DOI: 10.1111/jpn.12113
ORIGINAL ARTICLE
Activity of glutamate dehydrogenase and protein content in the breast of broilers fed diets containing different sources and levels of glycerine V. M. P. Bernardino1, P. B. Rodrigues1, L. de Paula Naves1, M. G. Zangeronimo2, R. R. Alvarenga1, P. V. Rosa1, L. M. Santos1 and L. V. Teixeira1 1 Department of Animal Science, Federal University of Lavras, Lavras, MG, Brazil, and 2 Department of Veterinary Science, Federal University of Lavras, Lavras, MG, Brazil
Summary According to scientific literature, glycerol in the diet can spare glucogenic amino acids by inhibiting the activity of enzymes, such as glutamate dehydrogenase, thereby promoting protein deposition in muscle tissues. Therefore, the objective of this study was to evaluate the effect of three sources of glycerine (crude glycerine from soybean oil – CGSO, mixed crude glycerine from frying oil and lard – MCG and a semipurified glycerine from soybean oil – SPGSO) in four concentrations in the diet (17.5, 35.0, 52.5 and 70.0 g of each type of glycerine/kg of feed) on the activity of hepatic glutamate dehydrogenase, performance and protein content in the breast of broilers, during 22–35 days of age (experiment I) and 33–43 days of age (experiment II). In both experiments, an increase in MCG induced a linear decline in glutamate dehydrogenase activity (p < 0.05). In contrast, increasing the concentration of SPGSO in the diet caused a linear increase in enzyme activity (p < 0.05). There was no (p > 0.05) isolated effect of glycerine on the enzyme activity in either evaluated phase; however, during 33–42 days of age, MCG inhibited (p < 0.05) the glutamate dehydrogenase activity by up to 34.43%. During 22–35 days of age, the diet containing SPGSO induced a higher protein content (p < 0.05) in the breast, and regardless of the source utilized, the maximum protein deposition was estimated (p < 0.05) when broilers were fed with 55.08 g glycerine/kg of diet. There was no (p > 0.05) interaction or isolated effects of the sources and levels of glycerine on the protein content in the breast of broilers at 33–42 days of age, and moreover, all diets containing glycerine promoted a similar protein deposition in the breast compared with birds that received the diet without glycerine. The bird age also showed to influence the feed intake and weight gain of broilers fed diet containing glycerine. It is concluded that for both rearing phases, an increase in glycerine in the diet did not necessarily reduce the glutamate dehydrogenase activity, and the protein deposition in the breast of broilers may not be strictly correlated with the activity of this enzyme. Keywords biodiesel, enzyme, glycerol, poultry Correspondence V. M. P. Bernardino, Department of Animal Science, Federal University of Lavras, Lavras, MG 37200000, Brazil. Tel: +55 35 3829 1691; Fax: +55 35 3829 1231; E-mail: [email protected] Received: 31 July 2012; accepted: 11 July 2013
Introduction Biodiesel production is on the increase, and consequently, the production of glycerine is also increased because it is a by-product of this important biofuel (Ma and Hanna, 1999; Van Gerpen, 2005). If the glycerine is refined, it can be used by the chemical, pharmaceutical and food industries to produce high-value coproducts. However, this purification process is expensive, and these industries do not require all amount of crude glycerine that has been produced (Thompson and He, 2006). Thus, it is necessary to
identify alternative markets to utilize the glycerine in order to add value to this coproduct and also to prevent its reverting to an environmentally detrimental waste product. Due to its high energetic value, the use of glycerine in the feeding of broilers has been considered and evaluated (Dozier et al., 2008; Lammers et al., 2008; Gianfelici et al., 2011; Guerra et al., 2011). The glycerol originating from the glycerine utilized in these diets could be used metabolically in different metabolic routes; for example, it can be converted enzymatically into glucose via gluconeo-
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genesis and afterwards can provide energy for cellular metabolism, or the glycerol can be directly oxidized for the production of energy via glycolysis and the Krebs cycle. Biodiesel can be produced from different sources including vegetable oils (cottonseed, peanut, babassu, canola, palm kernel, sunflower, castor oil plant and soy, among others), animal fats (bovine tallow, fish oil and lard) or oils or residual fats (from domestic, commercial or industrial processes) (Garcia and Tookuni, 2006; ANP, 2012), and the source affects the chemical composition of the glycerine present. Furthermore, the process of biofuel production itself can also alter the type of glycerine produced, and the use of different types of glycerine in poultry feed can induce different responses from the bird. Moreover, the levels of glycerine in the diet of broilers during different rearing phases should be evaluated with an aim towards establishing the content that produces optimal results. Glutamate dehydrogenase (EC 1.4.1.2) is an enzyme that catalyses the reversible deamination of the glutamate into a-ketoglutarate and requires NAD+ (or NADP+) or NADH (or NADPH) as cofactor, depending on the direction of the reaction. Therefore, it represents an important link between the metabolism of carbon and of nitrogen. According to Steele et al. (1971), glycerol in the diet can spare glucogenic amino acids by inhibiting the activity of enzymes, such as glutamate dehydrogenase, thereby promoting protein deposition in muscle tissues. In the case of protein deposition in broilers, it is important to consider that the meat from the breast represents approximately 30% of the total meat in the carcass of this bird, and it also accounts for approximately 50% of the total protein in the edible carcass (Summers et al., 1988). Thus, two experiments were conducted to evaluate the effects of different sources and concentrations of glycerine in the diet on the activity of hepatic glutamate dehydrogenase, performance and the protein content in the breast of broilers. One experiment was conducted when birds were 22–35 days of age and the other during the period from 33 to 42 days of age.
V. M. P. Bernardino et al.
other. Broilers were evaluated in the period from 22 to 35 days of age (experiment I) or 33 to 42 days of age (experiment II). All procedures were approved by the Committee of Bioethics of the Federal University of Lavras. Experimental procedures, birds, diets and performance measurements
Two experiments were conducted in the aviculture sector of the Department of Animal Science of the Federal University of Lavras to evaluate the effects of different sources and levels of glycerine in the diet on the activity of hepatic glutamate dehydrogenase, performance and the protein content in the breast of broilers. The experiments were conducted separately to avoid residual effects of one experiment on the
For experiment I, a total of 1144 male broiler chickens of the Cobb-500â strain were acquired at 1 day of age and reared in a conventional shed for broilers, where they received a basal diet until 21 days of age that was formulated to meet the requirements of the age, sex and strain of birds (Rostagno et al., 2005). For experiment II, 1300 male chickens of the same strain were reared from the 1st to the 32nd day of age and received a basal diet to meet their nutritional requirements (Rostagno et al., 2005). At the beginning of each experimental phase (22nd or 33rd day of age), the birds were weighed individually, separated by weight ranges and housed in masonry sheds such that each experimental unit (3-m2 pens that were covered with wood shavings) showed an initial similar mean weight. The number of birds per pen in the experiments I and II was 22 and 25 broilers respectively. Birds had free access to feed and water; each pen had a tubular feeder and a pendular drinking system. The experimental design of both experiments was completely randomized and used a 3 9 4 + 1 factorial arrangement; three types of glycerine were supplemented in the diet at four concentrations, which, together with a control diet without glycerine, totalled 13 treatments. These treatments were administered to four replicates of 22 birds (experiment I) or 25 birds (experiment II) per experimental unit. Three sources of glycerine were evaluated: crude from soybean oil (CGSO), mixed crude glycerine from frying oil and lard (MCG) and a semipurified glycerine from soybean oil (SPGSO). The chemical compositions of the types of glycerine are displayed in Table 1. In each experiment, concentrations of 17.5, 35.0, 52.5 and 70.0 g of each type of glycerine/kg of diet were used. The composition of the experimental diets utilized in experiments I and II is presented in Tables 2 and 3 respectively. Diets contained the same amount of nutrients and were formulated to meet the nutritional requirements of birds during each phase, based on the recommendations of Rostagno et al. (2005). Because of the elevated concentration of sodium in the types of glycerine utilized, the inclusion of salt in the diet was adjusted to maintain sodium and chloride levels at the nutritionally recommended
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Materials and methods
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Table 1 Chemical composition of the different glycerine types*
Parameter
Crude glycerine from soybean oil
Mixed glycerine†
Semipurified glycerine from soybean oil‡
Moisture and volatiles (g/kg) Karl Fischer moisture (g/kg) Glycerol (g/kg) Gross energy (MJ/kg) Crude protein (g/kg) Methanol§ Sodium (g/kg) Total phosphorus (g/kg) Potassium (g/kg) pH in aqueous solution
167.5 124.5 700.0 15.33 0.3 181.3 23.8 0.0 0.6 6.05
554.4 389.5 99.2 17.23 2.3 111.9 15.1 0.0 0.4 9.85
110.8 101.5 793.1 15.48 0.4 20.6 21.6 0.3 0.9 5.72
*Chemical analyses performed by CBO laboratory analyses, Campinas/ S~ao Paulo, Brazil. †Mixed glycerine from frying oil and lard. stria, ‡Semipurified glycerine from soybean oil (GENPAâ, Granol Indu rcio e Exportacß~ao S/A). Come §Units are mg/l for the glycerine from crude soybean and semipurified sources and mg/kg for the mixed glycerine.
levels for each phase by adding calcium chloride and sodium bicarbonate while maintaining the same electrolytic balance in each experimental diet. For the formulation of diets, the mean values of apparent metabolizable energy corrected for the retained nitrogen (AMEn) that had been previously determined by Lima et al. (2012) for each evaluated glycerine were utilized: 13.73 MJ AMEn/kg CGSO, 13.51 MJ AMEn/ kg MCG and 13.83 MJ AMEn/kg SPGSO. The feed intake and weight gain of the broilers were measured in the period from 22 to 35 (experiment I) and 33 to 42 days of age (experiment II). Moreover, at 35 and 42 days of age (experiments I and II, respectively), two birds from each replicate (totalling 104 chickens in each experiment) were slaughtered by cervical dislocation, and the liver and breast were utilized to determine hepatic glutamate dehydrogenase activity and the crude breast protein content. Chemical analyses Preparation of the liver extract and determination of glutamate dehydrogenase activity
After slaughter, the liver of birds was collected and immediately frozen in liquid nitrogen. For the extraction of glutamate dehydrogenase, 1.5 g of frozen liver was macerated with 3 ml Tris–HCl 0.05 M buffer, pH 7.6. After this tissue homogenization, the sample was centrifuged (15 000 g for 20 min at 5 °C), and the supernatant was collected. The determination of enzymatic activity in the supernatant was performed using Journal of Animal Physiology and Animal Nutrition © 2013 Blackwell Verlag GmbH
a commercial kit (Abcam, Code: ab102527, Cambridge, UK). The enzymatic assay was conducted based on the action of glutamate dehydrogenase on glutamate resulting in the stoichiometric generation of NADH, which can than react with one colour reagent, thereby generating a characteristic colour that can be quantified at 450 nm (PowerWaveTM XS Microplate Scanning Spectrophotometer; Bio-Tek Instruments, Potton, UK). The volumetric activity was calculated from a linear standard curve of NADH. Moreover, a blank sample was prepared in parallel to each sample by replacing the substrate with buffer. Protein content in the samples (mg protein/mL liver extract) was determined according to Bradford method (1976) that was performed by the dye-binding assay and utilizing bovine serum albumin as a standard. The specific activity of glutamate dehydrogenase (U/mg of protein) was calculated by dividing volumetric activity by protein content in the sample, so that U is the quantity of enzyme that generates 1 nmol NADH during 1 min of reaction performed at 37 °C in a solution of 2 M glutamate, pH 7.6. Determination of the crude protein content in the breast
Following slaughter, the chicken breast was collected, and an aliquot of 100.0 g was removed and lyophilized to a constant weight. The crude protein content in the breast was then determined according to method 988.05 (N 9 6.25) of Association of Official Analytical Chemists (AOAC) (1990), and the results were expressed in g crude protein/100 g dry matter. Statistical analysis
For each experiment, the data were submitted to variance analysis using the general linear model in SAS statistical software (2004). When the models were statistically significant, the control diet was compared with the other experimental diets using the Dunnett’s test at 5% probability. The sources of glycerine were compared with each other using the Student– Newman–Keuls mean comparison test at 5% probability. Polynomial regression models (p < 0.05) were utilized to evaluate the effect of the increasing concentration of glycerine on glutamate dehydrogenase activity and on crude protein content in the breast. Results Experiment I (22–35 days of age)
There was an interaction (p < 0.05) between the source and level of glycerine in the diet on the enzymatic activity of hepatic glutamate dehydrogenase 561
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Table 2 Ingredient and calculated nutritional composition of the diets (g/kg as fed basis) during the period from 22 to 35 days of age (experiment I)
Ingredient
Control
Crude glycerine from soybean oil
Mixed glycerine*
17.5
35.0
Corn 617.39 597.48 577.55 Soybean meal 316.02 319.63 323.24 Soybean oil 29.53 29.91 30.29 Glycerine 0.00 17.5 35.00 Dicalcium phosphate 16.47 16.52 16.57 Limestone 8.36 8.32 8.28 Common salt 2.24 2.25 2.26 DL-Methionine 2.23 2.25 2.28 L-Lysine HCl 1.75 1.68 1.62 L-Threonine 0.36 0.35 0.35 Calcium chloride 0.00 0.00 0.00 Sodium bicarbonate 3.61 2.06 0.51 Lasalocid 0.60 0.60 0.60 Mineral supplement‡ 0.50 0.50 0.50 Vitamin supplement§ 0.30 0.30 0.30 Choline chloride 0.40 0.40 0.40 Zinc bacitracin 0.25 0.25 0.25 Calculated nutrient composition (g/kg as fed basis) ME (MJ/kg) 12.98 12.98 12.98 Crude protein 197.27 197.27 197.27 Glycerol¶ 0.00 12.25 24.5 Calcium 8.24 8.24 8.24 Available phosphorus 4.11 4.11 4.11 Sodium 20.50 20.50 20.50 Chlorine 1.80 1.80 1.80 Lysine 10.73 10.73 10.73 Methionine + cystine 77.30 77.30 77.30 Threonine 69.70 69.70 69.70 D.E.B (mEq/kg)** 231 231 231
Semipurified glycerine†
52.5
70.0
17.5
35.0
52.5
70.0
17.5
35.0
52.5
70.0
556.37 327.08 31.09 52.50 16.63 7.61 1.57 2.31 1.55 0.35 0.89 0.00 0.60 0.50 0.30 0.40 0.25
534.60 331.03 32.11 70.0 16.68 6.63 0.53 2.33 1.48 0.34 2.22 0.00 0.60 0.50 0.30 0.40 0.25
596.23 319.85 30.36 17.50 16.53 8.32 2.25 2.25 1.68 0.35 0.00 2.63 0.60 0.50 0.30 0.40 0.25
575.06 323.69 31.19 35.00 16.58 8.28 2.27 2.28 1.61 0.35 0.00 1.64 0.60 0.50 0.30 0.40 0.25
553.90 327.53 32.01 52.50 16.64 8.24 2.28 2.31 1.54 00.35 0.00 0.65 0.60 0.50 0.30 0.40 0.25
532.34 331.44 32.98 70.00 16.69 7.99 2.07 2.34 1.47 0.34 0.29 0.00 0.60 0.50 0.30 0.40 0.25
597.44 319.64 29.80 17.50 16.52 8.32 2.25 2.25 1.68 0.35 0.00 2.20 0.60 0.50 0.30 0.40 0.25
577.47 323.26 30.06 35.00 16.57 8.28 2.26 2.28 1.62 0.35 0.00 0.80 0.60 0.50 0.30 0.40 0.25
556.77 327.01 30.58 52.50 16.63 7.87 1.86 2.30 1.55 0.35 0.53 0.00 0.60 0.50 0.30 0.40 0.25
535.13 330.94 31.42 70.00 16.68 6.98 0.92 2.33 1.48 0.34 1.73 0.00 0.60 0.50 0.30 0.40 0.25
12.98 197.27 36.75 8.24 4.11 20.50 1.80 10.73 77.30 69.70 231
12.98 197.27 49.00 8.24 4.11 20.50 1.80 10.73 77.30 69.70 231
12.98 197.27 1.74 8.24 4.11 20.50 1.80 10.73 77.30 69.70 231
12.98 197.27 3.47 8.24 4.11 20.50 1.80 10.73 77.30 69.70 231
12.98 197.27 5.21 8.24 4.11 20.50 1.80 10.73 77.30 69.70 231
12.98 197.27 6.94 8.24 4.11 20.50 1.80 10.73 77.30 69.70 231
12.98 197.27 13.88 8.24 4.11 20.50 1.80 10.73 77.30 69.70 231
12.98 197.27 27.76 8.24 4.11 20.50 1.80 10.73 77.30 69.70 231
12.98 197.27 41.64 8.24 4.11 20.50 1.80 10.73 77.30 69.70 231
12.98 197.27 55.52 8.24 4.11 20.50 1.80 10.73 77.30 69.70 231
*Crude glycerine from frying oil and lard. rcio e Exportacß~ao S/A). stria, Come †Semipurified glycerine from soybean oil (GENPAâ, Granol Indu ‡Supplied per kg of the diet: Zn, 55 mg; Se, 0.18 mg; I, 0.70 mg; Cu, 10 mg; Mn, 78 mg; Fe, 48 mg. §Supplied per kg of the diet: folic acid, 0.48 mg; pantothenic acid, 8.7 mg; biotin, 0.018 mg; butylated hydroxytoluene (BHT), 1.5 mg; niacin, 11.1 mg; vitamin A, 6000 IU; vitamin B1, 0.9 mg; vitamin E, 12.15 IU; vitamin B12, 8.1 lg; vitamin B2, 3.6 mg; vitamin B6, 1.8 mg; vitamin D3, 1,500 IU; vitamin K3, 1.44 mg. ¶Glycerol from glycerine supplementation. **Dietary electrolyte balance (D.E.B.) calculated using the equation proposed by Mongin (1981), which correlates the calculated concentrations of sodium, potassium and chloride (Na+ + K+ Cl). ME: metabolizable energy.
(Table 4). Increases in mixed crude glycerine (MCG) caused a linear reduction in the activity of the enzyme (y = 0.1235x + 16.880; R2 = 0.90), whereas for the semipurified glycerine from soybean oil (SPGSO), glutamate dehydrogenase showed up to a 42.5% increase in activity (y = 0.0970x + 9.475; R2 = 0.97) with increasing glycerine. However, the concentration of crude glycerine from soybean oil (CGSO) did not affect the activity of glutamate dehydrogenase. Moreover, 70.0 g glycerine/kg of diet, derived from CGSO and SPGSO, induced the greatest activity of glutamate dehydrogenase, whereas in the case of MCG, this dose induced the lowest activity. The glutamate dehydroge-
nase activity in the liver of birds that received different concentrations and sources of glycerine did not differ (p > 0.05) from the activity of birds fed the control diet. There was no interaction (p > 0.05) between the source or concentration of glycerine in the diet and the protein content in the breast of broilers (Table 4). However, there was an isolated effect of the source and concentration of glycerine for this parameter (p < 0.05). The highest mean content of protein deposition in the breast (70.3 g/100 g dry matter) was found in birds fed the diet containing SPGSO, whereas the lowest protein content (66.6 g/100 g dry matter)
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Glutamate dehydrogenase and protein content in broilers
Table 3 Ingredient and calculated nutritional composition of the diets (g/kg as fed basis) during the period from 33 to 42 days of age (experiment II)
Ingredient
Control
Crude glycerine from soybean oil
Mixed glycerine*
17.5
35.0
Corn 647.53 627.33 607.14 Soybean meal 288.27 291.93 295.60 Soybean oil 30.70 31.30 31.90 Glycerine 0.00 17.50 35.00 Dicalcium phosphate 14.94 14.99 15.04 Limestone 7.92 7.88 7.84 Common salt 2.07 2.08 2.10 DL-Methionine 2.02 2.05 2.07 L-Lysine HCl 1.85 1.79 1.72 L-Threonine 0.35 0.35 0.34 Calcium chloride 0.00 0.00 0.00 Sodium bicarbonate 3.45 1.90 0.35 Mineral supplement‡ 0.50 0.50 0.50 Vitamin supplement§ 0.20 0.20 0.20 Choline chloride 0.20 0.20 0.20 Calculated nutrient composition (g/kg as fed basis) ME (MJ/kg) 13.19 13.19 13.19 Crude protein 187.15 187.15 187.15 Glycerol¶ 0.00 12.25 24.5 Calcium 7.63 7.63 7.63 Available phosphorus 3.80 3.80 3.80 Sodium 1.94 1.94 1.94 Chloride 1.70 1.70 1.70 Lysine 10.17 10.17 10.17 Methionine + cystine 7.32 7.32 7.32 Threonine 6.61 6.61 6.61 D.E.B. (mEq/kg)** 219 219 219
Semipurified glycerine†
52.5
70.0
17.5
35.0
52.5
70.0
17.5
35.0
52.5
70.0
585.52 299.52 32.99 52.50 15.09 7.07 1.29 2.10 1.65 0.34 1.03 0.00 0.50 0.20 0.20
563.44 303.52 34.24 70.00 15.15 6.09 0.25 2.13 1.58 0.34 2.36 0.00 0.50 0.20 0.20
625.21 292.32 32.48 17.50 14.99 7.87 2.08 2.05 1.78 0.35 0.00 2.47 0.50 0.20 0.20
602.87 296.37 34.27 35.00 15.05 7.83 2.10 2.08 1.71 0.34 0.00 1.48 0.50 0.20 0.20
580.54 300.42 36.06 52.50 15.11 7.78 2.11 2.11 1.64 0.34 0.00 0.49 0.50 0.20 0.20
557.60 304.58 38.05 70.00 15.17 7.44 1.79 2.14 1.56 0.34 0.43 0.00 0.50 0.20 0.20
627.94 291.82 30.66 17.50 14.99 7.88 2.08 2.05 1.79 0.35 0.00 2.04 0.50 0.20 0.20
608.34 295.38 30.63 35.00 15.04 7.84 2.09 2.07 1.73 0.34 0.00 0.64 0.50 0.20 0.20
587.83 299.10 30.91 52.50 15.09 7.33 1.58 2.10 1.66 0.34 0.66 0.00 0.50 0.20 0.20
566.53 302.96 31.46 70.00 15.14 6.44 0.64 2.13 1.59 0.34 1.87 0.00 0.50 0.20 0.20
13.19 187.15 36.75 7.63 3.80 1.94 1.70 10.17 7.32 6.61 219
13.19 187.15 49.00 7.63 3.80 1.94 1.70 10.17 7.32 6.61 219
13.19 187.15 1.74 7.63 3.80 1.94 1.70 10.17 7.32 6.61 219
13.19 187.15 3.47 7.63 3.80 1.94 1.70 10.17 7.32 6.61 219
13.19 187.15 5.21 7.63 3.80 1.94 1.70 10.17 7.32 6.61 219
13.19 187.15 6.94 7.63 3.80 1.94 1.70 10.17 7.32 6.61 219
13.19 187.15 13.88 7.63 3.80 1.94 1.70 10.17 7.32 6.61 219
13.19 187.15 27.76 7.63 3.80 1.94 1.70 10.17 7.32 6.61 219
13.19 187.15 41.64 7.63 3.80 1.94 1.70 10.17 7.32 6.61 219
13.19 187.15 55.52 7.63 3.80 1.94 1.70 10.17 7.32 6.61 219
*Crude glycerine from frying oil and lard. rcio e Exportacß~ao S/A). stria, Come †Semipurified glycerine from soybean oil (GENPAâ, Granol Indu ‡Supplied per kg of the diet: Zn, 55 mg; Se, 0.18 mg; I, 0.70 mg; Cu, 10 mg; Mn, 78 mg; Fe, 48 mg. §Supplied per kg of the diet: folic acid, 0.32 mg; pantothenic acid, 5.8 mg; biotin, 0.012 mg; butylated hydroxytoluene (BHT), 1.0 mg; niacin, 7.4 mg; vitamin A, 4000 IU; vitamin B1, 0.6 mg; vitamin E, 8.1 IU; vitamin B12, 5.4 lg; vitamin B2, 2.4 mg; vitamin B6, 1.2 mg; vitamin D3, 1000 IU; vitamin K3, 0.96 mg. ¶Glycerol from glycerine supplementation. **Dietary electrolyte balance (D.E.B.) calculated using the equation proposed by Mongin (1981), which correlates the calculated concentrations of sodium, potassium and chloride (Na+ + K+ Cl). ME: metabolizable energy.
was observed in birds fed the other types of glycerine (CGSO and MCG). However, regardless of the source utilized, there was a quadratic effect of the glycerine concentration on the protein content in the breast of broilers (y = 0.0036x2 + 0.3966x + 58.7; R2 = 0.99). A maximum protein deposition of 69.62% was estimated for a diet containing 55.08 g glycerine/kg feed. The use of 52.5 and 70.0 g SPGSO/kg of diet promoted a greater (p < 0.05) percentage deposition of protein in the breast of broilers when compared with the content determined in birds that received the control diet without glycerine. There was no interaction or isolated effects (p > 0.05) between the sources and levels of glycerine on the feed intake, and so an overall intake of
2.26 kg/bird was observed in the period from 22 to 35 days of age (Table 5). Moreover, inclusion of MCG at the concentrations of 35.0, 52.5 and 70.0 g/kg resulted in higher (p < 0.05) feed intake than the recorded for broilers fed the control diet. There was an interaction (p < 0.05) between the source and level of glycerine in the diet on the weight gain of broilers (Table 5). When glycerine was included at concentration of 35.0 g/kg, the CGSO provided the highest weight gain (1.45 kg/bird), and SPGSO, the lowest value (1.34 kg/bird), whereas MCG resulted in intermediate weight gain (1.43 kg/ bird). However, for the concentration of inclusion of glycerine of 70.0 g/kg, the highest weight gain (1.47 kg/bird) was obtained with the use of MCG in
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Table 4 Glutamate dehydrogenase activity and protein content in the breast of broilers fed different sources of glycerine during the period from 22 to 35 days of age (experiment I) Glutamate dehydrogenase activity (U/mg protein)
Crude protein in the breast (g/100 g dry matter)
Glycerine content (g/kg)
Glycerine content (g/kg)
Source of glycerine
17.5
35.0
52.5
70.0
Mean
17.5
35.0
52.5
70.0
Mean
Crude glycerine from soybean oil Mixed glycerine*,† Semipurified glycerine‡,§ Mean¶ Control diet Coefficient of variation (%) p-value SG 9 LIG SG LIG
12.81 15.18 11.27 13.09
11.90 11.40 12.47 11.92
12.00 11.33 15.07 12.80
11.96a 8.00b 16.06a 12.01
12.17 11.48 13.72
64.3 61.2 68.3 64.6
67.6 66.5 69.8 68.0
67.8 69.3 72.4** 69.8
66.6 69.0 70.9** 68.8
66.6b 66.5b 70.3a
10.49
57.7
12.63
6.85
p < 0.05 p > 0.05 p > 0.05
p > 0.05 p < 0.05 p < 0.05
SG, source of glycerine; LIG, level of inclusion of glycerine. Means with different letters in the same column are significantly different from each other (p < 0.05, determined using the Student–Newman–Keuls test). Each value represents the mean obtained using two birds that were slaughtered for each one of the four replicates performed. *Mixed glycerine from frying oil and lard. †Linear effect of mixed glycerine content on the activity of glutamate dehydrogenase (y = 0.1235x + 16.880; R2 = 0.90). rcio e Exportacß~ao S/A). stria, Come ‡Semipurified glycerine from soybean oil (GENPAâ, Granol Indu §Linear effect of semipurified glycerine on the activity of glutamate dehydrogenase (y = 0.0970x + 9.475; R2 = 0.97). ¶Quadratic effect of glycerine on the crude protein content in the breast (y = 0.0036x2 + 0.3966x + 58.7; R2 = 0.99). **Significantly different from the control using the Dunnett’s test (p < 0.05).
Table 5 Feed intake and weight gain of broilers fed different sources of glycerine during the period from 22 to 35 days of age (experiment I) Feed intake (kg/bird)
Weight gain (kg/bird)
Glycerine content (g/kg)
Glycerine content (g/kg)
Source of glycerine
17.5
35.0
52.5
70.0
Mean
17.5
35.0
52.5
70.0
Mean
Crude glycerine from soybean oil† Mixed glycerine* Semipurified glycerine Mean Control diet Coefficient of variation (%) p-value SG 9 LIG SG LIG
2.21 2.26 2.22 2.23
2.25 2.30** 2.24 2.26
2.26 2.31** 2.24 2.27
2.25 2.35** 2.26 2.29
2.24 2.31 2.24
1.41 1.42 1.39 1.41
1.45a 1.43a,b 1.34b 1.43
1.45 1.41 1.41 1.42
1.26**b 1.47a 1.44b 1.39
1.39 1.43 1.39
2.19
1.43
3.71
5.49
p > 0.05 p > 0.05 p > 0.05
p < 0.05 p > 0.05 p > 0.05
SG: source of glycerine, LIG: level of inclusion of glycerine. Means with different letters in the same column are significantly different from each other (p < 0.05, determined using the Student–Newman–Keuls test). Each value represents the mean obtained using two birds that were slaughtered for each one of the four replicates performed. *Mixed glycerine from frying oil and lard. †Quadratic effect of crude glycerine from soybean oil on the weight gain (y = 0.0193x2 + 0.1423x + 1.2119; R2 = 0.95). **Significantly different from the control using the Dunnett’s test (p < 0.05).
the diet. Moreover, the increase in the concentration of CGSO caused a quadratic effect on the weight gain (y = 0.0193x2 + 0.1423x + 1.2119; R2 = 0.95). The inclusion of 70.0 g of CGSO/kg of diet worsened (p < 0.05) weight gain when compared with the broilers fed the control diet. 564
Experiment II (33–42 days of age)
There was an interaction (p < 0.05) between the source and concentration of glycerine in the diet on the enzymatic activity of hepatic glutamate dehydrogenase (Table 6). Increasing concentrations of MCG Journal of Animal Physiology and Animal Nutrition © 2013 Blackwell Verlag GmbH
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Table 6 Glutamate dehydrogenase activity and protein content in the breast of broilers fed different sources of glycerine during the period from 33 to 42 days of age (experiment II) Glutamate dehydrogenase activity (U/mg protein)
Crude protein in the breast (g/100 g dry matter)
Glycerine content (g/kg)
Glycerine content (g/kg)
Source of glycerine
17.5
35.0
52.5
70.0
Mean
17.5
35.0
52.5
70.0
Mean
Crude glycerine from soybean oil Mixed glycerine*,† Semipurified glycerine‡,§ Mean Control diet Coefficient of variation (%) p-value SG 9 LIG SG LIG
11.66 11.37 10.38 11.14
11.56 8.57 11.72 10.62
11.34a, b 7.60b 14.71a 11.22
11.45b 7.42c 16.52a 11.80
11.50a 8.74b 13.33a
81.45 79.69 80.59 80.57
81.82 80.22 81.46 81.17
82.13 80.63 82.40 81.72
82.22 81.66 84.35 82.74
81.90 80.55 82.20
11.76
82.66
11.99
3.76
p < 0.05 p < 0.05 p > 0.05
p > 0.05 p > 0.05 p > 0.05
SG, source of glycerine; LIG, level of inclusion of glycerine. Means with different letters in the same column are significantly different from each other (p < 0.05, determined using the Student–Newman–Keuls test). Each value represents the mean obtained using two birds that were slaughtered for each one of the four replicates performed. *Mixed glycerine from frying oil and lard. †Linear effect of mixed glycerine content on the activity of glutamate dehydrogenase (y = 0.0733x + 11.945; R2 = 0.82). rcio e Exportacß~ao S/A). stria, Come ‡Semipurified glycerine from soybean oil (GENPAâ, Granol Indu §Linear effect of semipurified glycerine on the activity of glutamate dehydrogenase (y = 0.1223x + 7.980; R2 = 0.98).
were inversely correlated with the activity of the enzyme (y = 0.0733x + 11.945; R2 = 0.82) and caused up to a 34.74% reduction in activity, whereas for SPGSO, the glutamate dehydrogenase activity increased linearly (y = 0.1223x + 7.980; R2 = 0.98) by up to 59.15%. However, the concentration of CGSO did not affect the glutamate dehydrogenase activity. Furthermore, at 52.5 and 70.0 g glycerine/kg of diet, the use of SPGSO promoted the greatest activity of glutamate dehydrogenase, but with MCG, these doses induced lower activity. The glutamate dehydrogenase activity in the liver of birds receiving different concentrations and sources of glycerine did not differ (p > 0.05) from the activity determined from the liver of birds fed the control diet. There was no interaction (p > 0.05) or isolated effect between the factors (sources and levels of glycerine) on the crude protein content in the breast of broilers, and a mean protein deposition of 81.55 g/ 100 g dry matter (Table 6) was observed. All diets containing glycerine promoted a similar level of protein deposition in the breast to that determined from birds fed the control diet without glycerine. There was an interaction (p < 0.05) between the source and concentration of glycerine in the diet on feed intake and weight gain of broilers (Table 7). Inclusion of 35.0 g of SPGSO/kg provided the highest feed intake (2.03 kg/bird), and the MCG, the lowest (1.88 kg/bird), whereas CGSO resulted in intermedi-
ate intake (1.94 kg/bird). For the weight gain, however, the lowest values were recorded for the birds fed diet containing 17.5 g of MCG or SPGSO/kg. Moreover, the increase in the concentration of CGSO increased linearly (y = 0.015x + 1.033; R2 = 0.84) the weight gain up to 8.32%.
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Discussion Experiment I (22–35 days of age)
During the period from 22 to 35 days of age, an increase in the glycerine concentration in the diet (and consequent increase in the concentration of glycerol) did not necessarily reduce the activity of hepatic glutamate dehydrogenase. These results opposed the initial hypothesis of this study, which was based on the report by Steele et al. (1971), who suggested that the glycerol present in the diet could spare glucogenic amino acids by reducing the activity of this enzyme, thereby enabling protein deposition in the muscle tissues of the bird. We did observe enzymatic inhibition with increasing concentrations of the MCG; however, against expectation, the increase in the concentration of semipurified glycerine from soybean oil (SPGSO) did not inhibit the enzyme, which was unexpected because this glycerine showed the highest percentage content of glycerol. Nevertheless, it is important to note that glutamate dehydrogenase converts glutamate into a-ketoglutarate and vice versa, thus
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Table 7 Feed intake and weight gain of broilers fed different sources of glycerine during the period from 33 to 42 days of age (experiment II) Feed intake (kg/bird)
Weight gain (kg/bird)
Glycerine content (g/kg)
Glycerine content (g/kg)
Source of glycerine
17.5
35.0
52.5
70.0
Mean
17.5
35.0
52.5
70.0
Mean
Crude glycerine from soybean oil† Mixed glycerine* Semipurified glycerine Mean Control diet Coefficient of variation (%) p-value SG 9 LIG SG LIG
1.94 1.95 2.05 1.98
1.94a, b 1.88b 2.03a 1.95
1.92 1.88 1.90 1.90
1.97 1.94 1.85 1.92
1.94 1.91 1.96
1.05b 1.12a 1.13a 1.10
1.11 1.09 1.10 1.10
1.12 1.10 1.12 1.11
1.13 1.10 1.08 1.11
1.10 1.10 1.11
1.92
1.09
4.57
3.91
p < 0.05 p > 0.05 p > 0.05
p < 0.05 p > 0.05 p > 0.05
SG, source of glycerine; LIG, level of inclusion of glycerine. Means with different letters in the same column are significantly different from each other (p < 0.05, determined using the Student–Newman–Keuls test). Each value represents the mean obtained using two birds that were slaughtered for each one of the four replicates performed. *Mixed glycerine from frying oil and lard. †Linear effect of crude glycerine from soybean oil on the weight gain (y = 0.0150x + 1.0330; R2 = 0.84). **Significantly different from the control using the Dunnett’s test (p < 0.05).
representing an important link between the metabolisms of carbon and nitrogen. Thus, it acts both on the degradation and on the synthesis of amino acids, and the direction of the reaction depends on the physiological condition of the animal (Champe et al., 2009). The adverse effects of the level of glycerine in the diet on the glutamate dehydrogenase activity that was observed in this study may be related to the glycerol content of the diets. Although the feed intake by broilers did not have been influenced by the inclusion level of glycerine in the diet, when SPGSO was utilized, the diets contained higher levels of glycerol than those calculated for the MCG diet (between 13.88 and 55.52 g of glycerol/kg of diet supplemented with SPGSO and between 1.74 and 6.94 g of glycerol/kg of diet supplemented with MCG). Thus, in the case of MCG, because the percentage glycerol content in this glycerine was lower, the largest portion of the glycerol from this source might have been utilized metabolically for the production of energy via glycolysis and the Krebs cycle, thus inhibiting glutamate dehydrogenase activity. This inhibition reduced the degradation of glucogenic amino acids, which were elevated because the diet was nutritionally adequate for broilers. In the diet containing SPGSO, the glycerol may have been converted into an intermediate of the glycolytic pathway being converted into pyruvate, which in turn may have been primarily utilized for the synthesis of a-ketoglutarate via several sequential biochemical reactions. a-ketoglutarate could then have been used as a substrate of glutamate dehydrogenase
in the synthesis of amino acids, as can be verified by the higher crude protein contents in the breast muscle of the broilers fed diets containing the SPGSO. Therefore, the up-regulation in catalytic activity of glutamate dehydrogenase could have been due to the increase in cellular concentration of a-ketoglutarate due to the higher content of glycerol in the diet. However, in this study, it was verified that glutamate dehydrogenase activity is regulated not only by the level of glycerol in the diet, but also by the source used as raw material for the production of biodiesel, because at 70.0 g glycerine/kg of diet, the use of CGSO and SPGSO increased the activity of glutamate dehydrogenase, whereas the MCG reduced enzymatic activity by up to 50%, showing that there is an interaction between the source and content of glycerine in the diet. The use of the SPGSO induced approximately 3.7% points more protein deposition in the breast than that of the other types of glycerine, possibly due to the higher catalytic activity of glutamate dehydrogenase and also the best quality of the SPGSO. Although the semipurification manner from SPGSO is not well described in the literature for this commercial product, this processing may have reduced the concentration of some residue that worsens the protein deposition, associated with their greater content of glycerol. Regardless of the source utilized, there was a quadratic effect of the concentration of glycerine in the diet on the percentage of protein in the breast of broilers. However, the protein deposition was not strictly correlated with glutamate dehydrogenase activity.
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The use of 52.5 and 70.0 g SPGSO/kg of diet induced a greater (p < 0.05) percentage of protein in the breast of broilers when compared with the content in birds that received the control diet without glycerine. These data are to some degree similar to those of Cerrate et al. (2006), who verified that breast yield was greater in birds fed diets containing 25 or 50 g of glycerine/kg of diet when compared with birds that received the control diet without glycerine. Based on the study by Steele et al. (1971), glycerol in the diet can spare glucogenic amino acids by reducing the activity of glutamate dehydrogenase, thus enabling protein deposition in the muscle tissues. However, according to Cryer and Bartley (1973), the sparing effect on glucogenic amino acids may also be due to the inhibition of another key enzyme of gluconeogenesis/ phosphoenolpyruvate carboxykinase (EC 4.1.1.32), which catalyses the conversion of oxaloacetate and GTP to phosphoenolpyruvate and GDP and CO2 (Champe et al., 2009). Therefore, to obtain more data on the relationship between the source and concentration of glycerine supplementation in the diet and the protein content in the breast of broilers, it is recommended that the activity of phosphoenolpyruvate carboxykinase be determined in future experiments. The lowest weight gain observed with the inclusion of 70.0 g of CGSO/kg of diet might probably be related to the higher methanol content present in this glycerine, associated with the influence of birds age and the more extensive rearing period evaluated (13 days). According to Macari et al. (2002), broilers from 22 to 35 days of age have still a low capacity to metabolize the methanol. Similarly, Cerrate et al. (2006) reported worse in the weight gain of birds fed diets containing high inclusion levels of CGSO, during the period from 1 to 35 days of age.
Glutamate dehydrogenase and protein content in broilers
reported by Ribeiro et al. (1995) (143.7 U/mg protein). However, Ribeiro et al. (1995) evaluated the effects of different concentrations of glutamine, alanine and proline in the diet. Therefore, the high levels determined by Ribeiro et al. (1995) may have been due to the conversion of the amino acids into glutamate, the levels of which then increase in the liver, thus requiring increased glutamate dehydrogenase activity to meet the requirement for transamination or oxidative deamination, that is, at the level of the degradation of glutamate to a-ketoglutarate and NHþ 4 . Contrary to that observed for the previous rearing period (experiment I), the different sources and concentrations of glycerine did not affect the protein content determined in the breast of broilers in the final rearing phase, demonstrating the influence of age of birds and duration of the rearing period (which in this case was shorter). Moreover, all diets containing glycerine promoted a similar deposition of protein in the breast to that determined from control-fed birds, with an average protein concentration of 81.55 g/100 g dry matter. Contrary to the observed for the previous rearing phase (experiment I), the increase in the concentration of CGSO in the diet improves the weight gain, which appears to demonstrate that in the rearing period from 33 to 42 days of age, the methanol present in the diet formulated with up to 70 g CGSO/kg can be properly metabolized by the bird. However, further specific studies still need to be conducted to evaluate and measure the real metabolic effects of the methanol at the different rearing ages of broilers fed diets containing glycerine. Conclusions
During the period from 33 to 42 days of age, glutamate dehydrogenase activity decreased linearly with increasing MCG and increased linearly with increasing SPGSO in the diet, which is similar to observations in experiment I (from 22 to 35 days of age) and may be due to the same previously discussed causes. Moreover, in this last rearing phase, the source of glycerine also affected glutamate dehydrogenase activity, which was lower when the diet was supplemented with MCG, possibly because of the lower percentage content of glycerol in this glycerine. Overall, glutamate dehydrogenase activities in the livers of birds that received the diet without glycerine (control) in both experiments were lower than those
For both rearing periods evaluated, an increase in glycerine in the diet did not necessarily reduce the activity of hepatic glutamate dehydrogenase; in addition, the deposition of protein in the breast of broilers is not strictly correlated with the activity of this enzyme. During the period from 22 to 35 days of age, the use of semipurified glycerine from soybean oil in the diet increased the protein content in the breast by up to 3.7% points more when compared with the crude glycerine from soybean oil and with the mixed glycerine from frying oil and lard. Moreover, regardless of the source of protein, the inclusion of 55.08 g of glycerine/kg of diet promoted the largest deposition of protein in the breast of broilers. However, during the period from 33 to 42 days of age, the protein content in the breast of broilers is not necessarily affected by
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the source or by the concentration of glycerine in the diet. The feed intake and weight gain also were influenced by age of broilers, demonstrating that when glycerine is used in the diet, the rearing period must be considered to allow the best performance results. The results of this study demonstrate that glycerine has potential to be used as an energetic ingredient in the broiler diets and can maintain or even improve the protein deposition in the breast of broilers. References ANP – National Agency of Petroleum, Natural Gas and Biofuels (Brazil). 2012: Available: http://www.anp.gov.br. Accessed April 14, 2012. Association of Official Analytical Chemists (AOAC), 1990: Official Methods of Analysis, 15th edn. Association of Official Analytical Chemists, Arlington, VA. Bradford, M. M., 1976: A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry 72, 248–254. Cerrate, S.; Yan, F.; Wang, Z.; Coto, C.; Sacakli, P.; Waldroup, P. W., 2006: Evaluation of glycerine from biodiesel production as a feed ingredient for broilers. International Journal of Poultry Science 5, 1001–1007. Champe, P. C.; Harvey, R. A.; Ferrier, D. R., 2009: Biochemistry: Lippincott’s Illustrated Reviews, 4th edn. Artmed, Porto Alegre, 528p. Cryer, A.; Bartley, W., 1973: Studies of the adaptation of rats to a diet high in glycerol. International Journal of Biochemistry 4, 293–308. Dozier, W. A.; Kerr, B. J.; Corzo, A.; Kidd, M. T.; Weber, T. E.; Bregendahl, K., 2008: Apparent metabolizable energy of glycerin for broiler chickens. Poultry Science 87, 317–322. Garcia, A. J. M.; Tookuni, J. P. M., 2006: Biodiesel from animal fat. Biodiesel Magazine BR. Available: http://www. biodieselbr.com/estudos/biodiesel/bio-
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Acknowledgements The authors acknowledge the Foundation for Research Support of Minas Gerais (FAPEMIG), the National Council for Scientific and Technological Development – Brazil (CNPq) and the National Institute of Technology Science of Animal Science (INCTCA) for the financial support, as well as Granol Ind. Exp. S/A for providing the semipurified glycerine from soybean oil.
diesel-sebo-gordura-animal.htm. Accessed April 14, 2012. Gianfelici, M. F.; Ribeiro, A. M. L.; Penz, A. M. Jr; Kessler, A. M.; Viira, M. M.; Manchisky, T., 2011: Determination of apparent metabolizable energy of crude glycerin in broilers chickens. Brazilian Journal of Poultry Science 13, 255–258. Guerra, R. L. H.; Murakami, A. E.; Garcia, A. F. Q. M.; Urgnani, F. J.; Moreira, I.; Picoli, K. P., 2011: Crude glycerine mixture in diets of broiler chickens (1 to 42 days). Revista Brasileira de Sa ude e Producß~ ao Animal 12, 1038–1050. Lammers, P. J.; Kerr, B. J.; Honeyman, M. S.; Stalder, K.; Dozier, W. A.; Weber, T. E.; Kidd, M. T.; Bregendahl, K., 2008: Nitrogen-corrected apparent metabolizable energy value of crude glycerol for laying hens. Poultry Science 87, 104–107. Lima, E. M. C.; Rodrigues, P. B.; Alvarenga, R. R.; Bernardino, V. M. P.; Makiyama, L.; Lima, R. R.; Cantarelli, V. S.; onimo, M. G. 2012: The energy Zanger^ value of biodiesel glycerine products fed to broilers at different ages. Journal of Animal Physiology and Animal Nutrition. doi: 10.1111/j.1439-0396.2012. 01335.x. [Epub ahead of print]. Ma, F.; Hanna, M. A., 1999: Biodiesel production: a review. Bioresource Technology 70, 1–15. Macari, M.; Furlan, R. L.; Gonzales, E., 2002: Avian Physiology Applied to Broilers. UNESP, FUNEP, S~ ao Paulo, 375p. Mongin, P., 1981: Recent advances in dietary anion-cation balance: application in
poultry. Proceedings of Nutrition Society 40, 285–294. Ribeiro, M.; Moraes, G. H. K.; Sant’anna, R.; Fonseca, J. B., 1995: Effects of dietary L-glutamic acid, L-alanine and Lproline on broiler chicks: II – liver L-glutamate dehydrogenase and serum amino acids and acid uric. Brazilian Journal of Animal Science 24, 778–787. Rostagno, H. S.; Albino, L. F. T.; Donzele, J. L.; Gomes, P. C.; Oliveira, R. F.; Lopes, D. C.; Ferreira, A. S.; Barreto, S. L. T., 2005: Brazilian Tables for Poultry and Swine: Composition of Feedstuffs and Nutritional Requirements, 2nd edn. Federal University of Vicßosa, Vicßosa 186p. Sas Institute, 2004: User’s Guide. Stat. Version 9.00, 4th edn. SAS Institute, Cary, NC. Steele, R.; Winkler, B.; Altszuler, N., 1971: Inhibition by infusion glycerol of gluconeogenesis from other precursors. American Journal of Physiology 221, 883– 888. Summers, J. D.; Leeson, S.; Spratt, D., 1988: Yield and composition of edible meat from male broilers as influenced by dietary protein level and amino acid supplementation. Canadian Journal of Animal Science 68, 241–248. Thompson, J. C.; He, B. B., 2006: Characterization of crude glycerol from biodiesel production from multiple feedstocks. Applied Engineering in Agriculture 22, 261–265. Van Gerpen, J., 2005: Biodiesel processing and production. Fuel Processing Technology 86, 1097–1107.
Journal of Animal Physiology and Animal Nutrition © 2013 Blackwell Verlag GmbH
ORIGINAL ARTICLE
Activity of glutamate dehydrogenase and protein content in the breast of broilers fed diets containing different sources and levels of glycerine V. M. P. Bernardino1, P. B. Rodrigues1, L. de Paula Naves1, M. G. Zangeronimo2, R. R. Alvarenga1, P. V. Rosa1, L. M. Santos1 and L. V. Teixeira1 1 Department of Animal Science, Federal University of Lavras, Lavras, MG, Brazil, and 2 Department of Veterinary Science, Federal University of Lavras, Lavras, MG, Brazil
Summary According to scientific literature, glycerol in the diet can spare glucogenic amino acids by inhibiting the activity of enzymes, such as glutamate dehydrogenase, thereby promoting protein deposition in muscle tissues. Therefore, the objective of this study was to evaluate the effect of three sources of glycerine (crude glycerine from soybean oil – CGSO, mixed crude glycerine from frying oil and lard – MCG and a semipurified glycerine from soybean oil – SPGSO) in four concentrations in the diet (17.5, 35.0, 52.5 and 70.0 g of each type of glycerine/kg of feed) on the activity of hepatic glutamate dehydrogenase, performance and protein content in the breast of broilers, during 22–35 days of age (experiment I) and 33–43 days of age (experiment II). In both experiments, an increase in MCG induced a linear decline in glutamate dehydrogenase activity (p < 0.05). In contrast, increasing the concentration of SPGSO in the diet caused a linear increase in enzyme activity (p < 0.05). There was no (p > 0.05) isolated effect of glycerine on the enzyme activity in either evaluated phase; however, during 33–42 days of age, MCG inhibited (p < 0.05) the glutamate dehydrogenase activity by up to 34.43%. During 22–35 days of age, the diet containing SPGSO induced a higher protein content (p < 0.05) in the breast, and regardless of the source utilized, the maximum protein deposition was estimated (p < 0.05) when broilers were fed with 55.08 g glycerine/kg of diet. There was no (p > 0.05) interaction or isolated effects of the sources and levels of glycerine on the protein content in the breast of broilers at 33–42 days of age, and moreover, all diets containing glycerine promoted a similar protein deposition in the breast compared with birds that received the diet without glycerine. The bird age also showed to influence the feed intake and weight gain of broilers fed diet containing glycerine. It is concluded that for both rearing phases, an increase in glycerine in the diet did not necessarily reduce the glutamate dehydrogenase activity, and the protein deposition in the breast of broilers may not be strictly correlated with the activity of this enzyme. Keywords biodiesel, enzyme, glycerol, poultry Correspondence V. M. P. Bernardino, Department of Animal Science, Federal University of Lavras, Lavras, MG 37200000, Brazil. Tel: +55 35 3829 1691; Fax: +55 35 3829 1231; E-mail: [email protected] Received: 31 July 2012; accepted: 11 July 2013
Introduction Biodiesel production is on the increase, and consequently, the production of glycerine is also increased because it is a by-product of this important biofuel (Ma and Hanna, 1999; Van Gerpen, 2005). If the glycerine is refined, it can be used by the chemical, pharmaceutical and food industries to produce high-value coproducts. However, this purification process is expensive, and these industries do not require all amount of crude glycerine that has been produced (Thompson and He, 2006). Thus, it is necessary to
identify alternative markets to utilize the glycerine in order to add value to this coproduct and also to prevent its reverting to an environmentally detrimental waste product. Due to its high energetic value, the use of glycerine in the feeding of broilers has been considered and evaluated (Dozier et al., 2008; Lammers et al., 2008; Gianfelici et al., 2011; Guerra et al., 2011). The glycerol originating from the glycerine utilized in these diets could be used metabolically in different metabolic routes; for example, it can be converted enzymatically into glucose via gluconeo-
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genesis and afterwards can provide energy for cellular metabolism, or the glycerol can be directly oxidized for the production of energy via glycolysis and the Krebs cycle. Biodiesel can be produced from different sources including vegetable oils (cottonseed, peanut, babassu, canola, palm kernel, sunflower, castor oil plant and soy, among others), animal fats (bovine tallow, fish oil and lard) or oils or residual fats (from domestic, commercial or industrial processes) (Garcia and Tookuni, 2006; ANP, 2012), and the source affects the chemical composition of the glycerine present. Furthermore, the process of biofuel production itself can also alter the type of glycerine produced, and the use of different types of glycerine in poultry feed can induce different responses from the bird. Moreover, the levels of glycerine in the diet of broilers during different rearing phases should be evaluated with an aim towards establishing the content that produces optimal results. Glutamate dehydrogenase (EC 1.4.1.2) is an enzyme that catalyses the reversible deamination of the glutamate into a-ketoglutarate and requires NAD+ (or NADP+) or NADH (or NADPH) as cofactor, depending on the direction of the reaction. Therefore, it represents an important link between the metabolism of carbon and of nitrogen. According to Steele et al. (1971), glycerol in the diet can spare glucogenic amino acids by inhibiting the activity of enzymes, such as glutamate dehydrogenase, thereby promoting protein deposition in muscle tissues. In the case of protein deposition in broilers, it is important to consider that the meat from the breast represents approximately 30% of the total meat in the carcass of this bird, and it also accounts for approximately 50% of the total protein in the edible carcass (Summers et al., 1988). Thus, two experiments were conducted to evaluate the effects of different sources and concentrations of glycerine in the diet on the activity of hepatic glutamate dehydrogenase, performance and the protein content in the breast of broilers. One experiment was conducted when birds were 22–35 days of age and the other during the period from 33 to 42 days of age.
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other. Broilers were evaluated in the period from 22 to 35 days of age (experiment I) or 33 to 42 days of age (experiment II). All procedures were approved by the Committee of Bioethics of the Federal University of Lavras. Experimental procedures, birds, diets and performance measurements
Two experiments were conducted in the aviculture sector of the Department of Animal Science of the Federal University of Lavras to evaluate the effects of different sources and levels of glycerine in the diet on the activity of hepatic glutamate dehydrogenase, performance and the protein content in the breast of broilers. The experiments were conducted separately to avoid residual effects of one experiment on the
For experiment I, a total of 1144 male broiler chickens of the Cobb-500â strain were acquired at 1 day of age and reared in a conventional shed for broilers, where they received a basal diet until 21 days of age that was formulated to meet the requirements of the age, sex and strain of birds (Rostagno et al., 2005). For experiment II, 1300 male chickens of the same strain were reared from the 1st to the 32nd day of age and received a basal diet to meet their nutritional requirements (Rostagno et al., 2005). At the beginning of each experimental phase (22nd or 33rd day of age), the birds were weighed individually, separated by weight ranges and housed in masonry sheds such that each experimental unit (3-m2 pens that were covered with wood shavings) showed an initial similar mean weight. The number of birds per pen in the experiments I and II was 22 and 25 broilers respectively. Birds had free access to feed and water; each pen had a tubular feeder and a pendular drinking system. The experimental design of both experiments was completely randomized and used a 3 9 4 + 1 factorial arrangement; three types of glycerine were supplemented in the diet at four concentrations, which, together with a control diet without glycerine, totalled 13 treatments. These treatments were administered to four replicates of 22 birds (experiment I) or 25 birds (experiment II) per experimental unit. Three sources of glycerine were evaluated: crude from soybean oil (CGSO), mixed crude glycerine from frying oil and lard (MCG) and a semipurified glycerine from soybean oil (SPGSO). The chemical compositions of the types of glycerine are displayed in Table 1. In each experiment, concentrations of 17.5, 35.0, 52.5 and 70.0 g of each type of glycerine/kg of diet were used. The composition of the experimental diets utilized in experiments I and II is presented in Tables 2 and 3 respectively. Diets contained the same amount of nutrients and were formulated to meet the nutritional requirements of birds during each phase, based on the recommendations of Rostagno et al. (2005). Because of the elevated concentration of sodium in the types of glycerine utilized, the inclusion of salt in the diet was adjusted to maintain sodium and chloride levels at the nutritionally recommended
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Table 1 Chemical composition of the different glycerine types*
Parameter
Crude glycerine from soybean oil
Mixed glycerine†
Semipurified glycerine from soybean oil‡
Moisture and volatiles (g/kg) Karl Fischer moisture (g/kg) Glycerol (g/kg) Gross energy (MJ/kg) Crude protein (g/kg) Methanol§ Sodium (g/kg) Total phosphorus (g/kg) Potassium (g/kg) pH in aqueous solution
167.5 124.5 700.0 15.33 0.3 181.3 23.8 0.0 0.6 6.05
554.4 389.5 99.2 17.23 2.3 111.9 15.1 0.0 0.4 9.85
110.8 101.5 793.1 15.48 0.4 20.6 21.6 0.3 0.9 5.72
*Chemical analyses performed by CBO laboratory analyses, Campinas/ S~ao Paulo, Brazil. †Mixed glycerine from frying oil and lard. stria, ‡Semipurified glycerine from soybean oil (GENPAâ, Granol Indu rcio e Exportacß~ao S/A). Come §Units are mg/l for the glycerine from crude soybean and semipurified sources and mg/kg for the mixed glycerine.
levels for each phase by adding calcium chloride and sodium bicarbonate while maintaining the same electrolytic balance in each experimental diet. For the formulation of diets, the mean values of apparent metabolizable energy corrected for the retained nitrogen (AMEn) that had been previously determined by Lima et al. (2012) for each evaluated glycerine were utilized: 13.73 MJ AMEn/kg CGSO, 13.51 MJ AMEn/ kg MCG and 13.83 MJ AMEn/kg SPGSO. The feed intake and weight gain of the broilers were measured in the period from 22 to 35 (experiment I) and 33 to 42 days of age (experiment II). Moreover, at 35 and 42 days of age (experiments I and II, respectively), two birds from each replicate (totalling 104 chickens in each experiment) were slaughtered by cervical dislocation, and the liver and breast were utilized to determine hepatic glutamate dehydrogenase activity and the crude breast protein content. Chemical analyses Preparation of the liver extract and determination of glutamate dehydrogenase activity
After slaughter, the liver of birds was collected and immediately frozen in liquid nitrogen. For the extraction of glutamate dehydrogenase, 1.5 g of frozen liver was macerated with 3 ml Tris–HCl 0.05 M buffer, pH 7.6. After this tissue homogenization, the sample was centrifuged (15 000 g for 20 min at 5 °C), and the supernatant was collected. The determination of enzymatic activity in the supernatant was performed using Journal of Animal Physiology and Animal Nutrition © 2013 Blackwell Verlag GmbH
a commercial kit (Abcam, Code: ab102527, Cambridge, UK). The enzymatic assay was conducted based on the action of glutamate dehydrogenase on glutamate resulting in the stoichiometric generation of NADH, which can than react with one colour reagent, thereby generating a characteristic colour that can be quantified at 450 nm (PowerWaveTM XS Microplate Scanning Spectrophotometer; Bio-Tek Instruments, Potton, UK). The volumetric activity was calculated from a linear standard curve of NADH. Moreover, a blank sample was prepared in parallel to each sample by replacing the substrate with buffer. Protein content in the samples (mg protein/mL liver extract) was determined according to Bradford method (1976) that was performed by the dye-binding assay and utilizing bovine serum albumin as a standard. The specific activity of glutamate dehydrogenase (U/mg of protein) was calculated by dividing volumetric activity by protein content in the sample, so that U is the quantity of enzyme that generates 1 nmol NADH during 1 min of reaction performed at 37 °C in a solution of 2 M glutamate, pH 7.6. Determination of the crude protein content in the breast
Following slaughter, the chicken breast was collected, and an aliquot of 100.0 g was removed and lyophilized to a constant weight. The crude protein content in the breast was then determined according to method 988.05 (N 9 6.25) of Association of Official Analytical Chemists (AOAC) (1990), and the results were expressed in g crude protein/100 g dry matter. Statistical analysis
For each experiment, the data were submitted to variance analysis using the general linear model in SAS statistical software (2004). When the models were statistically significant, the control diet was compared with the other experimental diets using the Dunnett’s test at 5% probability. The sources of glycerine were compared with each other using the Student– Newman–Keuls mean comparison test at 5% probability. Polynomial regression models (p < 0.05) were utilized to evaluate the effect of the increasing concentration of glycerine on glutamate dehydrogenase activity and on crude protein content in the breast. Results Experiment I (22–35 days of age)
There was an interaction (p < 0.05) between the source and level of glycerine in the diet on the enzymatic activity of hepatic glutamate dehydrogenase 561
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Table 2 Ingredient and calculated nutritional composition of the diets (g/kg as fed basis) during the period from 22 to 35 days of age (experiment I)
Ingredient
Control
Crude glycerine from soybean oil
Mixed glycerine*
17.5
35.0
Corn 617.39 597.48 577.55 Soybean meal 316.02 319.63 323.24 Soybean oil 29.53 29.91 30.29 Glycerine 0.00 17.5 35.00 Dicalcium phosphate 16.47 16.52 16.57 Limestone 8.36 8.32 8.28 Common salt 2.24 2.25 2.26 DL-Methionine 2.23 2.25 2.28 L-Lysine HCl 1.75 1.68 1.62 L-Threonine 0.36 0.35 0.35 Calcium chloride 0.00 0.00 0.00 Sodium bicarbonate 3.61 2.06 0.51 Lasalocid 0.60 0.60 0.60 Mineral supplement‡ 0.50 0.50 0.50 Vitamin supplement§ 0.30 0.30 0.30 Choline chloride 0.40 0.40 0.40 Zinc bacitracin 0.25 0.25 0.25 Calculated nutrient composition (g/kg as fed basis) ME (MJ/kg) 12.98 12.98 12.98 Crude protein 197.27 197.27 197.27 Glycerol¶ 0.00 12.25 24.5 Calcium 8.24 8.24 8.24 Available phosphorus 4.11 4.11 4.11 Sodium 20.50 20.50 20.50 Chlorine 1.80 1.80 1.80 Lysine 10.73 10.73 10.73 Methionine + cystine 77.30 77.30 77.30 Threonine 69.70 69.70 69.70 D.E.B (mEq/kg)** 231 231 231
Semipurified glycerine†
52.5
70.0
17.5
35.0
52.5
70.0
17.5
35.0
52.5
70.0
556.37 327.08 31.09 52.50 16.63 7.61 1.57 2.31 1.55 0.35 0.89 0.00 0.60 0.50 0.30 0.40 0.25
534.60 331.03 32.11 70.0 16.68 6.63 0.53 2.33 1.48 0.34 2.22 0.00 0.60 0.50 0.30 0.40 0.25
596.23 319.85 30.36 17.50 16.53 8.32 2.25 2.25 1.68 0.35 0.00 2.63 0.60 0.50 0.30 0.40 0.25
575.06 323.69 31.19 35.00 16.58 8.28 2.27 2.28 1.61 0.35 0.00 1.64 0.60 0.50 0.30 0.40 0.25
553.90 327.53 32.01 52.50 16.64 8.24 2.28 2.31 1.54 00.35 0.00 0.65 0.60 0.50 0.30 0.40 0.25
532.34 331.44 32.98 70.00 16.69 7.99 2.07 2.34 1.47 0.34 0.29 0.00 0.60 0.50 0.30 0.40 0.25
597.44 319.64 29.80 17.50 16.52 8.32 2.25 2.25 1.68 0.35 0.00 2.20 0.60 0.50 0.30 0.40 0.25
577.47 323.26 30.06 35.00 16.57 8.28 2.26 2.28 1.62 0.35 0.00 0.80 0.60 0.50 0.30 0.40 0.25
556.77 327.01 30.58 52.50 16.63 7.87 1.86 2.30 1.55 0.35 0.53 0.00 0.60 0.50 0.30 0.40 0.25
535.13 330.94 31.42 70.00 16.68 6.98 0.92 2.33 1.48 0.34 1.73 0.00 0.60 0.50 0.30 0.40 0.25
12.98 197.27 36.75 8.24 4.11 20.50 1.80 10.73 77.30 69.70 231
12.98 197.27 49.00 8.24 4.11 20.50 1.80 10.73 77.30 69.70 231
12.98 197.27 1.74 8.24 4.11 20.50 1.80 10.73 77.30 69.70 231
12.98 197.27 3.47 8.24 4.11 20.50 1.80 10.73 77.30 69.70 231
12.98 197.27 5.21 8.24 4.11 20.50 1.80 10.73 77.30 69.70 231
12.98 197.27 6.94 8.24 4.11 20.50 1.80 10.73 77.30 69.70 231
12.98 197.27 13.88 8.24 4.11 20.50 1.80 10.73 77.30 69.70 231
12.98 197.27 27.76 8.24 4.11 20.50 1.80 10.73 77.30 69.70 231
12.98 197.27 41.64 8.24 4.11 20.50 1.80 10.73 77.30 69.70 231
12.98 197.27 55.52 8.24 4.11 20.50 1.80 10.73 77.30 69.70 231
*Crude glycerine from frying oil and lard. rcio e Exportacß~ao S/A). stria, Come †Semipurified glycerine from soybean oil (GENPAâ, Granol Indu ‡Supplied per kg of the diet: Zn, 55 mg; Se, 0.18 mg; I, 0.70 mg; Cu, 10 mg; Mn, 78 mg; Fe, 48 mg. §Supplied per kg of the diet: folic acid, 0.48 mg; pantothenic acid, 8.7 mg; biotin, 0.018 mg; butylated hydroxytoluene (BHT), 1.5 mg; niacin, 11.1 mg; vitamin A, 6000 IU; vitamin B1, 0.9 mg; vitamin E, 12.15 IU; vitamin B12, 8.1 lg; vitamin B2, 3.6 mg; vitamin B6, 1.8 mg; vitamin D3, 1,500 IU; vitamin K3, 1.44 mg. ¶Glycerol from glycerine supplementation. **Dietary electrolyte balance (D.E.B.) calculated using the equation proposed by Mongin (1981), which correlates the calculated concentrations of sodium, potassium and chloride (Na+ + K+ Cl). ME: metabolizable energy.
(Table 4). Increases in mixed crude glycerine (MCG) caused a linear reduction in the activity of the enzyme (y = 0.1235x + 16.880; R2 = 0.90), whereas for the semipurified glycerine from soybean oil (SPGSO), glutamate dehydrogenase showed up to a 42.5% increase in activity (y = 0.0970x + 9.475; R2 = 0.97) with increasing glycerine. However, the concentration of crude glycerine from soybean oil (CGSO) did not affect the activity of glutamate dehydrogenase. Moreover, 70.0 g glycerine/kg of diet, derived from CGSO and SPGSO, induced the greatest activity of glutamate dehydrogenase, whereas in the case of MCG, this dose induced the lowest activity. The glutamate dehydroge-
nase activity in the liver of birds that received different concentrations and sources of glycerine did not differ (p > 0.05) from the activity of birds fed the control diet. There was no interaction (p > 0.05) between the source or concentration of glycerine in the diet and the protein content in the breast of broilers (Table 4). However, there was an isolated effect of the source and concentration of glycerine for this parameter (p < 0.05). The highest mean content of protein deposition in the breast (70.3 g/100 g dry matter) was found in birds fed the diet containing SPGSO, whereas the lowest protein content (66.6 g/100 g dry matter)
562
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Glutamate dehydrogenase and protein content in broilers
Table 3 Ingredient and calculated nutritional composition of the diets (g/kg as fed basis) during the period from 33 to 42 days of age (experiment II)
Ingredient
Control
Crude glycerine from soybean oil
Mixed glycerine*
17.5
35.0
Corn 647.53 627.33 607.14 Soybean meal 288.27 291.93 295.60 Soybean oil 30.70 31.30 31.90 Glycerine 0.00 17.50 35.00 Dicalcium phosphate 14.94 14.99 15.04 Limestone 7.92 7.88 7.84 Common salt 2.07 2.08 2.10 DL-Methionine 2.02 2.05 2.07 L-Lysine HCl 1.85 1.79 1.72 L-Threonine 0.35 0.35 0.34 Calcium chloride 0.00 0.00 0.00 Sodium bicarbonate 3.45 1.90 0.35 Mineral supplement‡ 0.50 0.50 0.50 Vitamin supplement§ 0.20 0.20 0.20 Choline chloride 0.20 0.20 0.20 Calculated nutrient composition (g/kg as fed basis) ME (MJ/kg) 13.19 13.19 13.19 Crude protein 187.15 187.15 187.15 Glycerol¶ 0.00 12.25 24.5 Calcium 7.63 7.63 7.63 Available phosphorus 3.80 3.80 3.80 Sodium 1.94 1.94 1.94 Chloride 1.70 1.70 1.70 Lysine 10.17 10.17 10.17 Methionine + cystine 7.32 7.32 7.32 Threonine 6.61 6.61 6.61 D.E.B. (mEq/kg)** 219 219 219
Semipurified glycerine†
52.5
70.0
17.5
35.0
52.5
70.0
17.5
35.0
52.5
70.0
585.52 299.52 32.99 52.50 15.09 7.07 1.29 2.10 1.65 0.34 1.03 0.00 0.50 0.20 0.20
563.44 303.52 34.24 70.00 15.15 6.09 0.25 2.13 1.58 0.34 2.36 0.00 0.50 0.20 0.20
625.21 292.32 32.48 17.50 14.99 7.87 2.08 2.05 1.78 0.35 0.00 2.47 0.50 0.20 0.20
602.87 296.37 34.27 35.00 15.05 7.83 2.10 2.08 1.71 0.34 0.00 1.48 0.50 0.20 0.20
580.54 300.42 36.06 52.50 15.11 7.78 2.11 2.11 1.64 0.34 0.00 0.49 0.50 0.20 0.20
557.60 304.58 38.05 70.00 15.17 7.44 1.79 2.14 1.56 0.34 0.43 0.00 0.50 0.20 0.20
627.94 291.82 30.66 17.50 14.99 7.88 2.08 2.05 1.79 0.35 0.00 2.04 0.50 0.20 0.20
608.34 295.38 30.63 35.00 15.04 7.84 2.09 2.07 1.73 0.34 0.00 0.64 0.50 0.20 0.20
587.83 299.10 30.91 52.50 15.09 7.33 1.58 2.10 1.66 0.34 0.66 0.00 0.50 0.20 0.20
566.53 302.96 31.46 70.00 15.14 6.44 0.64 2.13 1.59 0.34 1.87 0.00 0.50 0.20 0.20
13.19 187.15 36.75 7.63 3.80 1.94 1.70 10.17 7.32 6.61 219
13.19 187.15 49.00 7.63 3.80 1.94 1.70 10.17 7.32 6.61 219
13.19 187.15 1.74 7.63 3.80 1.94 1.70 10.17 7.32 6.61 219
13.19 187.15 3.47 7.63 3.80 1.94 1.70 10.17 7.32 6.61 219
13.19 187.15 5.21 7.63 3.80 1.94 1.70 10.17 7.32 6.61 219
13.19 187.15 6.94 7.63 3.80 1.94 1.70 10.17 7.32 6.61 219
13.19 187.15 13.88 7.63 3.80 1.94 1.70 10.17 7.32 6.61 219
13.19 187.15 27.76 7.63 3.80 1.94 1.70 10.17 7.32 6.61 219
13.19 187.15 41.64 7.63 3.80 1.94 1.70 10.17 7.32 6.61 219
13.19 187.15 55.52 7.63 3.80 1.94 1.70 10.17 7.32 6.61 219
*Crude glycerine from frying oil and lard. rcio e Exportacß~ao S/A). stria, Come †Semipurified glycerine from soybean oil (GENPAâ, Granol Indu ‡Supplied per kg of the diet: Zn, 55 mg; Se, 0.18 mg; I, 0.70 mg; Cu, 10 mg; Mn, 78 mg; Fe, 48 mg. §Supplied per kg of the diet: folic acid, 0.32 mg; pantothenic acid, 5.8 mg; biotin, 0.012 mg; butylated hydroxytoluene (BHT), 1.0 mg; niacin, 7.4 mg; vitamin A, 4000 IU; vitamin B1, 0.6 mg; vitamin E, 8.1 IU; vitamin B12, 5.4 lg; vitamin B2, 2.4 mg; vitamin B6, 1.2 mg; vitamin D3, 1000 IU; vitamin K3, 0.96 mg. ¶Glycerol from glycerine supplementation. **Dietary electrolyte balance (D.E.B.) calculated using the equation proposed by Mongin (1981), which correlates the calculated concentrations of sodium, potassium and chloride (Na+ + K+ Cl). ME: metabolizable energy.
was observed in birds fed the other types of glycerine (CGSO and MCG). However, regardless of the source utilized, there was a quadratic effect of the glycerine concentration on the protein content in the breast of broilers (y = 0.0036x2 + 0.3966x + 58.7; R2 = 0.99). A maximum protein deposition of 69.62% was estimated for a diet containing 55.08 g glycerine/kg feed. The use of 52.5 and 70.0 g SPGSO/kg of diet promoted a greater (p < 0.05) percentage deposition of protein in the breast of broilers when compared with the content determined in birds that received the control diet without glycerine. There was no interaction or isolated effects (p > 0.05) between the sources and levels of glycerine on the feed intake, and so an overall intake of
2.26 kg/bird was observed in the period from 22 to 35 days of age (Table 5). Moreover, inclusion of MCG at the concentrations of 35.0, 52.5 and 70.0 g/kg resulted in higher (p < 0.05) feed intake than the recorded for broilers fed the control diet. There was an interaction (p < 0.05) between the source and level of glycerine in the diet on the weight gain of broilers (Table 5). When glycerine was included at concentration of 35.0 g/kg, the CGSO provided the highest weight gain (1.45 kg/bird), and SPGSO, the lowest value (1.34 kg/bird), whereas MCG resulted in intermediate weight gain (1.43 kg/ bird). However, for the concentration of inclusion of glycerine of 70.0 g/kg, the highest weight gain (1.47 kg/bird) was obtained with the use of MCG in
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Glutamate dehydrogenase and protein content in broilers
Table 4 Glutamate dehydrogenase activity and protein content in the breast of broilers fed different sources of glycerine during the period from 22 to 35 days of age (experiment I) Glutamate dehydrogenase activity (U/mg protein)
Crude protein in the breast (g/100 g dry matter)
Glycerine content (g/kg)
Glycerine content (g/kg)
Source of glycerine
17.5
35.0
52.5
70.0
Mean
17.5
35.0
52.5
70.0
Mean
Crude glycerine from soybean oil Mixed glycerine*,† Semipurified glycerine‡,§ Mean¶ Control diet Coefficient of variation (%) p-value SG 9 LIG SG LIG
12.81 15.18 11.27 13.09
11.90 11.40 12.47 11.92
12.00 11.33 15.07 12.80
11.96a 8.00b 16.06a 12.01
12.17 11.48 13.72
64.3 61.2 68.3 64.6
67.6 66.5 69.8 68.0
67.8 69.3 72.4** 69.8
66.6 69.0 70.9** 68.8
66.6b 66.5b 70.3a
10.49
57.7
12.63
6.85
p < 0.05 p > 0.05 p > 0.05
p > 0.05 p < 0.05 p < 0.05
SG, source of glycerine; LIG, level of inclusion of glycerine. Means with different letters in the same column are significantly different from each other (p < 0.05, determined using the Student–Newman–Keuls test). Each value represents the mean obtained using two birds that were slaughtered for each one of the four replicates performed. *Mixed glycerine from frying oil and lard. †Linear effect of mixed glycerine content on the activity of glutamate dehydrogenase (y = 0.1235x + 16.880; R2 = 0.90). rcio e Exportacß~ao S/A). stria, Come ‡Semipurified glycerine from soybean oil (GENPAâ, Granol Indu §Linear effect of semipurified glycerine on the activity of glutamate dehydrogenase (y = 0.0970x + 9.475; R2 = 0.97). ¶Quadratic effect of glycerine on the crude protein content in the breast (y = 0.0036x2 + 0.3966x + 58.7; R2 = 0.99). **Significantly different from the control using the Dunnett’s test (p < 0.05).
Table 5 Feed intake and weight gain of broilers fed different sources of glycerine during the period from 22 to 35 days of age (experiment I) Feed intake (kg/bird)
Weight gain (kg/bird)
Glycerine content (g/kg)
Glycerine content (g/kg)
Source of glycerine
17.5
35.0
52.5
70.0
Mean
17.5
35.0
52.5
70.0
Mean
Crude glycerine from soybean oil† Mixed glycerine* Semipurified glycerine Mean Control diet Coefficient of variation (%) p-value SG 9 LIG SG LIG
2.21 2.26 2.22 2.23
2.25 2.30** 2.24 2.26
2.26 2.31** 2.24 2.27
2.25 2.35** 2.26 2.29
2.24 2.31 2.24
1.41 1.42 1.39 1.41
1.45a 1.43a,b 1.34b 1.43
1.45 1.41 1.41 1.42
1.26**b 1.47a 1.44b 1.39
1.39 1.43 1.39
2.19
1.43
3.71
5.49
p > 0.05 p > 0.05 p > 0.05
p < 0.05 p > 0.05 p > 0.05
SG: source of glycerine, LIG: level of inclusion of glycerine. Means with different letters in the same column are significantly different from each other (p < 0.05, determined using the Student–Newman–Keuls test). Each value represents the mean obtained using two birds that were slaughtered for each one of the four replicates performed. *Mixed glycerine from frying oil and lard. †Quadratic effect of crude glycerine from soybean oil on the weight gain (y = 0.0193x2 + 0.1423x + 1.2119; R2 = 0.95). **Significantly different from the control using the Dunnett’s test (p < 0.05).
the diet. Moreover, the increase in the concentration of CGSO caused a quadratic effect on the weight gain (y = 0.0193x2 + 0.1423x + 1.2119; R2 = 0.95). The inclusion of 70.0 g of CGSO/kg of diet worsened (p < 0.05) weight gain when compared with the broilers fed the control diet. 564
Experiment II (33–42 days of age)
There was an interaction (p < 0.05) between the source and concentration of glycerine in the diet on the enzymatic activity of hepatic glutamate dehydrogenase (Table 6). Increasing concentrations of MCG Journal of Animal Physiology and Animal Nutrition © 2013 Blackwell Verlag GmbH
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Glutamate dehydrogenase and protein content in broilers
Table 6 Glutamate dehydrogenase activity and protein content in the breast of broilers fed different sources of glycerine during the period from 33 to 42 days of age (experiment II) Glutamate dehydrogenase activity (U/mg protein)
Crude protein in the breast (g/100 g dry matter)
Glycerine content (g/kg)
Glycerine content (g/kg)
Source of glycerine
17.5
35.0
52.5
70.0
Mean
17.5
35.0
52.5
70.0
Mean
Crude glycerine from soybean oil Mixed glycerine*,† Semipurified glycerine‡,§ Mean Control diet Coefficient of variation (%) p-value SG 9 LIG SG LIG
11.66 11.37 10.38 11.14
11.56 8.57 11.72 10.62
11.34a, b 7.60b 14.71a 11.22
11.45b 7.42c 16.52a 11.80
11.50a 8.74b 13.33a
81.45 79.69 80.59 80.57
81.82 80.22 81.46 81.17
82.13 80.63 82.40 81.72
82.22 81.66 84.35 82.74
81.90 80.55 82.20
11.76
82.66
11.99
3.76
p < 0.05 p < 0.05 p > 0.05
p > 0.05 p > 0.05 p > 0.05
SG, source of glycerine; LIG, level of inclusion of glycerine. Means with different letters in the same column are significantly different from each other (p < 0.05, determined using the Student–Newman–Keuls test). Each value represents the mean obtained using two birds that were slaughtered for each one of the four replicates performed. *Mixed glycerine from frying oil and lard. †Linear effect of mixed glycerine content on the activity of glutamate dehydrogenase (y = 0.0733x + 11.945; R2 = 0.82). rcio e Exportacß~ao S/A). stria, Come ‡Semipurified glycerine from soybean oil (GENPAâ, Granol Indu §Linear effect of semipurified glycerine on the activity of glutamate dehydrogenase (y = 0.1223x + 7.980; R2 = 0.98).
were inversely correlated with the activity of the enzyme (y = 0.0733x + 11.945; R2 = 0.82) and caused up to a 34.74% reduction in activity, whereas for SPGSO, the glutamate dehydrogenase activity increased linearly (y = 0.1223x + 7.980; R2 = 0.98) by up to 59.15%. However, the concentration of CGSO did not affect the glutamate dehydrogenase activity. Furthermore, at 52.5 and 70.0 g glycerine/kg of diet, the use of SPGSO promoted the greatest activity of glutamate dehydrogenase, but with MCG, these doses induced lower activity. The glutamate dehydrogenase activity in the liver of birds receiving different concentrations and sources of glycerine did not differ (p > 0.05) from the activity determined from the liver of birds fed the control diet. There was no interaction (p > 0.05) or isolated effect between the factors (sources and levels of glycerine) on the crude protein content in the breast of broilers, and a mean protein deposition of 81.55 g/ 100 g dry matter (Table 6) was observed. All diets containing glycerine promoted a similar level of protein deposition in the breast to that determined from birds fed the control diet without glycerine. There was an interaction (p < 0.05) between the source and concentration of glycerine in the diet on feed intake and weight gain of broilers (Table 7). Inclusion of 35.0 g of SPGSO/kg provided the highest feed intake (2.03 kg/bird), and the MCG, the lowest (1.88 kg/bird), whereas CGSO resulted in intermedi-
ate intake (1.94 kg/bird). For the weight gain, however, the lowest values were recorded for the birds fed diet containing 17.5 g of MCG or SPGSO/kg. Moreover, the increase in the concentration of CGSO increased linearly (y = 0.015x + 1.033; R2 = 0.84) the weight gain up to 8.32%.
Journal of Animal Physiology and Animal Nutrition © 2013 Blackwell Verlag GmbH
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Discussion Experiment I (22–35 days of age)
During the period from 22 to 35 days of age, an increase in the glycerine concentration in the diet (and consequent increase in the concentration of glycerol) did not necessarily reduce the activity of hepatic glutamate dehydrogenase. These results opposed the initial hypothesis of this study, which was based on the report by Steele et al. (1971), who suggested that the glycerol present in the diet could spare glucogenic amino acids by reducing the activity of this enzyme, thereby enabling protein deposition in the muscle tissues of the bird. We did observe enzymatic inhibition with increasing concentrations of the MCG; however, against expectation, the increase in the concentration of semipurified glycerine from soybean oil (SPGSO) did not inhibit the enzyme, which was unexpected because this glycerine showed the highest percentage content of glycerol. Nevertheless, it is important to note that glutamate dehydrogenase converts glutamate into a-ketoglutarate and vice versa, thus
V. M. P. Bernardino et al.
Glutamate dehydrogenase and protein content in broilers
Table 7 Feed intake and weight gain of broilers fed different sources of glycerine during the period from 33 to 42 days of age (experiment II) Feed intake (kg/bird)
Weight gain (kg/bird)
Glycerine content (g/kg)
Glycerine content (g/kg)
Source of glycerine
17.5
35.0
52.5
70.0
Mean
17.5
35.0
52.5
70.0
Mean
Crude glycerine from soybean oil† Mixed glycerine* Semipurified glycerine Mean Control diet Coefficient of variation (%) p-value SG 9 LIG SG LIG
1.94 1.95 2.05 1.98
1.94a, b 1.88b 2.03a 1.95
1.92 1.88 1.90 1.90
1.97 1.94 1.85 1.92
1.94 1.91 1.96
1.05b 1.12a 1.13a 1.10
1.11 1.09 1.10 1.10
1.12 1.10 1.12 1.11
1.13 1.10 1.08 1.11
1.10 1.10 1.11
1.92
1.09
4.57
3.91
p < 0.05 p > 0.05 p > 0.05
p < 0.05 p > 0.05 p > 0.05
SG, source of glycerine; LIG, level of inclusion of glycerine. Means with different letters in the same column are significantly different from each other (p < 0.05, determined using the Student–Newman–Keuls test). Each value represents the mean obtained using two birds that were slaughtered for each one of the four replicates performed. *Mixed glycerine from frying oil and lard. †Linear effect of crude glycerine from soybean oil on the weight gain (y = 0.0150x + 1.0330; R2 = 0.84). **Significantly different from the control using the Dunnett’s test (p < 0.05).
representing an important link between the metabolisms of carbon and nitrogen. Thus, it acts both on the degradation and on the synthesis of amino acids, and the direction of the reaction depends on the physiological condition of the animal (Champe et al., 2009). The adverse effects of the level of glycerine in the diet on the glutamate dehydrogenase activity that was observed in this study may be related to the glycerol content of the diets. Although the feed intake by broilers did not have been influenced by the inclusion level of glycerine in the diet, when SPGSO was utilized, the diets contained higher levels of glycerol than those calculated for the MCG diet (between 13.88 and 55.52 g of glycerol/kg of diet supplemented with SPGSO and between 1.74 and 6.94 g of glycerol/kg of diet supplemented with MCG). Thus, in the case of MCG, because the percentage glycerol content in this glycerine was lower, the largest portion of the glycerol from this source might have been utilized metabolically for the production of energy via glycolysis and the Krebs cycle, thus inhibiting glutamate dehydrogenase activity. This inhibition reduced the degradation of glucogenic amino acids, which were elevated because the diet was nutritionally adequate for broilers. In the diet containing SPGSO, the glycerol may have been converted into an intermediate of the glycolytic pathway being converted into pyruvate, which in turn may have been primarily utilized for the synthesis of a-ketoglutarate via several sequential biochemical reactions. a-ketoglutarate could then have been used as a substrate of glutamate dehydrogenase
in the synthesis of amino acids, as can be verified by the higher crude protein contents in the breast muscle of the broilers fed diets containing the SPGSO. Therefore, the up-regulation in catalytic activity of glutamate dehydrogenase could have been due to the increase in cellular concentration of a-ketoglutarate due to the higher content of glycerol in the diet. However, in this study, it was verified that glutamate dehydrogenase activity is regulated not only by the level of glycerol in the diet, but also by the source used as raw material for the production of biodiesel, because at 70.0 g glycerine/kg of diet, the use of CGSO and SPGSO increased the activity of glutamate dehydrogenase, whereas the MCG reduced enzymatic activity by up to 50%, showing that there is an interaction between the source and content of glycerine in the diet. The use of the SPGSO induced approximately 3.7% points more protein deposition in the breast than that of the other types of glycerine, possibly due to the higher catalytic activity of glutamate dehydrogenase and also the best quality of the SPGSO. Although the semipurification manner from SPGSO is not well described in the literature for this commercial product, this processing may have reduced the concentration of some residue that worsens the protein deposition, associated with their greater content of glycerol. Regardless of the source utilized, there was a quadratic effect of the concentration of glycerine in the diet on the percentage of protein in the breast of broilers. However, the protein deposition was not strictly correlated with glutamate dehydrogenase activity.
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The use of 52.5 and 70.0 g SPGSO/kg of diet induced a greater (p < 0.05) percentage of protein in the breast of broilers when compared with the content in birds that received the control diet without glycerine. These data are to some degree similar to those of Cerrate et al. (2006), who verified that breast yield was greater in birds fed diets containing 25 or 50 g of glycerine/kg of diet when compared with birds that received the control diet without glycerine. Based on the study by Steele et al. (1971), glycerol in the diet can spare glucogenic amino acids by reducing the activity of glutamate dehydrogenase, thus enabling protein deposition in the muscle tissues. However, according to Cryer and Bartley (1973), the sparing effect on glucogenic amino acids may also be due to the inhibition of another key enzyme of gluconeogenesis/ phosphoenolpyruvate carboxykinase (EC 4.1.1.32), which catalyses the conversion of oxaloacetate and GTP to phosphoenolpyruvate and GDP and CO2 (Champe et al., 2009). Therefore, to obtain more data on the relationship between the source and concentration of glycerine supplementation in the diet and the protein content in the breast of broilers, it is recommended that the activity of phosphoenolpyruvate carboxykinase be determined in future experiments. The lowest weight gain observed with the inclusion of 70.0 g of CGSO/kg of diet might probably be related to the higher methanol content present in this glycerine, associated with the influence of birds age and the more extensive rearing period evaluated (13 days). According to Macari et al. (2002), broilers from 22 to 35 days of age have still a low capacity to metabolize the methanol. Similarly, Cerrate et al. (2006) reported worse in the weight gain of birds fed diets containing high inclusion levels of CGSO, during the period from 1 to 35 days of age.
Glutamate dehydrogenase and protein content in broilers
reported by Ribeiro et al. (1995) (143.7 U/mg protein). However, Ribeiro et al. (1995) evaluated the effects of different concentrations of glutamine, alanine and proline in the diet. Therefore, the high levels determined by Ribeiro et al. (1995) may have been due to the conversion of the amino acids into glutamate, the levels of which then increase in the liver, thus requiring increased glutamate dehydrogenase activity to meet the requirement for transamination or oxidative deamination, that is, at the level of the degradation of glutamate to a-ketoglutarate and NHþ 4 . Contrary to that observed for the previous rearing period (experiment I), the different sources and concentrations of glycerine did not affect the protein content determined in the breast of broilers in the final rearing phase, demonstrating the influence of age of birds and duration of the rearing period (which in this case was shorter). Moreover, all diets containing glycerine promoted a similar deposition of protein in the breast to that determined from control-fed birds, with an average protein concentration of 81.55 g/100 g dry matter. Contrary to the observed for the previous rearing phase (experiment I), the increase in the concentration of CGSO in the diet improves the weight gain, which appears to demonstrate that in the rearing period from 33 to 42 days of age, the methanol present in the diet formulated with up to 70 g CGSO/kg can be properly metabolized by the bird. However, further specific studies still need to be conducted to evaluate and measure the real metabolic effects of the methanol at the different rearing ages of broilers fed diets containing glycerine. Conclusions
During the period from 33 to 42 days of age, glutamate dehydrogenase activity decreased linearly with increasing MCG and increased linearly with increasing SPGSO in the diet, which is similar to observations in experiment I (from 22 to 35 days of age) and may be due to the same previously discussed causes. Moreover, in this last rearing phase, the source of glycerine also affected glutamate dehydrogenase activity, which was lower when the diet was supplemented with MCG, possibly because of the lower percentage content of glycerol in this glycerine. Overall, glutamate dehydrogenase activities in the livers of birds that received the diet without glycerine (control) in both experiments were lower than those
For both rearing periods evaluated, an increase in glycerine in the diet did not necessarily reduce the activity of hepatic glutamate dehydrogenase; in addition, the deposition of protein in the breast of broilers is not strictly correlated with the activity of this enzyme. During the period from 22 to 35 days of age, the use of semipurified glycerine from soybean oil in the diet increased the protein content in the breast by up to 3.7% points more when compared with the crude glycerine from soybean oil and with the mixed glycerine from frying oil and lard. Moreover, regardless of the source of protein, the inclusion of 55.08 g of glycerine/kg of diet promoted the largest deposition of protein in the breast of broilers. However, during the period from 33 to 42 days of age, the protein content in the breast of broilers is not necessarily affected by
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the source or by the concentration of glycerine in the diet. The feed intake and weight gain also were influenced by age of broilers, demonstrating that when glycerine is used in the diet, the rearing period must be considered to allow the best performance results. The results of this study demonstrate that glycerine has potential to be used as an energetic ingredient in the broiler diets and can maintain or even improve the protein deposition in the breast of broilers. References ANP – National Agency of Petroleum, Natural Gas and Biofuels (Brazil). 2012: Available: http://www.anp.gov.br. Accessed April 14, 2012. Association of Official Analytical Chemists (AOAC), 1990: Official Methods of Analysis, 15th edn. Association of Official Analytical Chemists, Arlington, VA. Bradford, M. M., 1976: A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry 72, 248–254. Cerrate, S.; Yan, F.; Wang, Z.; Coto, C.; Sacakli, P.; Waldroup, P. W., 2006: Evaluation of glycerine from biodiesel production as a feed ingredient for broilers. International Journal of Poultry Science 5, 1001–1007. Champe, P. C.; Harvey, R. A.; Ferrier, D. R., 2009: Biochemistry: Lippincott’s Illustrated Reviews, 4th edn. Artmed, Porto Alegre, 528p. Cryer, A.; Bartley, W., 1973: Studies of the adaptation of rats to a diet high in glycerol. International Journal of Biochemistry 4, 293–308. Dozier, W. A.; Kerr, B. J.; Corzo, A.; Kidd, M. T.; Weber, T. E.; Bregendahl, K., 2008: Apparent metabolizable energy of glycerin for broiler chickens. Poultry Science 87, 317–322. Garcia, A. J. M.; Tookuni, J. P. M., 2006: Biodiesel from animal fat. Biodiesel Magazine BR. Available: http://www. biodieselbr.com/estudos/biodiesel/bio-
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Acknowledgements The authors acknowledge the Foundation for Research Support of Minas Gerais (FAPEMIG), the National Council for Scientific and Technological Development – Brazil (CNPq) and the National Institute of Technology Science of Animal Science (INCTCA) for the financial support, as well as Granol Ind. Exp. S/A for providing the semipurified glycerine from soybean oil.
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