versión impresa ISSN 0375-1589
S. Afr. j. anim. sci. vol.40 no.3 Pretoria 2010
N.F. Namroud#; M. Shivazad; M. Zaghari; A. Zare Shahneh
Department of Animal Science, University of Tehran, Karaj, Tehran, Iran. Department of Animal Science, University of Tehran, University Street, Karaj, Iran
Consumption of low crude protein (CP) diets causes elevation in fat accumulation in chickens, and this effect is independent of dietary essential amino acid levels. Thyroid hormones, because of their metabolic regulatory characteristics, might be an effective factor in lipogenesis. Therefore, a study was conducted to investigate the influence of low CP diets on hormonal function of the thyroid. Two hundred and sixteen male broiler chicks, 10 - 25 days of age, were fed experimental diets that contained 230, 210, 190 and 170 g CP/kg with adequate amounts of total and digestible essential amino acids at or above NRC (1994) recommendations. Two other diets were the same as the 190 and 170 g CP/kg diets but contained additional glycine and glutamic acid. Reduction in CP below 190 g/kg led to a decline in body weight and feed intake and an increase in fat deposition in body, as would be expected. Plasma T4 concentration decreased significantly in the birds on the 170 g CP/kg diet and supplementation of glycine and glutamic acid had no effect on hormonal levels of the thyroid despite the reduction in whole-body and abdominal cavity fat deposition. Plasma T3 concentration was not affected. Therefore, it is concluded that a reduction in circulatory levels of T4 that occurs in broiler chicks fed diets containing below 190 g CP/kg though with adequate essential amino acids, may be an effective method of increasing fat deposition.
Keywords: Crude protein, nonessential amino acids, T3, T4, lipid deposition
Distinct effects of dietary protein on thyroid gland function especially circulating levels of triiodothyronine (T3) and thyroxine (T4) have become obvious in many investigations (Buyse et al., 1992; Carew & Alster, 1997). Protein restricted chickens are characterized by a reduced plasma T4 and increased plasma T3 level (Buyse et al., 2001). Carew et al. (1983; 1997) reported that dietary deficiencies of some individual essential amino acids may affect thyroid function in chicks. Carew et al. (1997) have categorized the mechanisms by which protein and essential amino acid deficiencies alter thyroid function in several animal species. The correlation between thyroid hormone levels and many biological factors has been noted. The concentrations of T3 and T4 in plasma have a direct correlation with growth of broiler cockerels (Chaisson et al., 1978). A low concentration of T4 induced by propylthiouracil reduced body weight, muscle weight and bone length (King & King, 1973). Thyroid hormones are involved in the regulation of growth, metabolism, heat production, gonadal development, moulting, migration and hatching in birds (McNabb, 2000). Hypothyroidism promotes fatness significantly (Decuypere et al., 1987). Direct actions of T3 have also been described on lipolysis (Harden & Oscar, 1993) and malic enzyme activity (Goodridge et al., 1989). Several studies assumed that the depressing influence of malnutrition on thyroid secretion is due to an alteration of the amount of thyroid stimulating hormone (TSH) secretion (Penn & Huston 1968; Abdullah & Falconer, 1977; Poczopko et al., 1977). The TSH is the primary regulating factor which controls production and secretion of T4. Triiodothyronine is believed to originate mainly from the peripheral (liver, kidneys, and intestines) deiodination of T4.
Although many studies have determined the minimal crude protein (CP) level of diets with adequate amounts of essential amino acids for maximum performance (Namroud et al., 2008), there is no acceptable explanation for the reason for increased lipid deposition in chicks fed diets with low CP, but adequate essential amino acid levels in comparison with diet containing higher levels of CP. The present experiment was carried out to investigate the effect of different dietary CP levels with almost equal amounts of essential digestible amino acid levels on growth, thyroid hormonal responses and fat deposition of growing broiler chicks. Furthermore, the effect of supplementing low CP diets with glycine and glutamic acid as sources of nonessential amino acids was examined. Nonessential amino acid supplementation was done on the assumption that in low CP diets, deficiency of nonessential amino acids may become a limiting factor in protein synthesis which could lead to fattening (Yamazaki et al., 1998). This study was conducted to investigate whether dietary CP and not essential amino acid levels could reduce the hormonal function of the thyroid and elevate fat accumulation in the body.
Materials and Methods
Three hundred and seventy two day-old male Ross 308 broiler chicks obtained from a local hatchery were housed in electrically heated battery cages (0.197 m2 per bird) and had free access to water and a commercial starter diet for 10 days. On the tenth day, 216 birds (215 ± 20 g) were selected and allotted to one of the six feeding treatments on the basis of body weight (BW). Each dietary treatment was applied randomly to six replicates of six chicks. The experimental birds were given ad libitum access to water and diet. The ambient temperature was gradually decreased from 34 ºC to 24 ºC over the period of 1 to 28 d of age. The birds were exposed to a 23 : 1, light : dark cycle. All procedures were approved by the Animal Care and Welfare Committee of Tehran University.
Maize, dehulled soyabean meal and maize gluten meal were sampled prior to diet formulation to determine their CP levels (Kjeldahl-N × 6.25), moisture and total amino acids content (Degussa AG, Germany.), after which the content of true digestible amino acids was calculated from standardized ileal digestibility coefficients listed by Lemme et al. (2004). The concentrations of calcium (method 968.08), phosphorus (method 965.17), potassium (method 966.03), sodium (method 966.03) and chloride (method 943.01) were analyzed in triplicate by the AOAC (1995) procedures for all feed ingredients. The dietary electrolyte balance was set at 280 mEq/kg. All diets were formulated to be isoenergetic (13.29 MJ MEn/kg). The amount of dietary calcium, available phosphorus and sodium were maintained constant in all treatments (Table 1). Four levels of CP were used in this study, viz. 230, 210, 190 and 170 g/kg with almost equal ileal digestible amounts of essential amino acids. Total essential amino acid levels in all treatments were maintained at or above NRC (1994) recommended levels (Table 2). Furthermore, one series of 190 and 170 g CP/kg diets were supplemented with Gly and Glu to reach the amounts in the high CP diets. Diets were supplemented with complete vitamin and trace mineral premixes. The L-Lys.HCl, DL-Met and L-Thr used in the diets were feed grade, whereas all other crystalline amino acids as well as K2CO3 were reagent grade (minimum 99.9% purify) and purchased from Degussa Iran AG.
At 25 days of age, three blood samples were taken per replicate from the brachial vein of the birds and placed into evacuated heparinized tubes. Samples were put on ice immediately and processed within 1.5 h of collection. Plasma was obtained by centrifuging the blood samples at 3000 × g for 15 min at 4 ºC and kept frozen at -20 ºC for later analysis. Plasma T3 and T4 concentrations were determined by radioimmunoassay. RIA was performed using the Coat-A-Count Free T4 and T3 RIA kit validated for use with chicken blood according to the manufacturer's protocol. Intra- and inter-assay coefficients of variation were 6.9 and 4.7% for T4 and 10.9 and 6.1 for T3, respectively. A different assay was used in an attempt to assess plasma levels of TSH, the human RIA immunoradiometric assay from Diagnostic Products Corporation (USA) but the TSH assay did not prove to be effective in measuring chicken TSH.
At the end of the experimental period (Day 28), two birds per replicate (with a BW close to the replicate mean), were slaughtered by cervical dislocation. The birds were killed 20 h after their last meal (with free access to water). One of the slaughtered birds was used to determine carcass characteristics including abdominal fat. The other euthanized chick per replicate was stored in airtight polyethylene bags at -22 ºC for later determination of whole-body composition. The whole body of chicks were thawed overnight at room temperature, homogenized with a Waring blender (Waring Products Division, New Hartford, CT.) for 2.5 min, and sampled according to procedures described by Barker & Sell (1994). Whole-body DM (Barker & Sell, 1994), nitrogen (N) content, and fat content (EE) were analyzed in triplicate subsequently. Whole-body CP was calculated as Kjeldahl N × 6.25.
Data were analyzed using the general linear models ANOVA with P <0.05 in a completely randomized design (SAS, 2004). Means were compared using Duncan's multiple range test.
The body weights of the birds on the 230, 210 and 190 g dietary protein/kg diets were not significantly different, but was significantly reduced with 170 g/kg (P <0.05). Supplementing low CP diets with Gly and Glu did not improve performance significantly (Table 3).
Abdominal fat and whole-body fat were affected by dietary CP (Table 4). High CP diets produced carcasses that contained lower levels of abdominal and total fat than the low CP diets. Fortifying low CP diets with Gly and Glu led to a reduction in whole-body and abdominal cavity fat content. No difference in percentage of whole-body CP was observed between different treatments.
Figure 1 shows that T4 concentration was affected (P <0.05) by CP level. Below the 19 g CP/kg level, plasma T4 concentration with and without nonessential amino acids supplementation was lower. Plasma concentration of T3 was not influenced significantly.
Although effects of individual amino acid deficiencies on hormonal function of thyroid gland were studied previously, the specific effects of CP and nonessential amino acid were not determined. This study has indicated that not only amino acid levels and pattern (Refetoff et al., 1970; Carew et al., 1997,) but also dietary CP by itself may reduce plasma thyroid hormone concentrations. In agreement with Namroud et al. (2008), in the present experiment a minimum CP level was recorded below which retardation in growth and appetite were experienced. Glycine and Glu supplementation to low CP diets did not improve performance except by reducing fat deposition in the abdominal cavity and whole-body. Namroud et al. (2008) reported that lower CP levels may increase fattening quantitatively. Triiodothyronine is the main metabolic stimulating hormone (McNabb & King, 1993; Gabarrou et al., 1997). Plasma T3 and T4 concentrations are associated strongly with metabolism regulation and they are important growth promoters in chickens (McNabb & King, 1993; Carew et al., 1998; Gonzales et al., 1999; Yahav, 2000). Sun et al. (2006) demonstrated that T4 is the most important hormone for predicting the percentage carcass fat in ad libitum-fed pullets. Furthermore, the low increase of circulating T4 does not appear to modify body composition, heart rate, metabolism rate or muscular function in the human (Dubois et al., 2008). The circulating concentration of T3 and probably T4 are positively correlated with feed intake (Yahav et al., 1996; 1998). Nevertheless, the low body weight of the birds on the 170 g CP/kg diet could not be the consequence of low hormonal thyroid actions because over a fairly wide range of thyroid activities, growth rate is independent of that, and affected mainly by feed intake and composition. However, just under severe conditions of hypo- or hyperthyroidism, growth retardation may occur. On the other hand, elevated fat deposition could not be the consequence of reduced dietary CP solely in view of the fact that all diets contained the same levels of digestible essential amino acids. Our conclusion is that reducing dietary CP increases fat accumulation partly by altering thyroid hormones metabolism. In the present study, although reducing dietary CP with a constant profile of essential amino acids could decrease circulating T4 concentration, even the fortifying of the low CP diet with nonessential amino acids could not compensate for this reduction. Despite the fact that nonessential amino acid (Gly and Glu) supplementation could reduce fat deposition, its effect on increasing the thyroid hormones (T3) concentration was not significant. Therefore, dietary Gly and Glu supplementation could not have an important effect on the hormone secretion of the thyroid. Unfortunately, we did not succeed in measuring plasma TSH concentration. Therefore it is not clear if a reduction in thyroid activity is solely the result of changes in T3 and T4 metabolism, or if thyrotrophs of the anterior pituitary interfered in that. Further research is needed to investigate the mechanism(s) of how dietary CP affects the thyroid.
The authors gratefully acknowledge the Degussa AG and its technical service manager in Iran, Ali Afsar, for conducting the amino acid analyses of our feedstuffs and providing crystalline amino acids.
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