Plasma Insulin and Glucagon Levels and Leg Measurements of Lame Turkeys 1

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1 Plasma Insulin and Glucagon Levels and Leg Measurements of Lame Turkeys 1 PATRICIA Y. HESTER and HENRY KOHL Department of Animal Sciences, Purdue University, West Lafayette, Indiana JOHN P. McMURTRY U.S. Department of Agriculture, Agricultural Research Service, Beltsville Agricultural Research Center, Livestock and Poultry Institute, Nonruminant Nutrition Laboratory, Beltsville, Maryland (Received for publication August 30, 1988) ABSTRACT As insulin is a regulator of cartilage and bone growth, plasma levels of pancreatic hormones were determined in 41 lame male turkeys with long bone distortion and 42 control toms without leg deformities. Blood samples were collected 3.5 h following the removal of feed and then 1 h after the return of feed. The day following blood collection, toms, 119 days of age, were slaughtered to obtain leg measurements. Body weight, width, and length of the tarsometatarsus, presence of tibial dyschondroplasia (TD) lesions, pancreas weight, and plasma glucose did not differ between lame and control turkeys. A significantly greater proportion of growth plates of the tarsometatarsus was closed in the control as compared to the lame turkeys. Pancreas weight relative to BW was significantly greater for lame as compared to control turkeys. Plasma insulin was undetectable in both fasted lame and control turkeys. When feed was returned to the turkeys, plasma insulin concentrations were significantly lower in lame compared to control turkeys. The return of feed had no effect on plasma glucagon levels in controls, but concentrations were decreased significantly in lame birds. These data suggest that the regulation of plasma insulin and glucagon may be altered in lame turkeys perhaps contributing to the cause of long bone distortion. (Key words: insulin, glucagon, glucose, lame, turkeys) INTRODUCTION Insulin is one of the more important hormones affecting bone formation (Canalis, 1983). Through its effects on chondrocytes and osteoblasts (Raisz and Kream, 1981), insulin stimulates the synthesis of collagen and to a lesser extent, noncollagen protein (Canalis, 1983). Although many of insulin's regulatory functions in bone formation may be mediated through somatomedins (Canalis, 1983), insulin receptors separate from those for somatomedins have been isolated in chick cartilage (Stuart et al., 1979). Insulin indirectly enhances bone mineralization by increasing the activity of renal 1 a-hydroxylase, thus increasing circulating levels of 1,25 dihydroxycholecalciferol (Nyomba et al., Journal Paper Number 11,495 of the Purdue University Agricultural Experiment Station Poultry Science 68: ). In contrast to insulin, glucagon's effect on bone is not as clearly defined. Some evidence indicates that glucagon is not physiologically important as a regulator of bone protein synthesis (Canalis et al., 1977), but it may affect bone resorption (Bell, 1970; Stern and Bell, 1970). The objective of the present study was to determine plasma levels of insulin and glucagon in lame vs. control turkeys during both fasted and fed conditions. After blood sampling, turkeys were slaughtered so that pancreas weights and various leg measurements could be made. MATERIALS AND METHODS Nicholas male turkeys used in the present study were from an experiment dealing with light schedules and time of hatch (Hester and Kohl, 1989). Only turkeys of control treat- 1294

2 PLASMA INSULIN AND GLUCAGON LEVELS OF LAME TURKEYS 1295 ments (i.e., step-down lighting and normal hatching time) were included in this study. At hatching, male poults were housed in two light-tight rooms each individually heated, ventilated, and lighted. A low intensity stepdown lighting program was used in these two rooms. A continuous photoperiod of 10 lx intensity was used the first 3 days of age. Beginning on the 4th day, light intensity was reduced to 2.5 lx, and light hours were decreased by 1 h each day until there were 15 h of light (at 12 days of age); this schedule was maintained until time of marketing. A light meter (Kalgo Electric Co., Inc., Bethlehem, PA) was used to monitor light intensity. Of the 12 floor pens in each room, three were assigned to each of the four hatching treatments. Only pens for normal hatching time were used in the present study. Each floor pen received 26 poults, was bedded with pine shavings, and was equipped with one flat tray for feed, one hanging bell-shaped drinker, two hanging feeders, and one fount type waterer. Feed trays and fount type waterers were removed after the 1st wk. Cardboard brooder guards were used the first 2 wk to keep poults close to the source of feed and water. Ambient temperatures were maintained at approximately 31 to 32 C from day of housing to Day 8, 29 C on Days 9 to 14, 27 C on Days 15 to 24, 24 C on Days 25 to 31, and 21 C on Day 32. After Day 32, temperatures fluctuated between 17 and 29 C depending on outside environmental conditions. All toms were fed prestarter, starter, grower, developer, and nonmedicated finisher diets (Hester et al., 1987) from 1 to 4, 5 to 8, 9 to 12, 13 to 15.4 and 15.5 to 17 wk of age, respectively. At the time of the experiment, each torn had.37 m 2 of floor space and 10.6 cm of feeder space. At 118 days of age (16.9 wk), 41 lame toms with long bone distortion and 42 control toms without leg deformities were leg banded and weighed. Long bone distortion was characterized by varus (bowed legs) and valgus (knock kneed) deformities and twisted leg (Riddell, 1983). No emaciated birds were used. Feed was removed at 0800 h. Heparinized blood samples were collected from the brachial vein at 3.5 h following removal of feed. Feed was returned at 1200 h, and the same birds were bled again 1 h later. Five-milliliter blood samples were collected on ice and centrifuged at 300 x g for 15 min at 4 C. Each plasma sample obtained was divided into two aliquots and frozen at -20 C. One aliquot was used for glucose determination by the glucose oxidase method (Hill and Kessler, 1961) and for insulin analysis by an homologous radioimmunoassay for chicken insulin (McMurtry et al., 1983). In McMurtry et al. (1983), the insulin assay was validated for turkey plasma. The other aliquot of plasma was stored in the presence of the protease inhibitor, aprotinin, and was used for glucagon determinations via radioimmunoassay (Allen and McMurtry, 1984). The glucagon radioimmunoassay for chicken plasma has been validated by Allen and McMurtry (1984). The glucagon radioimmunoassay for turkey plasma has also been validated by McMurtry (unpublished data); the structures of glucagon in chickens and turkeys are identical. The day following blood collection, all toms were processed. The following variables were measured: absolute and relative pancreas and testes weights, absolute and relative tarsometatarsal lengths and widths, and the presence or absence of the growth plate and tibial dyschondroplasia (TD) lesions of the proximal end of the right tarsometatarsal bone. All data were subjected to an analysis of variance (Anderson and McLean, 1974). Glucagon and glucose were analyzed as a two-way analysis of variance with leg status and time of bleeding (fasted and refed) as fixed effects and birds within leg status and time of bleeding as a random effect. As insulin levels were not detected following feed removal, concentrations were analyzed as a one-way analysis of variance. All other variables were statistically evaluated through a one-way analysis of variance. Log transformations of all data did not alter statistical interpretation; therefore, untransformed data will be presented in the tables. Newman-Keuls' sequential range test was used to partition differences among means (Steel and Torrie, 1960). RESULTS AND DISCUSSION Pancreas weights and plasma levels of insulin, glucagon, and glucose of lame and control turkeys are shown in Table 1. Absolute pancreas weights of lame and control turkeys were not significantly different. However, when expressed relative to BW, pancreas weights were significantly greater for lame turkeys (P<.03).

3 1296 HESTER ET AL. TABLE 1. Pancreas weights and plasma levels of insulin, glucagon, and glucose of lame versus control turkeys Dependent variable Lame 1 Control SEM Pancreas weight, g Relative pancreas weight, g/100 kg BW Plasma insulin-fasted, pg/ml Plasma insulin-refed, pg/ml Plasma glucagon-fasted, pg/ml Plasma glucagon-refed, pg/ml Plasma glucose-fasted, mg/dl Plasma glucose-refed, mg/dl * ND * a b ND ab 361.l ab a ' b Denotes a leg status x time of bleeding interaction for glucagon (P<.07). 'Numbers of observations equal 41 and 42 for lame and control turkeys, respectively. ND = Not detected; assay sensitivity = 30 pg per tube or 60 pg/ml. *Lame birds are significantly different from controls (P<.03) Male turkeys deprived of feed for 3.5 h had undetectable levels of plasma insulin. Such low levels were not anticipated, as turkey laying hens showed plasma insulin values of 190 pg/ml after 48 h without feed using the same chicken radioimmunoassay (McMurtry et al., 1983). McMurtry et al. (1983) reported the sensitivity of the radioimmunoassay to be 30 pg/tube at 95% binding (60 pg/ml). As expected, the return of feed caused plasma insulin levels to increase in male turkeys (Table 1). A similar increase in plasma insulin occurred with the return of feed to turkey laying hens, but the increase was greater in hens (540 pg/ml, McMurtry et al., 1983). Hazelwood (1984) reported that insulin values double or triple within minutes of a blood glucose increase in the chicken. Plasma insulin values were significantly lower for lame turkeys as compared to controls after 1 h of feeding (P<.03). A leg status x bleeding time interaction occurred with plasma glucagon (P<.07) (Table 1). The return of feed had no effect on plasma glucagon levels in controls, but concentrations decreased (P<.07) in lame birds. The main effects of leg status and bleeding time were nonsignificant for glucagon. Plasma glucose levels increased significantly after feeding for 1 h (P<.0001), but concentrations in birds with long bone distortion and controls were not significantly different. Seven week-old broilers with long bone distortion, specifically valgus-varus bone deformity, showed histomorphometric characteristics of reduced bone formation and bone resorption. Likewise, plasma levels of 1,25 dihydroxycholecalciferol, but not 25 hydroxycholecalciferol, were reduced in these lame broilers (Newbrey et al., 1988). Newbrey et al. (1988) hypothesized that the activity of renal 1 a-hydroxylase may have been reduced, perhaps due to a deficiency of parathyroid hormone or prostaglandin E2 (Trechsel et al., 1980; Wark et al, 1981). The possibility also exists that reduced levels of insulin as observed in the lame turkeys of the present study could affect the activity of renal 1 ochydroxylase (Nyomba et al., 1987), thus indirectly affecting bone mineralization. Although the organic matrix of bones of lame birds has not yet been studied, the possibility exists that collagen and noncollagen protein synthesis is adversely affected in birds with long bone distortion, perhaps as a result of altered hormonal patterns. The glucagon profile of lame birds was also different from that of controls, with a reduction in plasma glucagon occurring with the return of feed in turkeys with long bone distortion but not in controls (Table 1). In light of studies conducted on the chicken, one could expect blood glucagon levels to decrease in response to increasing glucose and insulin (Hazelwood, 1984). However, pancreatic responses to known secretagogues may differ in the chicken and turkey. In addition, data in the present experiment suggest that pancreatic function may be altered in the lame turkeys as evidenced by the smaller insulin response to refeeding (increased glucose) and increased pancreatic weights relative to BW. Although glucagon does not appear to affect collagen synthesis in bone (Canalis et al., 1977), in vitro studies have shown that glucagon inhibits bone resorption directly (Stern and Bell, 1970)

4 PLASMA INSULIN AND GLUCAGON LEVELS OF LAME TURKEYS 1297 TABLE 2. Body and testes weight and leg measurements of lame versus control turkeys Dependent variable Lame 1 Control SEM BW, kg Total testes weight, g Relative testes weight, g/100 kg BW Length of left tarsometatarsus, cm Relative length of left tarsometatarsus, cm/kg BW Width of left tarsometatarsus, cm Relative width of left tarsometatarsus, cm/kg BW Presence of tibial dyschondroplasia lesion 2 Presence of growth plate *.93.63* 'Numbers of observations equal 41 and 42 for lame and control turkeys, respectively. Score of 1 = presence and score of 0 = absence of lesion or growth plate. *Lame different from control (P<.02). and indirectly through enhanced secretion of calcitonin (Bell, 1970). Larger fluctuations in plasma glucagon of lame turkeys, as compared with those in controls, could result in less finetuned control of bone modeling and mineralization. Body weight, testes weight, and leg measurements of lame vs. control turkeys are shown in Table 2. As emaciated lame birds were avoided in the present study, it was not unexpected that BW did not differ with respect to leg status. Testes weights and lengths and widths of the left tarsometatarsus, with the exception of relative widths, did not differ in lame and control turkeys. For some unknown reason, the relative width of the left tarsometatarsus was significantly greater for lame than control turkeys (P<.02). This pattern is especially perplexing, as Nestor et al. (1985) have reported that the walking ability of male turkeys can be improved by selecting turkeys with wider shanks. The presence of TD lesions was unaffected by leg status. The lack of correlation between TD lesions and long bone distortion is not unexpected as many birds with such lesions are not lame (Riddell, 1975; 1976). More unclosed growth plates were present in the lame birds at 119 days of age than in controls (P<.02) (Table 2). Evidence from a high intensity step-up lighting program indicates that earlier closure of the growth plate through earlier sexual maturity may be beneficial in reducing leg abnormalities among turkey toms (Hester et al., 1983; 1986; Klingensmith et al, 1986), but the results have not been consistent with other lighting regimens (Hester et al., 1987; Hester and Kohl, ). Delayed closure of the growth plate in lame turkeys of the present study did not result in longer shank lengths. Lame turkeys may have been less aggressive and may not have consumed feed as soon as control birds during the hour of refeeding. A delay in feed intake could have caused the observed pancreatic hormonal responses of lame turkeys, but this does not explain the larger relative pancreas weights. As feeder space was 2.8 times the minimum recommended by the Guide for the Care of Agricultural Animals (1988), each turkey should have had ample opportunity for feeding within a very short period of time. Similar plasma glucose levels of lame and control turkeys suggest comparable feeding behavior. Therefore, it is concluded that long bone distortion in male turkeys may be influenced by altered levels of plasma insulin and glucagon. Pancreatic hormones could affect both the organic and inorganic matrix of bone either directly or indirectly via other hormones such as somatomedins, parathyroid hormone, calcitonin, 1,25-dihydroxycholecalciferol, and prostaglandins. ACKNOWLEDGMENTS Appreciation is extended to Donna Brocht and Natalie F. Newlon for excellent technical assistance. REFERENCES Allen, P. C, and J. P. McMurtry, Changes in pancreatic hormones associated with coccidiosis. Poultry Sci. 63:

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