with different superscripts differ significantly (P < 0.05). View Large Table 4. Effect of dietary supplementation with salinomycin from d 21 to 38 on the relative weights of liver, heart, spleen, proventriculus, gizzard, pancreas, and bursa of Fabricius of broiler chickens.1 Relative organ weight (% of body weight) Salinomycin level (ppm) Liver Heart Spleen Proventriculus Gizzard Pancreas Bursa of Fabricius 0 1.83 ± 0.06a 0.54 ± 0.03a 0.101 ± 0.006ab 0.32 ± 0.01b 1.00 ± 0.04b 0.164 ± 0.007b 0.056 ± 0.004a 60 1.81 ± 0.05a 0.59 ± 0.03a 0.094 ± 0.006b 0.31 ± 0.01b 1.01 ± 0.04b 0.182 ± 0.006b 0.063 ± 0.004a 120 1.87 ± 0.05a 0.61 ± 0.05a 0.084 ± 0.006b 0.33 ± 0.01b 0.97 ± 0.04b 0.174 ± 0.006b 0.056 ± 0.004a 180 1.96 ± 0.05a 0.62 ± 0.03a 0.118 ± 0.006a 0.43 ± 0.01a 1.26 ± 0.04a 0.246 ± 0.006a 0.067 ± 0.004a P 0.174 0.274 0.001 0.000 0.000 0.000 0.064 Relative organ weight (% of body weight) Salinomycin level (ppm) Liver Heart Spleen Proventriculus Gizzard Pancreas Bursa of Fabricius 0 1.83 ± 0.06a 0.54 ± 0.03a 0.101 ± 0.006ab 0.32 ± 0.01b 1.00 ± 0.04b 0.164 ± 0.007b 0.056 ± 0.004a 60 1.81 ± 0.05a 0.59 ± 0.03a 0.094 ± 0.006b 0.31 ± 0.01b 1.01 ± 0.04b 0.182 ± 0.006b 0.063 ± 0.004a 120 1.87 ± 0.05a 0.61 ± 0.05a 0.084 ± 0.006b 0.33 ± 0.01b 0.97 ± 0.04b 0.174 ± 0.006b 0.056 ± 0.004a 180 1.96 ± 0.05a 0.62 ± 0.03a 0.118 ± 0.006a 0.43 ± 0.01a 1.26 ± 0.04a 0.246 ± 0.006a 0.067 ± 0.004a P 0.174 0.274 0.001 0.000 0.000 0.000 0.064 1Values are mean ± SEM of 4 replicate pens per treatment. Within a column, means with different superscripts differ significantly (P < 0.05). View Large The analysis of the liver, breast muscle, and serum samples for salinomycin showed no detectable levels in muscle or liver except for the samples from the birds receiving 180 ppm salinomycin. The mean ± SD values of salinomycin in these samples were 8.9 ± 1.2 and 13.3 ± 6.5 μg/kg for breast muscle and liver, respectively. In contrast with the tissue samples, salinomycin was detected in the serum samples of all birds receiving salinomycin in the diet. The mean ± SD of salinomycin in serum samples were N.D. (not detected), 24.4 ± 7.9, 61.4 ± 18.9, and 94.5 ± 9.1 μg/L for salinomycin levels in the diet of 0, 60, 120, and 180 ppm, respectively. Serum salinomycin content followed a linear dose relationship with salinomycin content in feed (r2 = 0.999, regression equation: y = 0.584x – 10) as shown in Figure 1. Chromatograms of the salinomycin standard and of serum samples from birds receiving 0 and 180 ppm salinomycin are shown in Figure 2. Figure 1. View largeDownload slide Salinomycin concentration in serum vs. salinomycin content of feed. Figure 1. View largeDownload slide Salinomycin concentration in serum vs. salinomycin content of feed. Figure 2. View largeDownload slide HPLC-MS/MS chromatograms: (A) salinomycin standard equivalent to 50 μg/L in sample; (B) serum from a control chicken with no detectable levels of salinomycin; (C) serum from a chicken fed 180 ppm salinomycin containing 77.5 μg/L of salinomycin. The retention time of salinomycin was 11.5 min. Figure 2. View largeDownload slide HPLC-MS/MS chromatograms: (A) salinomycin standard equivalent to 50 μg/L in sample; (B) serum from a control chicken with no detectable levels of salinomycin; (C) serum from a chicken fed 180 ppm salinomycin containing 77.5 μg/L of salinomycin. The retention time of salinomycin was 11.5 min. The mortality registered during the 18 experimental d was 14, 22, 7, and 0 birds, from the total of 88, for salinomycin levels of 0, 60, 120, and 180 ppm, respectively. Post-mortem examination established that in all cases, the chickens died from right ventricular hypertrophy, with or without hydropericardium or ascites. Histological examination of the tissues collected at the end of the experiment showed cellular necrosis in pectoral and cranial iliotibial muscles as the major findings in chickens exposed to 180 ppm salinomycin (Figure 3). No alterations were seen in the other groups. Figure 3. View largeDownload slide Cellular necrosis in breast muscle (A) and cranial iliotibial muscle (B) in a chicken fed 180 ppm salinomycin in the diet from d 21 to 38. Bar = 50 μm. Figure 3. View largeDownload slide Cellular necrosis in breast muscle (A) and cranial iliotibial muscle (B) in a chicken fed 180 ppm salinomycin in the diet from d 21 to 38. Bar = 50 μm. DISCUSSION Although the signs and lesions of ionophore toxicosis are not pathognomonic, the toxicosis is clinically characterized by anorexia, diarrhea, dyspnea, ataxia, depression, recumbence, and death, and pathologically by focal degenerative cardiomyopathy, skeletal muscle necrosis, and congestive heart failure (Novilla, 1992). With the exception of anorexia (shown as decreased feed intake) none of the clinical signs of ionophore toxicosis were observed in the present experiment; however, histological examination revealed skeletal muscle necrosis. Our findings indicate that exposure to salinomycin at 3 times the recommended prophylactic level for 18 d does not cause overt toxicity in chickens. However, all salinomycin levels tested, even at the recommended dosage, caused at least one adverse effect on performance. At the recommended level of 60 ppm no differences in body weight, feed intake, or feed conversion were recorded on d 1, 15, or 18 of the experiment. However, the 18-d body weight gain was significantly lower in all chickens receiving salinomycin, although this difference was only 5% compared to the chickens receiving the recommended level and in contrast with the findings of Keshavarz and McDougald (1982), who reported no effects on performance at the recommended salinomycin level. In regard to the decrease in body weight and increase in feed conversion caused by 2 and 3 times the recommended salinomycin level, our results agree with those of Keshavarz and McDougald (1982) who reported decreased body weight in one-week-old chickens fed salinomycin levels 1.5, 2, 2.5, and 3 times the recommended level for 3 weeks; at 2.5 and 3 times the recommended levels the feed conversion was affected as well. Our results also agree with those of Migaki and Babcock (1979) who reported decreased growth with dietary salinomycin levels >80 ppm and partially agree with the results of Rizvi and Anjum (1999), who reported decreased body weight and increased feed conversion with 120 and 180 ppm salinomycin in the diet. In the present study, feed conversion was affected by 180 ppm but not by 120 ppm. Further, in our experiment chickens fed 180 ppm salinomycin had a feed conversion rate of 1.7 compared to 1.52 in the control group. This difference was much lower than the one reported by Rizvi and Anjum (1999) who observed feed conversion values of 2.21, 3.25, 3.85, and 4.16 with salinomycin levels of 0, 60, 120, and 180 ppm, respectively. These differences can be the result of the different exposure times (2.5 weeks in the present study vs. 8 weeks in the one from Rizvi and Anjum) and/or the difference in broiler performance between the current strains of chickens and those from 1999. In almost 3 decades, genetic selection have reduced significantly the time required to reach the target body weight with better feed conversion rates. The clinical enzymology results were contradictory. AST is a cytosolic enzyme present in many tissues and it is considered to be a non-specific but highly sensitive marker of muscle damage; even though CK is more specific for muscle damage, AST is often used to confirm changes in CK (Kramer and Hoffmann, 1997). In the present study, a significant increase in AST activity was seen after 7 d of exposure to 180 ppm salinomycin, confirming the sensitivity of this enzyme to muscular damage; however, no difference was observed after 14 d of exposure. A possible explanation for this finding could be the high prevalence of pulmonary hypertension syndrome (which affects soft tissues like the heart and the liver) that affected the birds growing faster (0 and 60 ppm salinomycin). CK increases are usually associated with damage to cardiac and/or skeletal muscle, the target tissues of ionophore antibiotics. In contrast to expectations, the group receiving the highest salinomycin level had the lowest CK serum levels after 14 d of exposure. This finding could also be related to the high incidence of right ventricular hypertrophy found in the birds receiving 0, 60, and 120 ppm salinomycin, all of which showed high mortality due to ascites syndrome. The CK kit used in the present study determines the isoenzyme CK-MB, which has traditionally been used as a marker protein for acute myocardial infarction (Adams et al., 1994); the damage to the myocardium from the pulmonary hypertension could have caused the increase in serum CK in the present study, not necessarily the exposure to salinomycin. The enzyme LDH has 2 main monomers found in heart (LDH-1) and liver and skeletal muscle (LDH-5); however, it is also found in kidneys and red blood cells (Kramer and Hoffmann, 1997). In broiler chickens, the ionophore monensin caused an increase in LDH activity, even at the prophylactic dose of 100 ppm (Dowling, 1992). In the present experiment, no difference in LDH activity among experimental groups was observed at any sampling time. It is possible that LDH is not a sensitive enough muscle damage marker or that the serum enzyme activity had also been influenced by the high morbidity of pulmonary hypertension and ascites as previously mentioned. Measurement of relative organ weights is common in toxico-pathological studies. In the present study a significant increase in the relative weights of proventriculus, gizzard, spleen, and pancreas were observed only in the birds fed 180 ppm salinomycin. Our results are in contrast with those of Rizvi and Anjum (1999), who found a significant decrease in the relative weight of the gizzard and kidneys and a significant increase in the relative heart weight in chickens that received 180 ppm salinomycin for 8 weeks. These differences could be due to different exposure times, since the effects of anticoccidials depend not only on the dose but also on the exposure time. No detectable levels of salinomycin were found in muscle or liver tissue from the birds fed 0, 60, or 120 ppm. Only the chickens receiving 3 times the prophylactic level showed residues in these tissues and in both cases they were above the MRL of 5 μg/kg recommended by the European Union (8.9 μg/kg in breast muscle and 13.3 μg/kg in liver). The results obtained confirm the current regulations regarding withdrawal times for salinomycin. In the United States there is no withdrawal time required, whereas the European Union requires withdrawal of salinomycin from the chicken diets at least 24 hours before slaughter. In the present study the feed was suspended 12 hours before the chickens were sacrificed and even at double the recommended prophylactic dose there were no detectable residues of salinomycin in muscle or liver. These results are in contrast with those from the serum samples taken 14 d after the beginning of the experiment, at which time the chickens were still being fed. All chickens receiving salinomycin had detectable levels in serum and the salinomycin concentration was linearly related to the dietary levels. This finding suggests that it is possible to measure salinomycin in serum as a marker of exposure and even estimate the concentration in feed by using the regression equation y = 0.584x – 10. No mortality associated with salinomycin was observed in any of the treatments. Mortality was found to be due only to ascitic syndrome. Interestingly, the growth decrease caused by 180 ppm salinomycin prevented the presentation of ascitic syndrome since no mortality was observed in this group. Our results agree with those of Rizvi and Anjum (1999), who reported no mortality in chickens fed 60, 120, or 180 ppm salinomycin for 8 weeks. In summary, the results of the present study showed that no mortality or clinical signs of toxicosis occur in broiler chickens fed 2 or 3 times the recommended prophylactic salinomycin dose. Even though chickens receiving 60 ppm salinomycin had a 5% decrease in 18-d body weight gain compared with controls, it is important to note that the birds were not exposed to coccidia as occurs under field conditions. Birds without a coccidiostat in their diet would experience severe adverse effects on their health and performance in field conditions. Clinical biochemistry showed AST as the most sensitive enzyme for muscle damage. It may be possible that the other enzymes measured (CK and LDH) might also be used as markers of muscular damage in situations where the confounding effect of ascites syndrome is not present (chickens are not raised at high altitude). The chemical analysis results confirm the fast elimination of salinomycin and the lack of the requirement for a withdrawal period. Determination of salinomycin in serum could be used as a marker of exposure and to predict the salinomycin level in the diet. FUNDING Partial funding was provided by Alimentos Balanceados Tequendama, Albateq, S.A. Bogotá, Colombia. DECLARATION OF CONFLICTING INTERESTS The authors declare no potential conflicts of interest with respect to the research, authorship, and/or publication of this article. REFERENCES Adams J. E. , Schechtman K. B. , Landt Y. , Ladenson J. H. , Jaffe A. S. . 1994 . Comparable detection of acute myocardial infarction by creatine kinase MB isoenzyme and cardiac troponin I . Clin. Chem. 40 : 1291 – 1295 . Google Scholar PubMed Blanchard J. 1981 . Evaluation of the relative efficacy of various techniques for deproteinizing plasma samples prior to high-performance liquid chromatographic analysis . J. of Chromatogr. B: Biomed. Sci. and Appl. 226 : 455 – 460 . Google Scholar CrossRef Search ADS Clarke L. , Fodey T. L. , Crooks S. R. H. , Moloney M. , O’Mahony J. , Delahaut P. , O’Kennedy R. , Danaher M. . 2014 . A review of coccidiostats and the analysis of their residues in meat and other food . Meat Sci. 97 : 358 – 374 . Google Scholar CrossRef Search ADS PubMed Commission Regulation (EC) No. 496/2007 of 4 May 2007 amending Regulation (EC) No. 600/2005 as regards the introduction of a maximum residue limit for the feed additive ‘Salinomax 120G’, belonging to the group of coccidiostats and other medicinal substances. Official Journal of the European Union L117/9, 5.5 . 2007 . Dowling L. 1992 . Ionophore toxicity in chickens: a review of pathology and diagnosis . Avian Pathol. 21 : 355 – 368 . Google Scholar CrossRef Search ADS PubMed Keshavarz K. , McDougald L. R. . 1982 . Anticoccidial Drugs: Growth and Performance Depressing Effects in Young Chickens . Poult. Sci. 61 : 699 – 705 . Google Scholar CrossRef Search ADS PubMed Kinashi H. , Otake N. , Yonehara H. , Sate S. , Saito Y. . 1973 . The structure of salinomycin, a new member of the polyether antibiotics . Tetrahedron Lett. 14 : 4955 – 4958 . Google Scholar CrossRef Search ADS Kramer J. W. , Hoffmann W. E. . 1997 . Clinical Enzymology . Pages 303 – 325 in Clinical Biochemistry of Domestic Animals , 5th Edn . Kaneko J. J. , Harvey J. W. , Bruss M. L. , eds. Academic Press , San Diego . Matabudul D. K. , Lumley I. D. , Points J. S. . 2002 . The determination of 5 anticoccidial drugs (nicarbazin, lasalocid, monensin, salinomycin and narasin) in animal livers and eggs by liquid chromatography linked with tandem mass spectrometry (LC-MS-MS) . Analyst . 127 : 760 – 768 . Google Scholar CrossRef Search ADS PubMed Migaki T. T. , Babcock W. E. . 1979 . Safety evaluation of salinomycin in broiler chickens reared in floor pens . Poult. Sci. 58 : 481 – 482 . Google Scholar CrossRef Search ADS PubMed Miyazaki Y. , Shibuya M. , Sugawara H. , Kawaguchi O. , Hirose C. , Nagatsu J. , Esumi S. . 1974 . Salinomycin, a new polyether antibiotic . J. Antibiot. 27 : 814 – 821 . Google Scholar CrossRef Search ADS PubMed Novilla M. 1992 . The veterinary importance of the toxic syndrome induced by ionophores . Vet. Hum. Toxicol. 34 : 66 – 70 . Google Scholar PubMed Rizvi F. , Anjum A. D. . 1999 . Effect of salinomycin on broiler health . Vet. Arh. 69 : 39 – 47 . Statistix 9 for Windows . 2008 . User's Manual. Tallahassee Analytical Software . © 2018 Poultry Science Association Inc. This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices)

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Poultry ScienceOxford University Press

Published: Mar 15, 2018

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