Effects of nicarbazin on the blood glucose and liver glycogen statuses of male broilers 1,2

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1 Effects of nicarbazin on the blood glucose and liver glycogen statuses of male broilers 1,2 E. D. Peebles,* 3 K. W. Bafundo, S. K. Womack,* W. Zhai,* R. Pulikanti,* and L. W. Bennett * Department of Poultry Science, Mississippi State University, Mississippi State 39762; Phibro Animal Health Corporation, Ridgefield Park, NJ 07660; and College of Veterinary Medicine, Mississippi State University, Mississippi State ABSTRACT Nicarbazin (NCZ), an effective anticoccidial widely used by the global broiler industry, is known to produce some side effects in broilers. Recent field observations have suggested that NCZ could be associated with spiking mortality syndrome, a disease of uncertain etiology that is most commonly characterized by hypoglycemia. In turn, 2 trials were conducted to investigate the effects of NCZ on the blood glucose levels and liver glycogen content of Ross Ross 708 male broilers. In 6 of 12 pens beginning at 1 d of age, NCZ was added to basal broiler diets at a rate of 125 mg/kg, and the other 6 pens were control pens in which birds received salinomycin instead of NCZ, at a rate of 66 mg/kg. Feed and water were provided ad libitum, feed was closely monitored in all pens to avoid shortages, no coccidial challenge was imposed, and room temperature never exceeded 29.4 C. At the end of the second trial, a photoperiod increase from 8L:16D to 24L:0D was imposed in an effort to induce stress due to feed engorgement. In response to NCZ in trial 1, 0 to 28 d feed conversion and relative liver weights on d 21 and 28 were higher, whereas 0 to 28 d cumulative BW gain was lower. In response to NCZ in trial 2, 0 to 13 d feed conversion was higher, whereas 0 to 13 d cumulative BW gain was lower. The added NCZ increased blood glucose on d 13 in trial 2, but did not affect at any time the liver constituents analyzed in both trials. In conclusion, the uninterrupted feeding of NCZ at 125 mg/kg in the starter and grower diets of male Ross Ross 708 broilers increased feed conversion and reduced BW gain, but despite an increase in relative liver weight on d 21 and 28 posthatch, liver glucose and glycogen concentrations were not affected. Key words: blood, broiler, glycogen, liver, nicarbazin 2012 Poultry Science 91 : INTRODUCTION 2012 Poultry Science Association Inc. Received March 26, Accepted May 29, Approved for publication as Journal Article No. J of the Mississippi Agricultural and Forestry Experiment Station, Mississippi State University. 2 Use of trade names in this publication does not imply endorsement by Mississippi Agricultural and Forestry Experiment Station of these products, nor similar ones not mentioned. 3 Corresponding author: dpeebles@poultry.msstate.edu Spiking mortality syndrome is a disease of uncertain etiology characterized by mortality (>0.5%) for approximately 3 consecutive days during the first 3 wk of life. Affected young broilers may experience tremors, blindness, ataxia, and coma; most are hypoglycemic (Davis et al., 1995). Because none of these clinical signs can be considered a specific identifying characteristic of the disease, spiking mortality is often confused with other conditions occurring in young broilers (Davis, 2003). For more than 50 yr, nicarbazin (NCZ) has been used as an effective anticoccidial agent. It is considered among the most effective and reliable anticoccidial products available (Chapman, 1994). Despite this reputation, NCZ is known to produce heat-induced side effects in broilers that increase mortality, often during the first 3 wk of life (McDougald and McQuiston, 1980; Beers et al., 1989). In recent years it has become clear that many broiler production personnel confuse the signs of NCZ exposure with those of spiking mortality. Glycogenolysis in the liver is used in preference to that in muscle for the release of glucose units into the circulation and subsequent transfer to surrounding tissues (Klasing, 1998; De Oliveira et al., 2008). Ruff (1982) examined the blood glucose and liver glycogen levels in broilers exhibiting high mortality, stunting, and leg weakness, which constitute a condition that has been referred to as infectious stunting or pale bird syndrome. In that report, it was noted that although the broilers did not exhibit decreased plasma glucose levels when compared with normal broilers, their liver glycogen levels were sometimes observed to be markedly elevated. In addition, although the glycogen content of the liver can be depleted within a few hours 2183

2 2184 Peebles et al. of fasting (Hazelwood and Lorenz, 1959), and blood glucose levels can decline in broilers experiencing food deprivation (van der Wal et al., 1999), Renner and Elcombe (1967) showed that while chicks fed carbohydrate-free diets were unable to maintain normal blood glucose levels, they showed a marked depression in liver glycogen concentrations. These previous experimental results indicate that liver glycogen and blood glucose levels may vary independently in response to diet and disease state, and may likewise potentially respond differently when subjected to dietary NCZ. In an effort to gain a better understanding of the glycogen and glucose statuses of NCZ-fed broilers, 2 trials were conducted in the current study to examine the effects of NCZ on the blood concentrations of glucose and of the liver concentrations of glycogen and glucose in male commercial broilers. Rapid increases in feed and NCZ intake have also been observed when lighting programs for young broilers are adjusted from extended dark to light periods. As a result, we hypothesized that these changes in feed and NCZ intake could have influenced the health and glucose status of broilers. Therefore, at the end of the second trial, a photoperiod increase to 24 h of light was used for the purpose of inducing stress in the birds due to the engorgement of feed and to determine the possible associated influences of NCZ on the energy status of the birds. In fulfillment of the overall objective of this study, results from these 2 trials should provide useful information as to the true association of dietary NCZ with the energy statuses of broilers. MATERIALS AND METHODS Bird Management General. The experimental protocols for trials 1 and 2 were approved by the Institutional Animal Care and Use Committee of Mississippi State University. In each trial, 1-d-old Ross Ross 708 male feather-sexed broilers from an older (peak) breeder flock were tagged and randomly allocated to 12 experimental pens in a lightand temperature-controlled facility that never exceeded 29.4 C. Within each trial, photoperiod changes were made at the same time on each of the days designated. In each trial, 6 replicate pens were assigned to each of 2 treatment groups. The NCZ was added to Mississippi State University basal diets at a rate of 125 mg/kg for 6 of the 12 pens, and salinomycin (control anticoccidial; SAL) was added at a rate of 66 mg/kg for the other 6 pens. The SAL treatment was used as a control because SAL is recognized as both the global and US standard for anticoccidial drugs (Chapman, 2001; AgriStats, 2011), is recognized globally as providing excellent performance responses (Lohner and Wilson, 1985), and has never been associated with adverse effects on carbohydrate metabolism (Austic and Smith, 1980). Birds remained on their experimental diets throughout each trial, and no coccidial challenge was imposed during the trials. Feed and water were provided for ad libitum consumption, and quantities of feed were monitored closely in all pens to avoid feed shortages. Trial 1. A total of 192 birds were placed in floor minipens (0.91 m 1.22 m) containing fresh pine shavings in a tunnel-ventilated broiler house. Lighting was set at a 24L:0D photoperiod from d 0 to 5 and at a 20L:4D photoperiod from d 6 to 28. Broiler starter feed containing either additive was provided from d 0 to 14, and broiler grower feed containing either additive was provided from d 15 to 28. Each pen contained 16 birds, and pens were randomly assigned to a treatment throughout the broiler house, with equal treatment representation on each of its north and south sides extending along its length from east to west. Trial 2. A total of 180 birds were placed in pens of a single Petersime brooder battery (2 pens per battery level) in an isolation room set at a 24L:0D photoperiod. At that time, broiler starter feed containing either additive was provided and was continued throughout the trial (d 0 to 14). Each pen contained 15 birds, and pens were randomly assigned to a treatment throughout the battery. The room photoperiod was decreased to 8L:16D on d 4 and was subsequently increased back to 24L:0D on d 12 of the trial. The 24L:0D photoperiod was continued through the end of the trial on d 13. Data Collection General. Within each trial, data collection was performed at the same time of day on the various days of age designated. Bird and feed weights were also recorded on a replicate pen basis for the calculation of BW gain, feed consumption, and feed conversion in each trial. Any feed added before the day designated for the weighing of feed was weighed and recorded. Only male birds were used in both trials for all data collection and analyses (BW, mortality, feed consumption, feed conversion, blood glucose, liver weight, and liver moisture, glucose, and glycogen concentration). Mortality was recorded on a daily basis, and dead birds were weighed and removed from their pens. All birds used for liver extraction were killed by cervical dislocation, and the sex of necropsied birds was confirmed by gonadal examination. Trial 1. On d 0, 7, 14, 21, and 28, bird body and feed weights on a pen basis were recorded. Cumulative BW gain, feed intake, and feed conversion ratios were calculated for the 0 to 28 d period, and cumulative percentage mortality was calculated for the 0 to 28 d period. At 7, 14, 21, and 28 d of age, 2 birds per pen were randomly selected, weighed, and necropsied for liver extraction for subsequent weight, moisture, and glycogen analysis. Trial 2. On d 0, 4, 12, and 13, bird and feed weights on a pen basis were recorded. Cumulative BW gain, feed intake, and feed conversion ratios were calculated for the 0 to 13 d period, and cumulative percentage mortality was calculated for the 0 to 13 d period. On d 12, 6 birds from each pen were randomly selected for

3 weighing and for blood and liver sample collections. Over a 12-h period on d 13, after the birds had been on 24L:0D, they were continuously monitored for observable symptoms including weakness, fatigue, and death. During that time, at least 6 birds, particularly those exhibiting the aforementioned symptoms, were weighed and sampled from each pen for the collection of blood and liver samples. On d 12 and 13, blood samples were used for serum glucose analysis, and liver weights were recorded and liver samples taken for glucose and glycogen analysis. Analysis of Blood Glucose and Liver Glycogen, Glucose, and Moisture Concentrations In trial 2, blood samples of approximately 100 µl were collected in nonheparinized tubes, and serum glucose was determined using an Ektachem DT-60 analyzer (Eastman Kodak Co., Rochester, NY) according to the procedures described by Latour et al. (1996). Serum glucose concentrations were expressed as milligrams per deciliter. Whole liver weights were recorded in both trials, and liver weight was expressed as an absolute weight and as a percentage of total bird BW (absolute liver weight normalized for BW; relative liver weight). Liver samples (0.25 g) were then taken from the same lobe for glycogen concentration analysis in trial 1, and for glucose and glycogen concentration analyses in trial 2. For the determination of liver moisture concentration in trial 1, liver samples were dried at 85 C for 96 h, or until moisture loss ceased. The dry liver samples were then allowed to reach room temperature for 2 h before a dry weight was obtained (Peebles et al., 1998). Liver moisture concentration was calculated as the difference between the liver s fresh and dry weights and was expressed as a percentage of its fresh weight (Peebles et al., 1999). For determination of liver glucose concentration in trial 2 and liver glycogen concentrations in trials 1 and 2, liver samples were preserved in 10% perchloric acid immediately after collection (Bennett et al., 2007). Analyses of liver glucose and glycogen concentrations were performed using the phenol-sulfuric acid method according to the procedures of Bennett et al. (2007). Liver glucose (trial 2) and glycogen (trials 1 and 2) concentrations were expressed as percentages of fresh sample weight (Pulikanti et al., 2010), and glycogen concentration was also expressed as a percentage of dry sample weight in trial 1. Statistical Analysis EFFECTS OF NICARBAZIN ON BROILER ENERGY STATUS In both trials, a completely randomized arrangement of treatment replicates was employed. A split-plot statistical design was used, with treatment as the main plot that was split on time (bird age), to evaluate the effects of bird age, treatment, and their interaction on those parameters evaluated at specific time periods (BW, liver weight, and glucose and glycogen concentrations), whereas a 1-way ANOVA was used to test for the effects of treatment on time interval data (mortality, BW gain, feed intake, and feed consumption). Angular transformation (arc sine of the square root of the proportion affected) of percentage mortality data was performed before the statistical analysis (Steel and Torrie, 1980). Least squares means were compared in the event of significant global effects (Steel and Torrie, 1980). Global effects and differences among least squares means were considered significant at P All data were analyzed using the MIXED procedure of SAS software (SAS Institute, 2003). Trial 1 RESULTS 2185 Bird mortality was low in each treatment group, and 0 to 28 d percentage cumulative mortality was not significantly affected by treatment. Cumulative percentage mortality in the control (SAL) and NCZ treatment groups was 3.03 and 3.79%, respectively (pooled SEM = 1.457%). There was a significant (P ) bird age treatment interaction for mean bird BW when determined on a replicate pen basis (Table 1). On d 21 and 28, the BW of birds provided dietary NCZ was significantly lower than that of those provided control diets containing SAL. had no significant effect on BW on d 0, 7, or 14. Furthermore, there was a significant treatment main effect for 0 to 28 d (P 0.001) cumulative BW gain. In response to the addition of dietary NCZ, 0 to 28 d cumulative BW gain was decreased. Means for 0 to 28 d cumulative BW gain in the control (SAL) and NCZ treatment groups were 1.33 and 1.16 kg, respectively (pooled SEM = kg). There was a significant treatment main effect for 0 to 28 d (P 0.005) feed conversion. In the 0 to 28 d period, feed conversion was significantly increased in the NCZ treatment group. Mean feed conversion for the 0 to 28 d period in the control (SAL) and NCZ treatment groups was 1.42 and 1.63 kg of feed intake per kg of BW gain, respectively (pooled SEM = kg of feed intake per kg of BW gain). However, compared with the control treatment, the feeding of NCZ had no significant effect on 0 to 28 d feed intake. There were significant bird age treatment interactions for the BW (P ) and relative liver weights (P 0.008) of the birds that were randomly selected for sampling (Table 2). In comparison with the control (SAL) diet, NCZ in the feed reduced BW but increased relative liver weight on d 21 and 28. had no significant effect on BW or relative liver weight on d 7 and 14. Absolute liver weight was not affected by treatment. However, there was a significant (P ) bird age main effect on absolute liver weight. Absolute liver weight increased significantly between each age period and was 4.92, 12.2, 22.7, and 35.7 g (pooled SEM = g) on d 7, 14, 21, and 28, respectively. There were significant bird age main effects for liver

4 2186 Peebles et al. Table 1. Mean BW of male broilers (kg), on a replicate pen basis, at 0, 7, 14, 21, and 28 d of age when provided added dietary salinomycin (SAL) or nicarbazin (NCZ) in trial 1 1,2 Day of age SAL a 1.58 a NCZ b 1.40 b a,b Means among treatments within a column with no common superscript differ significantly (P 0.05). 1 Pooled SEM = Six replicate units (pens), each containing approximately 16 birds, were used for the calculation of each mean. moisture concentration (P ), and fresh (P 0.003) and dry (P 0.003) liver glycogen concentrations (data not shown). Liver moisture concentration was higher on d 7 in comparison with that on d 14, 21, and 28, and was higher on d 14 in comparison with that on d 21 and 28. Conversely, fresh and dry liver glycogen concentrations were higher on d 21 in comparison with those on d 7 and 28, and were higher on d 14 in comparison with those on d 7. However, compared with the control treatment, the feeding of NCZ had no significant effects on liver moisture concentration or on fresh and dry liver glycogen concentrations. Trial 2 Similar to that in trial 1, percentage cumulative mortality was not significantly affected by treatment. Cumulative percentage mortality through d 13 in the control (SAL) and NCZ treatment groups was and 0.188%, respectively (pooled SEM = %). Furthermore, the birds in both treatment groups appeared normal, and none of the outward symptoms indicative of stress, including weakness and fatigue, were exhibited in the birds during the 12-h period after they had been subjected to a 16-h photoperiod increase beginning on d 12. There was a significant (P 0.003) bird age treatment interaction for mean bird BW when determined on a replicate pen basis (Table 3). On d 12 and 13, the BW of birds provided dietary NCZ was significantly lower than that of those provided control diets containing SAL. had no significant effect on BW on d 0 and 4. There was also a significant treatment main effect for 0 to 13 d (P 0.02) cumulative BW gain. In response to the addition of dietary NCZ, 0 to 13 d cumulative BW gain was decreased. Mean BW gain for the 0 to 13 d period in the control (SAL) and NCZ treatment groups was and kg, respectively (pooled SEM = kg). In addition, there was a significant treatment main effect for 0 to 13 d (P ) feed conversion. In the 0 to 13 d period, feed conversion was significantly increased in the NCZ treatment group. Mean feed conversion for the 0 to 13 d period in the control (SAL) and NCZ treatment groups was 1.22 and 1.33 kg of feed intake per kg of BW gain, respectively (pooled SEM = kg of feed intake per kg of BW gain). However, compared with the control treatment, the feeding of NCZ had no significant effect on 0 to 13 d feed intake. There were significant main effects due to bird age (P ) and treatment (P 0.05) for the BW of those birds that were randomly selected for sampling. Body weight was lower on d 12 than on d 13, and was also lower in birds provided dietary NCZ in comparison with those that received control diets containing SAL. Mean ± SEM BW was ± kg and ± kg on d 12 and 13, respectively, and was ± kg and ± kg in the SAL and NCZ treatment groups, respectively. There were no bird age or treatment effects on absolute liver weight. However, there was a significant (P ) main effect due to bird age on relative liver weight. Relative liver weight was higher on d 12 than on d 13. Mean relative liver weight was 3.86 and 3.36% (pooled SEM = 0.052%) on d 12 and 13, respectively. Relative liver weight was not significantly affected by treatment. There was a significant (P 0.003) bird age treatment interaction for serum glucose concentration (Table 4). On d 13, serum glucose concentrations were higher in the NCZ treatment group in comparison with the control (SAL) group. did not Table 2. Mean BW 1 and absolute liver weight normalized for BW (percentage liver weight; PLW) 2 of male broilers at 7, 14, 21, and 28 d of age when provided either added dietary salinomycin (SAL) or nicarbazin (NCZ) in trial d of age 14 d of age 21 d of age 28 d of age BW (kg) PLW (%) BW (kg) PLW (%) BW (kg) PLW (%) BW (kg) PLW (%) SAL a 2.63 b a 2.13 b NCZ b 2.92 a b 2.50 a a,b Means among treatments within a column with no common superscript differ significantly (P 0.05). 1 Pooled SEM = Pooled SEM = Two birds from each of 6 replicate units (pens) were sampled for the calculation of each mean.

5 Table 3. Mean BW of male broilers (kg), on a replicate pen basis, at 0, 4, 12, and 13 d of age when provided either added dietary salinomycin (SAL) or nicarbazin (NCZ) in trial 2 1,2 Day of age SAL a a NCZ b b a,b Means among treatments within a column with no common superscript differ significantly (P 0.05). 1 Pooled SEM = Six replicate units (pens), each containing approximately 15 birds, were used for the calculation of each mean. significantly affect serum glucose concentration on d 12. However, as in trial 1, the feeding of NCZ had no significant effect on fresh liver glycogen concentration. Furthermore, as in trial 1, there was a significant (P ) bird age main effect for fresh liver glycogen concentration. Fresh liver glycogen concentration was higher on d 12 in comparison with that on d 13. Mean fresh liver glycogen concentrations were 3.02 and 1.48% (pooled SEM = 0.277%) on d 12 and 13, respectively. Lastly, there were no significant effects due to bird age or treatment on fresh liver glucose concentration. DISCUSSION Davis (2008) has indicated that a high spike in mortality at 7 to 14 d of age is suggestive of but not diagnostic of hypoglycemia-spiking mortality syndrome (HSMS). Despite this report, the current results indicate that when provided at a rate of 125 mg/kg, NCZ did not increase the mortality of Ross Ross 708 male broilers that were housed at temperatures below 29.4 C and that did not receive a coccidial challenge through 28 d posthatch. However, the addition of NCZ to the diet caused a decrease in bird BW toward the end of each trial in association with a decrease in cumulative BW gain and an increase in feed conversion over the complete period of each trial. Conversely, previous research has indicated that when used at normal levels (125 mg/kg) in diets, NCZ may allow for normal growth and feed conversion (Ott et al., 1956; Newberne and Buck, 1957), and Penz et al. (1999) showed that commercial use of dietary NCZ (100 or 125 mg/kg) Table 4. Mean ± SEM serum glucose concentrations (mg/dl) of male broilers at 12 and 13 d of age when provided either added dietary salinomycin (SAL) or nicarbazin (NCZ) in trial 2 1 EFFECTS OF NICARBAZIN ON BROILER ENERGY STATUS Day of age SAL 356 ± 10.3 a 256 ± 9.60 c NCZ 352 ± 10.8 a 302 ± 9.52 b a c Means among treatments with no common superscript differ significantly (P 0.05). 1 Six birds in each of 6 replicate units were used for the calculation of each mean leads to tissue residues (liver and muscle) of NCZ that are much lower than the limits that are established to be safe for human consumption. Decreased BW gain (Newberne and Buck, 1957) and increased feed conversion (Keshavarz and McDougald, 1982) may occur in response to higher dietary NCZ concentrations. Nevertheless, because NCZ is widely recognized for its broadspectrum anticoccidial activity in commercial environments where broilers are continuously subjected to field infections, these negative responses are not usually observed (Chapman, 1992, 1994). Upon consideration of the results of these previous studies, it is therefore suggested that the negative performance responses in this study are due in large part to the fact that no coccidiosis challenge was imposed. Bafundo et al. (2008) have shown that under challenge conditions, NCZ potentiates broiler performance. Results from that work showed that 125 mg/kg of NCZ in diets significantly improved BW gain and feed conversion in infected birds. Watkins and Bafundo (1993) also showed that even during a mild coccidial challenge, NCZ provided a similar positive effect during a complete broiler growout period. Furthermore, Bafundo (1989) reported that although NCZ, up to 160 mg/kg in the diet, improved pigmentation in coccidia-free and coccidia-infected broilers, the amount of improvement was observed to be greater in those that were infected. Although the feeding of NCZ at 125 mg/kg increased relative liver weight on d 21 and 28 in trial 1, it had no effect on liver moisture concentration at any time in either trial. Also, despite noted age-related changes in liver glycogen concentration, liver glycogen concentrations were not found in either trial of this study to be influenced by NCZ. Furthermore, liver glucose concentrations of the birds in trial 2 were not affected by the addition of 125 mg/kg of NCZ to the diet. These results suggest that NCZ does not alter the energy status of Ross Ross 708 broilers, in terms of their liver glycogen or glucose concentrations, when they are not subjected to heat stress or a coccidial challenge. On average, serum glucose concentration was lower on d 13 than on d 12. Similarly, Peebles et al. (1997) also observed fluctuating serum glucose levels of broilers between d 14 and 42 of age, with significant decreases occurring between d 14 and 21 and between d 28 and 35. The reason for these age-related changes is uncertain, although Peebles et al. (1997) suggested that the observed changes in serum glucose concentration were not related to relative pancreas weight. It is of further interest to note that not only was serum glucose not decreased due to NCZ treatment in trial 2, but that it was actually observed to be higher on d 13 in the NCZtreated birds compared with controls. Davis (2008) has discussed that a diagnosis of HSMS is based on clinical findings including huddling, trembling, blindness, loud chirping, litter eating, ataxia, prostration with outstretched legs, and coma, and the demonstration of hypoglycemia (blood glucose concentration <150 mg/ dl). However, the birds belonging to the NCZ treat-

6 2188 Peebles et al. ment group did not display any of the clinical signs noted above in either trial, even in trial 2, in which birds were subjected to a photoperiod increase. Furthermore, the lowest mean blood glucose levels observed were 256 mg/dl, and were found in the SAL control birds on d 13. Blood glucose levels in birds on d 12 belonging to the NCZ treatment were as high as 356 mg/dl, which is more than twice the upper concentration in birds diagnosed as hypoglycemic. These data would certainly indicate that the birds provided 125 mg/kg of dietary NCZ did not become hypoglycemic. Therefore, it is important to be careful not to confuse any possible physiological effects that NCZ may have in broilers during the first 3 wk of their life with those that are associated with HSMS. REFERENCES AgriStats AgriStats Monthly Live Production. November, AgriStats Inc., Fort Wayne, IN. Austic, D. E., and J. B. Smith Interaction of ionophores with nutrients. Proc. GA. Nutr. Conf.:2 10. 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