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Effect of Early Castration on Body Weight, Muscle Growth, and Bone Characteristics of Male Nicholas Strain Turkeys1
PHYSIOLOGY AND REPRODUCTION Effect of Early Castration on Body Weight, Muscle Growth, and Bone Characteristics of Male Nicholas Strain Turkeys1 W. H. BURKE and H. M. EDWARDS, JR. Department of Poultry Science, The University of Georgia, Athens, Georgia 30602-2772
1994 Poultry Science 73:457-463
INTRODUCTION Marked sex differences in BW and muscle mass exist in many avian species. Male chickens and turkeys are heavier than females and have greater muscle mass, although females of many other avian species are larger than males (Dunning, 1993). The physiological bases for these differences are unknown but gonadal secretions may play a role. A number of workers have studied the effects of castration on growth of male chickens and turkeys. In many cases, sham-operated controls, the most appropriate control for these experiments, have not been used, and the castrations have not been done until the birds were weeks old, leaving open the possibility that testicular secretions early in life might affect later growth. It was the purpose of this study to
Received for publication September 7, 1993. Accepted for publication November 12, 1993. iSupported in part by state and Hatch funds allocated to the Georgia Agricultural Experiment Stations of the University of Georgia.
determine the effect of early castration on body growth, muscle mass, and bone characteristics of male turkeys. MATERIALS AND METHODS Day old Nicholas commercial cross males were obtained from a commercial hatchery. They were brooded and reared in a single 3.2 x 10.4 m pine shavings litter-floored pen in a force-ventilated light-tight house. A photoperiod of 24 h light:0 h dark was provided throughout the experiment with an average light intensity of 47 lx measured 30 cm above the floor. From 0 to 4 wk a ration with 30% CP and 2,828 kcal ME/kg was fed. The protein level was reduced to 26% and 22% and energy was increased to 2,878 and 2,983 kcal/kg at 4 and 8 wk, respectively. Water, provided by dome waterers, was continuously available. No medication or vaccinations were given. Heat was provided by gas-fired forced-air furnaces maintaining a room temperature of 31 to 32 C during the first 5 d and reducing it about 3 C/wk until a temperature of 20 to
457
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ABSTRACT Body weight of male Nicholas strain turkeys, castrated at 8 to 10 d of age, averaged .60,2.20,5.26,8.26, and 10.83 kg at 3,6,9,12, and 15 wk of age, respectively. These weights did not differ significantly from those of shamoperated control birds, but were significantly less than those of unoperated controls at 12 and 15 wk of age. Pectoralis major weights of 15-wk-old castrated turkeys were not significantly different than the sham-operated controls, but were less than those of intact controls. Pectoralis weights, expressed as a percentage of BW, did not differ among these groups. There were no differences in tarsometatarsal lengths, weight, or percentage ash among the groups. The incidence of tibial dyschondroplasia ranged from 66.7 to 80.0%, with average scores ranging from 1.00 to 1.42, with no differences among groups. Plasma testosterone concentrations of unoperated and sham-operated controls averaged .16 and .26 ng/mL at 15 wk, whereas concentrations in 7 of 12 castrates were undetectable and amounts in the other 5 castrates averaged .03 ng/mL. (Key words: turkey, castration, body weight, muscle growth, bone growth)
458
BURKE AND EDWARDS, JR.
23 C was reached and maintained for the duration of the experiment. Castration and Sham-Operation
Measurements Individual BW were recorded at 3, 6, 9, 12, and 15 wk of age. Blood was collected from the brachial vein of each bird at 15 wk of age using a syringe prerinsed in a .15 M NaCl solution containing 10 m g / m L of EDTA. It was then placed into a tube containing 10 iiL of a 100 m g / m L EDTA solution per milliliter of blood. The blood was kept on ice, centrifuged at 5 C, and the recovered plasma was stored at -20 C until assayed for testosterone. Testosterone was measured in duplicate on 50-/iL aliquots of plasma using the Coat-A-Count 2 testosterone radioimmunoassay following procedures and using the zero reference and
diagnostic Products Corp., Los Angeles, CA 90045.
The viscera were exposed by reflection of the sternum and laid aside to expose the region of normal testicular attachment. Testicular tissue, when present, was removed and weighed. After being stored frozen for approximately 3 mo, the tarsometatarsal-foot segments were thawed and the tarsometatarsus was dissected free from the foot. Soft tissue was removed from the tarsometatar-
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At 8, 9, or 10 d of age males were randomly chosen from the population, deprived of feed for 1 to 3 h, and castrated or subjected to a sham operation. The birds were anesthetized by intramuscular injection of a mixture consisting of equal volumes of xylazine (20 mg/mL) and ketamine HC1 (100 mg/mL). Down feathers were removed from the lateral region just anterior to the thigh. The region was swabbed with a dilute disinfectant and the skin was incised. An incision was then made between the last two ribs and widened by a small spreader. The testis was exposed by blunt dissection and removed by simultaneously teasing its connective tissue supports free and applying gentle suction. The incision was closed using surgical silk and the operation was then repeated on the opposite side. The birds were kept warm in an electrically heated battery brooder until they were standing, after which they were returned to the floor pen. Sham-operated poults were similarly treated to the point of gentle manipulation of the testis with forceps. Closing sutures were then applied and birds were allowed to recover as above.
standard hormone preparations provided by the manufacturer. Cross-reactivity of other steroid hormones, as stated by the supplier are: 5a-dihydrotestosterone, 3.2%; a n d r o s t a n e d i o n e , . 5 % ; 5/3-androstan3a,17/3-diol, .4%; 5a-androstan-3,17 dione, .2%. Other androgens, adrenal glucocorticoids, and endogenous estrogens crossreact to much lesser degrees. The testosterone concentration at the 50% bound point was 1.5 n g / m L with intrassay coefficients of variation averaging 4.1% over the whole range of the standard curve. The antiserum used in this assay was raised in rabbits that had been immunized against testosterone coupled to a carrier protein. At 15 wk of age all 17 of the surviving castrated turkeys, 10 of the sham-operated controls, and 10 unoperated controls were stunned by an electric shock and killed by exsanguination. The controls were randomly selected from their respective groups. They were scalded and plucked free of feathers. The left Pectoralis major muscle was carefully dissected free and weighed. The left tarsometatarsus with the foot attached was severed free from its articulation with the tibiotarsus and stored frozen for later analysis. The incidence and severity of tibial dyschondroplasia (TD) was determined as described by Sanders and Edwards (1991) using each bird's right tibiotarsus. Two cuts were made through the proximal end of the tibiotarsus. The first was made on the cranial surface of the bone, cutting through the tibial crest region. The second was made on the medial-caudal surface, cutting toward the proximal end of the bone and through the ridge that articulates with the medial condyle of the femur. All of the lesions that were given a score of 3 were characterized by a large opaque mass of cartilage that was exposed by the second cut.
CASTRATION AND GROWTH OF TURKEYS
sus after it had been placed in boiling water for 10 min. The ash weight of the cleaned, defatted bone was then determined (Association of Official Agricultural Chemists, 1955) and expressed as a percentage of the dry, defatted bone weight. Statistical analyses were performed using procedures of SAS® (SAS Institute, 1985). Body weight data were analyzed using a model for repeated measures that included the following sources of variability: treatment, birds (treatment), age, and age by treatment. A significant age by treatment interaction was observed and body weights were then analyzed within age using a one-way ANOVA. If P values < .05 were obtained, mean comparisons were made using Duncan's multiple range test at an a value of .05. In addition, relationships between BW and age were calculated within treatments by regression analysis. Treatment differences in slopes were tested using the standard error of the differences between slopes.
459
died at later ages, with 16 surviving for the entire experiment. One of 13 unoperated controls died during the experiment. At the time of necropsy, 5 of the 17 surviving castrated birds were found to have residual pieces of testicular tissue. For purposes of data analysis, these birds were placed into a group termed "partially castrated". Body Weight
TABLE 1. Body weight of 3- to 15-wk-old castrated and intact male Nicholas turkeys Treatment
Age
Intact control (n = 12 to 13)
(wk) 3 .65 6 2.32 9 5.80 12 9.51 15 12.63 Regression analysis Slope1 1,036.8 a b
Sham-operated control (n = 16 to 19)
Partially castrated (n = 5)
Castrated (n = 12)
P
Mrrt (''•bl
± ± ± ± ±
.03 .14 .16 .21* .30"
.64 2.33 5.43 8.66 11.57 937.0
± .02 ± .08 ± .14 ± .21b ± .34ab
.60 2.20 5.26 8.26 10.83 883.8
± ± ± ± ±
.02 .08 .16 .21b .29b
.57 ± 2.12 ± 5.10 ± 8.48 ± 11.71 ±
.03 .19 .35 .48b .58ab
.23 .61 .09 .004 .006
954.7
- Means within ages with no common superscript differ significantly (P = .05). linear regression coefficient between body weight and age within treatment. Slopes of all operated groups were significantly (P £ .05) different than the unoperated control but not different than each other.
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There were no differences in BW among the treatment groups at 3, 6, or 9 wk of age (Table 1). At 12 wk, the intact controls were significantly heavier than all three groups of birds that had undergone surgery. Average BW of the castrated birds was significantly less than that of the intact controls at 15 wk of age but not different than the sham-operated or partially castrated groups. Body weights of the sham-operated or partially castrated group did not differ from those of the unoperated group at this age. Mean BW of castrated birds with detectable testosterone levels (see below) were slightly lower, but not significantly RESULTS lower, than BW of birds with undetectable levels. Statistical analysis with the castrates Castrations subdivided into two groups did not alter Twenty-six poults were castrated, of the results described above. The growth which 6 died from excessive bleeding rate of intact controls, as revealed by during or shortly after the surgery, 2 died regression analysis (Table 1), was signifiwithin 48 h, 1 died after 5 d, and 17 survived cantly greater than that of all other groups, for the duration of the experiment. Two of but there were no significant differences in 21 sham-operated controls died at the time the regression coefficients of the other three of surgery or within the next 72 h, and 3 groups.
460 Tissue
BURKE AND EDWARDS, JR.
Measurements
Testes and
Testosterone
The residual testicular tissue in the partially castrated birds weighed between .12 and 3.13 g, averaging 1.03 + .56 g (Table 2). Combined testicular weights in the unoperated controls averaged 5.32 + .89 g and those in the sham-operated controls averaged 3.50 ± .68 g. The difference between the latter two groups was not significant, but both were significantly heavier than those of the partially castrated group. The lowest point on the testosterone standard curve was .2 n g / m L , and the least detectable level, being defined as the lowest concentration of testosterone at which the counts of tracer bound were two SD less than the counts found in the zero reference tubes that contained no unlabeled testosterone, was .004 n g / m L . Plasma testosterone concentrations were undetectable in 7 of 12 castrated individuals (Table 2) and averaged .03 n g / m L in the other 5. One of the birds designated as a castrate had a testosterone concentration of .11 ng/mL. Four of the five partially castrated birds had undetectable testosterone values and one had a concentration of .02 ng/mL. Testosterone was measurable in all of the sham-operated controls and in 10 out of 11 unoperated birds, with average concentrations of .26 and .16 ng/mL, respectively. The high incidence of undetectable values made statistical analysis of these data impossible.
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The Pectoralis major weight of castrated males was less than that of unoperated controls (Table 2) but not different from the sham-operated or partially castrated groups. The differences in Pectoralis major weight, expressed as a percentage of BW, approached, but did not reach, statistical significance. Tarsometatarsal length, weight, or percentage ash were unaffected by castration or by the sham castration (Table 2). Tibial dyschondroplasia was noted in 66.7 to 80.0% of birds in the various groups with TD scores averaging 1.00 to 1.42. One-third of the castrates had severity scores of 3, whereas 0 to 11% of birds in other groups exhibited lesions of this severity.
CASTRATION AND GROWTH OF TURKEYS
DISCUSSION
operated controls, Ross et al. (1984) stated, in an abstract, that castration at 3 d of age resulted in a significant reduction in BW through 4 wk of age, but by implication, not thereafter. No effect on "skeletal growth parameters" were noted at 12 or 24 wk of age. To the authors' knowledge these data have not been published in full. Castration of male poults at 3.5 wk of age (Pierson et al, 1981) had no effect on BW at 5, 10, 15, or 20 wk, nor did s u p p l e m e n t a t i o n of the feed w i t h 17-a-methyltestosterone affect growth of intact controls. Caponized toms and shamoperated controls had more leg abnormalities than unoperated controls or unoperated birds fed 17-a-methyltestosterone. These data indicate that leg abnormalities were the result of surgery per se and were unrelated to plasma testosterone, because testosterone concentrations did not differ between the sham-operated and unoperated controls, but leg abnormalities did. Castration of poults at 4 wk (Fennel and Scanes, 1992b) had no effect on BW at 6, 7, or 8 wk, whereas castrates were significantly heavier than controls at 9 wk. Although castration did not suppress BW or alter shank-toe length or breast muscle mass in this study, implantation of 19-nortestosterone into castrates resulted in a significant increase in BW and muscle mass. In summary then, these earlier studies indicate that physiological concentrations of testosterone play no role in growth, muscle mass development, or in the leg abnormalities that are commonly encountered in turkeys. The initial aim of the present study was to determine the influence of very early castration on growth, muscle mass, and bone characteristics. In preliminary efforts, castration at 3 d of age resulted in nearly 100% mortality but about 75% of birds castrated at 8 to 10 d survived. Although it would be desirable to study the effects of androgen deprivation from an even earlier age, the present study provides the first data on castration of turkeys at this age. In essence these data support the findings that have resulted from castrations of older turkeys: presence of testes and normal testicular secretions do not affect body growth, muscle growth, or bone growth of this species. By implica-
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There are few reports of the effects of castration in male turkeys and of these, fewer still in which appropriate shamoperated controls were used. The completeness of castration and, consequently, the plasma concentrations of testosterone are of crucial importance in the interpretation of experiments such as this. Five of the 12 castrates in this study had low but detectable concentrations of testosterone, using as a definition of the minimum detectable value the lowest concentration of cold hormone at which the counts of testosterone bound were two or more SD below that of the zero reference (B0) tubes. That amount was calculated to equal 97.6% of the B 0 tubes and represents a concentration of .004 n g / m L , which is 10-fold more sensitive than the value provided by the manufacturer. Reanalysis of all samples from castrates and sham-operated controls, using a pool of testosterone-free castrate male turkey plasma as the zero reference point and as a diluent for the standards, indicated that 3 of the 12 castrates had detectable testosterone. One of them, with a value of .11 ng/mL, probably had an undetected piece of residual testis in the body cavity. The Coat-A-Count testosterone assay has very low cross-reactivity with other physiologically important steroids so it is unlikely, but possible, that they might contribute to these low concentrations of "testosterone" being detected. Testosterone concentrations of castrated turkeys averaged 206 p g / m L in the study of Pierson et al. (1981), a value not significantly different than the 356 p g / m L value in sham-operated controls in that study. Post-mortem examination for residual testicular tissue was not done in that study. Testicular remnants were not observed and plasma testosterone concentrations were not presented by Fennel and Scanes (1992b). Testosterone measurements were similarly not reported by Marion et al. (1972), Smith and Smyth (1963), or Ross et al. (1984). Marion et al. (1972) and Smith and Smyth (1963) found that castrated male turkeys were lighter than unoperated controls at ages ranging from 20 to 30 wk. Comparing castrated poults with sham-
461
462
BURKE AND EDWARDS, JR.
quently cannot be used as a method to study androgenic influences on growth. Injection of flutamide, an antiandrogen, into chicken eggs on Day 11 of incubation, reduced the weight of 7-wk-old male broilers but had no effect on females (Burke, unpublished data). In ovo injection of estradiol-17/3 had no effect on posthatching growth of chickens or turkeys (Freeman and Burke, 1986) or Japanese quail (Burke, unpublished data). REFERENCES Association of Official Agricultural Chemists, 1955. Official Methods of Analysis, 8th ed. Association of Official Agricultural Chemists, Washington, DC. Burke, W. H., K. D. Arbtan, and N. Snapir, 1990a. The role of plasma thyroid hormones in the regulation of body weight of Single Comb White Leghorn and broiler embryos. Poultry Sci. 69:1388-1393. Burke, W. H., and P. J. Sharp, 1989. Sex differences in body weight of chicken embryos. Poultry Sci. 68:805-810. Burke, W. H., F. T. Shultz, and S. W. Bielfelt, 1990b. Plasma hormones and growth of turkeys. Poultry Sci. 69(Suppl. l):27.(Abstr.) Cason, J. A., D. L. Fletcher, and W. H. Burke, 1988. Research note: Effects of caponization on broiler growth. Poultry Sci. 67:979-981. Dunning, J. B., 1993. CRC Handbook of Avian Body Masses. CRC Press Inc., Boca Raton, FL. Fennell, M. J., and C. G. Scanes, 1992a. Inhibition of growth in chickens by testosterone, 5adihydrotestosterone, and 19-nortestosterone. Poultry Sci. 71:357-366. Fennell, M. J., and C. G. Scanes, 1992b. Effects of androgen (testosterone, 5a-dihydrotestosterone, 19-nortestosterone) administration on growth in turkeys. Poultry Sci. 71:539-547. Freeman, R. M., and W. H. Burke, 1986. Effect of in ovo administration of estradiol 17/3 on postembryonic sexual dimorphism in body weight of chickens and turkeys. Poultry Sci. 65(Suppl. l):44.(Abstr.) Marion, W. W., R. J. Paulson, and D. L. Hatcher, 1972. The effect of caponization on the chemical composition of the turkey. Poultry Sci. 51: 1831.(Abstr.) Mast, M. G., H. C. Jordan, and J. H. MacNeil, 1981. The effect of partial and complete caponization on growth rate, yield, and selected physical and sensory attributes of cockerels. Poultry Sci. 60: 1827-1833. Munro, S. S., and J. L. Kosin, 1940. The existence of a sex difference in the weight of day-old chicks, with further data on the egg weight-chick weight relationship. Sci. Agric. 20:586-591. Pierson, F. W., P. Y. Hester, and Ellen K. Wilson, 1981. The effect of caponization and dietary 17 a-methyltestosterone on the incidence of leg abnormalities in turkeys. Poultry Sci. 60: 2144-2149.
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tion, because marked sex differences in BW and muscle mass exist well before 15 wk of age in commercial turkeys, it appears that posthatching testicular secretions are not responsible for the sexual dimorphism in BW of this species. Similar findings have been reported in chickens when caponized birds were compared with sham-operated controls (Mast et al, 1981; Rahman et al, 1984; Cason et al, 1988; Fennel and Scanes, 1992a), indicating that testicular secretions in the posthatching period are not required for the development of sexual dimorphism in BW of Gallinaceous birds reared for meat production. The age of onset of posthatching sex differences in BW are controversial, but some workers have reported them to be significant at 1 d of age in chickens (Munro and Kosin, 1940; Zawalsky, 1962; Whiting and Pesti, 1983), and we (Burke et al, 1990b) have noted them at 1 d of age in one strain of turkeys but not another. The recent findings (Burke and Sharp, 1989; Burke et al, 1990a) that male chicken embryos are heavier than females by midincubation and that male turkey embryos are heavier than females by 12 d of incubation (unpublished data) suggest that the underlying cause for the development of posthatching sexual dimorphism may actually act well before hatching, be masked by egg size limitations around the time of hatching, and then be re-expressed after hatching. The role of early embryonic sex steroids in embryonic and posthatching growth has not been extensively investigated. Immunohistochemical studies have demonstrated the presence of testosterone (Woods and Podczaski, 1974) and estrogens (Woods and Erton, 1978) in the testes and both ovaries of chick embryos as early as 3.5 d of incubation. Testosterone was present in chick embryo blood by 5.5 d of incubation in both sexes, with concentrations in males exceeding those of females at 7.5 d of incubation and at all later embryonic ages (Woods et al, 1975). There is little information on the effects of embryonic sex steroid hormones on posthatching growth. In ovo testosterone administration causes bursal involution and immunoincompetence and conse-
CASTRATION AND GROWTH OF TURKEYS Rahman, M. A., S. D. Ross, R. C. Fanguy, and D. T. Hyatt, 1984. The influence of gonadal development on lipid accretion in commercial broilers. Poultry Sci. 63(Suppl. l):167.(Abstr.) Ross, S. D., R. C. Fanguy, M. A. Rahman, and D. T. Hyatt, 1984. The effects of unilateral and bilateral gonadectomy on performance of male and female turkeys. Poultry Sci. 63(Suppl. 1): 171.(Abstr.) Sanders, A. M., and H. M. Edwards, Jr., 1991. The effects of 1,25-dihydroxycholecalciferol on performance and bone development in the turkey poult. Poultry Sci. 70:853-866. SAS Institute, 1985. SAS® User's Guide: Statistics. SAS Institute Inc., Cary, NC. Smith, R. T., and J. R. Smyth, Jr., 1963. Effects of castration on growth and development of
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turkey males. Poultry Sci. 42:418-423. Whiting, T. S., and G. M. Pesti, 1983. Effects of the dwarfing gene (dw) on egg weight, chick weight and chick weight:egg weight ratio in a commercial broiler strain. Poultry Sci. 62:2297-2302. Woods, J. E., and L. H. Erton, 1978. The synthesis of estrogens in the gonads of the chick embryo. Gen. Comp. Endocrinol. 36:360-370. Woods, J. E., and E. S. Podczaski, 1974. Androgen synthesis in the gonads of the chick embryo. Gen. Comp. Endocrinol. 24:413-423. Woods, J. E., R. M. Simpson, and P. L. Moore, 1975. Plasma testosterone levels in the chick embryo. Gen. Comp. Endocrinol. 27:543-547. Zawalsky, M., 1962. The effect of sex, egg weight and preincubation storage on hatching time and chick weight. Poultry Sci. 41:1697.(Abstr.) Downloaded from http://ps.oxfordjournals.org/ at Carleton University on May 8, 2015
1994 Poultry Science 73:457-463
INTRODUCTION Marked sex differences in BW and muscle mass exist in many avian species. Male chickens and turkeys are heavier than females and have greater muscle mass, although females of many other avian species are larger than males (Dunning, 1993). The physiological bases for these differences are unknown but gonadal secretions may play a role. A number of workers have studied the effects of castration on growth of male chickens and turkeys. In many cases, sham-operated controls, the most appropriate control for these experiments, have not been used, and the castrations have not been done until the birds were weeks old, leaving open the possibility that testicular secretions early in life might affect later growth. It was the purpose of this study to
Received for publication September 7, 1993. Accepted for publication November 12, 1993. iSupported in part by state and Hatch funds allocated to the Georgia Agricultural Experiment Stations of the University of Georgia.
determine the effect of early castration on body growth, muscle mass, and bone characteristics of male turkeys. MATERIALS AND METHODS Day old Nicholas commercial cross males were obtained from a commercial hatchery. They were brooded and reared in a single 3.2 x 10.4 m pine shavings litter-floored pen in a force-ventilated light-tight house. A photoperiod of 24 h light:0 h dark was provided throughout the experiment with an average light intensity of 47 lx measured 30 cm above the floor. From 0 to 4 wk a ration with 30% CP and 2,828 kcal ME/kg was fed. The protein level was reduced to 26% and 22% and energy was increased to 2,878 and 2,983 kcal/kg at 4 and 8 wk, respectively. Water, provided by dome waterers, was continuously available. No medication or vaccinations were given. Heat was provided by gas-fired forced-air furnaces maintaining a room temperature of 31 to 32 C during the first 5 d and reducing it about 3 C/wk until a temperature of 20 to
457
Downloaded from http://ps.oxfordjournals.org/ at Carleton University on May 8, 2015
ABSTRACT Body weight of male Nicholas strain turkeys, castrated at 8 to 10 d of age, averaged .60,2.20,5.26,8.26, and 10.83 kg at 3,6,9,12, and 15 wk of age, respectively. These weights did not differ significantly from those of shamoperated control birds, but were significantly less than those of unoperated controls at 12 and 15 wk of age. Pectoralis major weights of 15-wk-old castrated turkeys were not significantly different than the sham-operated controls, but were less than those of intact controls. Pectoralis weights, expressed as a percentage of BW, did not differ among these groups. There were no differences in tarsometatarsal lengths, weight, or percentage ash among the groups. The incidence of tibial dyschondroplasia ranged from 66.7 to 80.0%, with average scores ranging from 1.00 to 1.42, with no differences among groups. Plasma testosterone concentrations of unoperated and sham-operated controls averaged .16 and .26 ng/mL at 15 wk, whereas concentrations in 7 of 12 castrates were undetectable and amounts in the other 5 castrates averaged .03 ng/mL. (Key words: turkey, castration, body weight, muscle growth, bone growth)
458
BURKE AND EDWARDS, JR.
23 C was reached and maintained for the duration of the experiment. Castration and Sham-Operation
Measurements Individual BW were recorded at 3, 6, 9, 12, and 15 wk of age. Blood was collected from the brachial vein of each bird at 15 wk of age using a syringe prerinsed in a .15 M NaCl solution containing 10 m g / m L of EDTA. It was then placed into a tube containing 10 iiL of a 100 m g / m L EDTA solution per milliliter of blood. The blood was kept on ice, centrifuged at 5 C, and the recovered plasma was stored at -20 C until assayed for testosterone. Testosterone was measured in duplicate on 50-/iL aliquots of plasma using the Coat-A-Count 2 testosterone radioimmunoassay following procedures and using the zero reference and
diagnostic Products Corp., Los Angeles, CA 90045.
The viscera were exposed by reflection of the sternum and laid aside to expose the region of normal testicular attachment. Testicular tissue, when present, was removed and weighed. After being stored frozen for approximately 3 mo, the tarsometatarsal-foot segments were thawed and the tarsometatarsus was dissected free from the foot. Soft tissue was removed from the tarsometatar-
Downloaded from http://ps.oxfordjournals.org/ at Carleton University on May 8, 2015
At 8, 9, or 10 d of age males were randomly chosen from the population, deprived of feed for 1 to 3 h, and castrated or subjected to a sham operation. The birds were anesthetized by intramuscular injection of a mixture consisting of equal volumes of xylazine (20 mg/mL) and ketamine HC1 (100 mg/mL). Down feathers were removed from the lateral region just anterior to the thigh. The region was swabbed with a dilute disinfectant and the skin was incised. An incision was then made between the last two ribs and widened by a small spreader. The testis was exposed by blunt dissection and removed by simultaneously teasing its connective tissue supports free and applying gentle suction. The incision was closed using surgical silk and the operation was then repeated on the opposite side. The birds were kept warm in an electrically heated battery brooder until they were standing, after which they were returned to the floor pen. Sham-operated poults were similarly treated to the point of gentle manipulation of the testis with forceps. Closing sutures were then applied and birds were allowed to recover as above.
standard hormone preparations provided by the manufacturer. Cross-reactivity of other steroid hormones, as stated by the supplier are: 5a-dihydrotestosterone, 3.2%; a n d r o s t a n e d i o n e , . 5 % ; 5/3-androstan3a,17/3-diol, .4%; 5a-androstan-3,17 dione, .2%. Other androgens, adrenal glucocorticoids, and endogenous estrogens crossreact to much lesser degrees. The testosterone concentration at the 50% bound point was 1.5 n g / m L with intrassay coefficients of variation averaging 4.1% over the whole range of the standard curve. The antiserum used in this assay was raised in rabbits that had been immunized against testosterone coupled to a carrier protein. At 15 wk of age all 17 of the surviving castrated turkeys, 10 of the sham-operated controls, and 10 unoperated controls were stunned by an electric shock and killed by exsanguination. The controls were randomly selected from their respective groups. They were scalded and plucked free of feathers. The left Pectoralis major muscle was carefully dissected free and weighed. The left tarsometatarsus with the foot attached was severed free from its articulation with the tibiotarsus and stored frozen for later analysis. The incidence and severity of tibial dyschondroplasia (TD) was determined as described by Sanders and Edwards (1991) using each bird's right tibiotarsus. Two cuts were made through the proximal end of the tibiotarsus. The first was made on the cranial surface of the bone, cutting through the tibial crest region. The second was made on the medial-caudal surface, cutting toward the proximal end of the bone and through the ridge that articulates with the medial condyle of the femur. All of the lesions that were given a score of 3 were characterized by a large opaque mass of cartilage that was exposed by the second cut.
CASTRATION AND GROWTH OF TURKEYS
sus after it had been placed in boiling water for 10 min. The ash weight of the cleaned, defatted bone was then determined (Association of Official Agricultural Chemists, 1955) and expressed as a percentage of the dry, defatted bone weight. Statistical analyses were performed using procedures of SAS® (SAS Institute, 1985). Body weight data were analyzed using a model for repeated measures that included the following sources of variability: treatment, birds (treatment), age, and age by treatment. A significant age by treatment interaction was observed and body weights were then analyzed within age using a one-way ANOVA. If P values < .05 were obtained, mean comparisons were made using Duncan's multiple range test at an a value of .05. In addition, relationships between BW and age were calculated within treatments by regression analysis. Treatment differences in slopes were tested using the standard error of the differences between slopes.
459
died at later ages, with 16 surviving for the entire experiment. One of 13 unoperated controls died during the experiment. At the time of necropsy, 5 of the 17 surviving castrated birds were found to have residual pieces of testicular tissue. For purposes of data analysis, these birds were placed into a group termed "partially castrated". Body Weight
TABLE 1. Body weight of 3- to 15-wk-old castrated and intact male Nicholas turkeys Treatment
Age
Intact control (n = 12 to 13)
(wk) 3 .65 6 2.32 9 5.80 12 9.51 15 12.63 Regression analysis Slope1 1,036.8 a b
Sham-operated control (n = 16 to 19)
Partially castrated (n = 5)
Castrated (n = 12)
P
Mrrt (''•bl
± ± ± ± ±
.03 .14 .16 .21* .30"
.64 2.33 5.43 8.66 11.57 937.0
± .02 ± .08 ± .14 ± .21b ± .34ab
.60 2.20 5.26 8.26 10.83 883.8
± ± ± ± ±
.02 .08 .16 .21b .29b
.57 ± 2.12 ± 5.10 ± 8.48 ± 11.71 ±
.03 .19 .35 .48b .58ab
.23 .61 .09 .004 .006
954.7
- Means within ages with no common superscript differ significantly (P = .05). linear regression coefficient between body weight and age within treatment. Slopes of all operated groups were significantly (P £ .05) different than the unoperated control but not different than each other.
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There were no differences in BW among the treatment groups at 3, 6, or 9 wk of age (Table 1). At 12 wk, the intact controls were significantly heavier than all three groups of birds that had undergone surgery. Average BW of the castrated birds was significantly less than that of the intact controls at 15 wk of age but not different than the sham-operated or partially castrated groups. Body weights of the sham-operated or partially castrated group did not differ from those of the unoperated group at this age. Mean BW of castrated birds with detectable testosterone levels (see below) were slightly lower, but not significantly RESULTS lower, than BW of birds with undetectable levels. Statistical analysis with the castrates Castrations subdivided into two groups did not alter Twenty-six poults were castrated, of the results described above. The growth which 6 died from excessive bleeding rate of intact controls, as revealed by during or shortly after the surgery, 2 died regression analysis (Table 1), was signifiwithin 48 h, 1 died after 5 d, and 17 survived cantly greater than that of all other groups, for the duration of the experiment. Two of but there were no significant differences in 21 sham-operated controls died at the time the regression coefficients of the other three of surgery or within the next 72 h, and 3 groups.
460 Tissue
BURKE AND EDWARDS, JR.
Measurements
Testes and
Testosterone
The residual testicular tissue in the partially castrated birds weighed between .12 and 3.13 g, averaging 1.03 + .56 g (Table 2). Combined testicular weights in the unoperated controls averaged 5.32 + .89 g and those in the sham-operated controls averaged 3.50 ± .68 g. The difference between the latter two groups was not significant, but both were significantly heavier than those of the partially castrated group. The lowest point on the testosterone standard curve was .2 n g / m L , and the least detectable level, being defined as the lowest concentration of testosterone at which the counts of tracer bound were two SD less than the counts found in the zero reference tubes that contained no unlabeled testosterone, was .004 n g / m L . Plasma testosterone concentrations were undetectable in 7 of 12 castrated individuals (Table 2) and averaged .03 n g / m L in the other 5. One of the birds designated as a castrate had a testosterone concentration of .11 ng/mL. Four of the five partially castrated birds had undetectable testosterone values and one had a concentration of .02 ng/mL. Testosterone was measurable in all of the sham-operated controls and in 10 out of 11 unoperated birds, with average concentrations of .26 and .16 ng/mL, respectively. The high incidence of undetectable values made statistical analysis of these data impossible.
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The Pectoralis major weight of castrated males was less than that of unoperated controls (Table 2) but not different from the sham-operated or partially castrated groups. The differences in Pectoralis major weight, expressed as a percentage of BW, approached, but did not reach, statistical significance. Tarsometatarsal length, weight, or percentage ash were unaffected by castration or by the sham castration (Table 2). Tibial dyschondroplasia was noted in 66.7 to 80.0% of birds in the various groups with TD scores averaging 1.00 to 1.42. One-third of the castrates had severity scores of 3, whereas 0 to 11% of birds in other groups exhibited lesions of this severity.
CASTRATION AND GROWTH OF TURKEYS
DISCUSSION
operated controls, Ross et al. (1984) stated, in an abstract, that castration at 3 d of age resulted in a significant reduction in BW through 4 wk of age, but by implication, not thereafter. No effect on "skeletal growth parameters" were noted at 12 or 24 wk of age. To the authors' knowledge these data have not been published in full. Castration of male poults at 3.5 wk of age (Pierson et al, 1981) had no effect on BW at 5, 10, 15, or 20 wk, nor did s u p p l e m e n t a t i o n of the feed w i t h 17-a-methyltestosterone affect growth of intact controls. Caponized toms and shamoperated controls had more leg abnormalities than unoperated controls or unoperated birds fed 17-a-methyltestosterone. These data indicate that leg abnormalities were the result of surgery per se and were unrelated to plasma testosterone, because testosterone concentrations did not differ between the sham-operated and unoperated controls, but leg abnormalities did. Castration of poults at 4 wk (Fennel and Scanes, 1992b) had no effect on BW at 6, 7, or 8 wk, whereas castrates were significantly heavier than controls at 9 wk. Although castration did not suppress BW or alter shank-toe length or breast muscle mass in this study, implantation of 19-nortestosterone into castrates resulted in a significant increase in BW and muscle mass. In summary then, these earlier studies indicate that physiological concentrations of testosterone play no role in growth, muscle mass development, or in the leg abnormalities that are commonly encountered in turkeys. The initial aim of the present study was to determine the influence of very early castration on growth, muscle mass, and bone characteristics. In preliminary efforts, castration at 3 d of age resulted in nearly 100% mortality but about 75% of birds castrated at 8 to 10 d survived. Although it would be desirable to study the effects of androgen deprivation from an even earlier age, the present study provides the first data on castration of turkeys at this age. In essence these data support the findings that have resulted from castrations of older turkeys: presence of testes and normal testicular secretions do not affect body growth, muscle growth, or bone growth of this species. By implica-
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There are few reports of the effects of castration in male turkeys and of these, fewer still in which appropriate shamoperated controls were used. The completeness of castration and, consequently, the plasma concentrations of testosterone are of crucial importance in the interpretation of experiments such as this. Five of the 12 castrates in this study had low but detectable concentrations of testosterone, using as a definition of the minimum detectable value the lowest concentration of cold hormone at which the counts of testosterone bound were two or more SD below that of the zero reference (B0) tubes. That amount was calculated to equal 97.6% of the B 0 tubes and represents a concentration of .004 n g / m L , which is 10-fold more sensitive than the value provided by the manufacturer. Reanalysis of all samples from castrates and sham-operated controls, using a pool of testosterone-free castrate male turkey plasma as the zero reference point and as a diluent for the standards, indicated that 3 of the 12 castrates had detectable testosterone. One of them, with a value of .11 ng/mL, probably had an undetected piece of residual testis in the body cavity. The Coat-A-Count testosterone assay has very low cross-reactivity with other physiologically important steroids so it is unlikely, but possible, that they might contribute to these low concentrations of "testosterone" being detected. Testosterone concentrations of castrated turkeys averaged 206 p g / m L in the study of Pierson et al. (1981), a value not significantly different than the 356 p g / m L value in sham-operated controls in that study. Post-mortem examination for residual testicular tissue was not done in that study. Testicular remnants were not observed and plasma testosterone concentrations were not presented by Fennel and Scanes (1992b). Testosterone measurements were similarly not reported by Marion et al. (1972), Smith and Smyth (1963), or Ross et al. (1984). Marion et al. (1972) and Smith and Smyth (1963) found that castrated male turkeys were lighter than unoperated controls at ages ranging from 20 to 30 wk. Comparing castrated poults with sham-
461
462
BURKE AND EDWARDS, JR.
quently cannot be used as a method to study androgenic influences on growth. Injection of flutamide, an antiandrogen, into chicken eggs on Day 11 of incubation, reduced the weight of 7-wk-old male broilers but had no effect on females (Burke, unpublished data). In ovo injection of estradiol-17/3 had no effect on posthatching growth of chickens or turkeys (Freeman and Burke, 1986) or Japanese quail (Burke, unpublished data). REFERENCES Association of Official Agricultural Chemists, 1955. Official Methods of Analysis, 8th ed. Association of Official Agricultural Chemists, Washington, DC. Burke, W. H., K. D. Arbtan, and N. Snapir, 1990a. The role of plasma thyroid hormones in the regulation of body weight of Single Comb White Leghorn and broiler embryos. Poultry Sci. 69:1388-1393. Burke, W. H., and P. J. Sharp, 1989. Sex differences in body weight of chicken embryos. Poultry Sci. 68:805-810. Burke, W. H., F. T. Shultz, and S. W. Bielfelt, 1990b. Plasma hormones and growth of turkeys. Poultry Sci. 69(Suppl. l):27.(Abstr.) Cason, J. A., D. L. Fletcher, and W. H. Burke, 1988. Research note: Effects of caponization on broiler growth. Poultry Sci. 67:979-981. Dunning, J. B., 1993. CRC Handbook of Avian Body Masses. CRC Press Inc., Boca Raton, FL. Fennell, M. J., and C. G. Scanes, 1992a. Inhibition of growth in chickens by testosterone, 5adihydrotestosterone, and 19-nortestosterone. Poultry Sci. 71:357-366. Fennell, M. J., and C. G. Scanes, 1992b. Effects of androgen (testosterone, 5a-dihydrotestosterone, 19-nortestosterone) administration on growth in turkeys. Poultry Sci. 71:539-547. Freeman, R. M., and W. H. Burke, 1986. Effect of in ovo administration of estradiol 17/3 on postembryonic sexual dimorphism in body weight of chickens and turkeys. Poultry Sci. 65(Suppl. l):44.(Abstr.) Marion, W. W., R. J. Paulson, and D. L. Hatcher, 1972. The effect of caponization on the chemical composition of the turkey. Poultry Sci. 51: 1831.(Abstr.) Mast, M. G., H. C. Jordan, and J. H. MacNeil, 1981. The effect of partial and complete caponization on growth rate, yield, and selected physical and sensory attributes of cockerels. Poultry Sci. 60: 1827-1833. Munro, S. S., and J. L. Kosin, 1940. The existence of a sex difference in the weight of day-old chicks, with further data on the egg weight-chick weight relationship. Sci. Agric. 20:586-591. Pierson, F. W., P. Y. Hester, and Ellen K. Wilson, 1981. The effect of caponization and dietary 17 a-methyltestosterone on the incidence of leg abnormalities in turkeys. Poultry Sci. 60: 2144-2149.
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tion, because marked sex differences in BW and muscle mass exist well before 15 wk of age in commercial turkeys, it appears that posthatching testicular secretions are not responsible for the sexual dimorphism in BW of this species. Similar findings have been reported in chickens when caponized birds were compared with sham-operated controls (Mast et al, 1981; Rahman et al, 1984; Cason et al, 1988; Fennel and Scanes, 1992a), indicating that testicular secretions in the posthatching period are not required for the development of sexual dimorphism in BW of Gallinaceous birds reared for meat production. The age of onset of posthatching sex differences in BW are controversial, but some workers have reported them to be significant at 1 d of age in chickens (Munro and Kosin, 1940; Zawalsky, 1962; Whiting and Pesti, 1983), and we (Burke et al, 1990b) have noted them at 1 d of age in one strain of turkeys but not another. The recent findings (Burke and Sharp, 1989; Burke et al, 1990a) that male chicken embryos are heavier than females by midincubation and that male turkey embryos are heavier than females by 12 d of incubation (unpublished data) suggest that the underlying cause for the development of posthatching sexual dimorphism may actually act well before hatching, be masked by egg size limitations around the time of hatching, and then be re-expressed after hatching. The role of early embryonic sex steroids in embryonic and posthatching growth has not been extensively investigated. Immunohistochemical studies have demonstrated the presence of testosterone (Woods and Podczaski, 1974) and estrogens (Woods and Erton, 1978) in the testes and both ovaries of chick embryos as early as 3.5 d of incubation. Testosterone was present in chick embryo blood by 5.5 d of incubation in both sexes, with concentrations in males exceeding those of females at 7.5 d of incubation and at all later embryonic ages (Woods et al, 1975). There is little information on the effects of embryonic sex steroid hormones on posthatching growth. In ovo testosterone administration causes bursal involution and immunoincompetence and conse-
CASTRATION AND GROWTH OF TURKEYS Rahman, M. A., S. D. Ross, R. C. Fanguy, and D. T. Hyatt, 1984. The influence of gonadal development on lipid accretion in commercial broilers. Poultry Sci. 63(Suppl. l):167.(Abstr.) Ross, S. D., R. C. Fanguy, M. A. Rahman, and D. T. Hyatt, 1984. The effects of unilateral and bilateral gonadectomy on performance of male and female turkeys. Poultry Sci. 63(Suppl. 1): 171.(Abstr.) Sanders, A. M., and H. M. Edwards, Jr., 1991. The effects of 1,25-dihydroxycholecalciferol on performance and bone development in the turkey poult. Poultry Sci. 70:853-866. SAS Institute, 1985. SAS® User's Guide: Statistics. SAS Institute Inc., Cary, NC. Smith, R. T., and J. R. Smyth, Jr., 1963. Effects of castration on growth and development of
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turkey males. Poultry Sci. 42:418-423. Whiting, T. S., and G. M. Pesti, 1983. Effects of the dwarfing gene (dw) on egg weight, chick weight and chick weight:egg weight ratio in a commercial broiler strain. Poultry Sci. 62:2297-2302. Woods, J. E., and L. H. Erton, 1978. The synthesis of estrogens in the gonads of the chick embryo. Gen. Comp. Endocrinol. 36:360-370. Woods, J. E., and E. S. Podczaski, 1974. Androgen synthesis in the gonads of the chick embryo. Gen. Comp. Endocrinol. 24:413-423. Woods, J. E., R. M. Simpson, and P. L. Moore, 1975. Plasma testosterone levels in the chick embryo. Gen. Comp. Endocrinol. 27:543-547. Zawalsky, M., 1962. The effect of sex, egg weight and preincubation storage on hatching time and chick weight. Poultry Sci. 41:1697.(Abstr.) Downloaded from http://ps.oxfordjournals.org/ at Carleton University on May 8, 2015