DOI: 10.1111/jpn.12217
ORIGINAL ARTICLE
Metabolic parameters in rats receiving different levels of oral glycerol supplementation K. G. Lisenko1, E. F. Andrade1, R. V. Lobato1, D. R. Orlando1, D. H. C. Damin2, A. C. Costa3, R. R. Lima4, R. R. Alvarenga3, M. G. Zangeronimo1, R. V. Sousa1 and L. J. Pereira1 1 2 3 4
Department of Veterinary Medicine, Federal University of Lavras Lavras, Brazil Department of Food Sciences, Federal University of Lavras Lavras, Brazil Department of Animal Sciences, Federal University of Lavras Lavras, Brazil, and Department of Exact Sciences, Federal University of Lavras Lavras, Brazil
Summary The use of glycerol in the diets for animals is of interest because it is a residue of biodiesel production and rich in energy. Thus, this study aimed to evaluate metabolic and physiological parameters of rats receiving supplemental pure glycerol by gavage. We used 30 Wistar rats (initial weight 202.7 29.98 g) receiving 0 (control/saline), 200, 400, 800 and 1600 mg glycerol/kg of body weight (bidistilled glycerine, 99.85% glycerol) beside food and water ad libitum for 28 days. We used a completely randomised design with five treatments and six replicates. At the end of the experiment, the animals were killed, and the results showed that there was no change (p > 0.05) in the intake and excretion of water, the average daily weight gain, dry matter, ash and crude protein in the carcass or plasma triacylglycerols. There was a beneficial effect (p < 0.05) up to a dose of 800 mg/kg glycerol on feed intake, percentage of carcass fat, plasma levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), high-density lipoprotein (HDLc) and low-/very low-density lipoprotein (LDLc + VLDLc). The levels of total cholesterol and glucose were increased with up to a dose of 800 mg/kg glycerol (but remained within the normal range); they were reduced with the dose of 1600 mg/kg. The total leucocyte count tended to be reduced, although it was within the reference values for rats. There were no renal or pancreatic lesions. In conclusion, glycerol presented as a safe supplement at the studied doses, even having some beneficial effects in a dose-dependent manner in rats. Keywords glycerine, metabolism, animal nutrition, supplementary feeding Correspondence Prof. Dr. L. J. Pereira, DMV – Department of Physiology and Pharmacology, University of Lavras – UFLA, PO Box 3037 – Campus, Lavras, Minas Gerais, Brazil. Tel: +55-35-38295211; Fax: +55-35-38291715; E-mail: [email protected] Received: 20 February 2014; accepted: 27 May 2014
Introduction The steady increase in the global consumption of fuel for engines in industrial, residential and automotive markets has generated a greater demand for energy production (Goldemberg, 2000). At present, the most used energy resources are derived from fossil oil compounds. However, the use of these products has become environmentally and economically unfavourable because they may cause major impacts that are difficult to predict (Crutzen et al., 2008). Thus, new sources of energy are required, creating conditions for biofuel development, production and research. Although biodiesel is considered an alternative form of energy, its production generates waste, such as glycerol, being necessary sustainable measures of reuse of these by-products. In recent years, several
studies have shown the effects of the use of residual glycerol as an energy source in diets of different animal species (Lammers et al., 2008; Berenchtein et al., 2010). Being a product that is rich in energy (approximately 4320 kcal of gross energy per kg of pure glycerol), the use of glycerol in the formulation of animal diets arouses immediate interest (Berenchtein et al., 2010) and has also been used as a regulator of body fluids during rehydration or exercise for horses (Schott et al., 2001) and even humans (Kavouras et al., 2006; Dini et al., 2007). According to the National Petroleum Agency (ANP, 2011) in Brazil, there are 66 plants used for the production of biodiesel, corresponding to a total production of 16 216 m3/day. In addition, pursuant to Resolution No. 6/2009 of the National Energy
Journal of Animal Physiology and Animal Nutrition 99 (2015) 265–272 © 2014 Blackwell Verlag GmbH
265
K. G. Lisenko et al.
Glycerol feeding and rats metabolism
Policy (CNPE), diesel oil sold from the year 2010 onwards throughout the Brazilian territory must contain 5% biodiesel (ANP, 2011). At the same time, Brazil has an annual production of 2.4 billion litres of biodiesel, with an installed capacity for the same year of approximately 5.8 billion litres (ANP, 2011). This production has placed the country among the largest producers and consumers of fuel in the world (ANP, 2011). As the by-products of biodiesel can be used in animal nutrition, this situation raises the need to check the toxicity of this substance, its optimal dose and its advantages as a substitute or supplement in food, as few studies have evaluated these variables. In this sense, the objective of this study was to evaluate the effect of pure glycerol as a feed supplement in order to find an optimal dose that is beneficial for the physiological and metabolic parameters of rats. Materials and methods Location of the experiment, animals and plants
The experiment was conducted in the period between July and August 2012, in the Department of Veterinary Medicine, Federal University of Lavras (UFLA), in Lavras, southern Minas Gerais State, Brazil. The experimental protocol was approved by the Ethics Committee on Animal Use (CEUA – protocol 022/12) of the Federal University of Lavras. We used 30 adult male healthy rats (Rattus norvegicus Albinus, Wistar), weighing 202.7 29.98 g. The animals were placed in individual metabolic cages and subjected to a period of 7 days of acclimatisation. The room was heated to a temperature of 22 2 °C and kept under 12-h/12-h light–dark cycles. A commercial diet and water were given ad libitum during the experimental period. Diets
The basal diet consisted of a commercial feed for rats (Nuvilabâ; Nuvital Feeding, Curitiba, PR, Brazil), containing by weight: 19% protein, 56% carbohydrate, 3.5% fat, 4.5% cellulose, 5.0% vitamins and minerals, 12% humidity and an energy content of 17.03 kJ/g. To supplement the diets, we used pure glycerol (glycerine with double distilled 99.85% of glycerol – ALMAD, Aracßatuba, SP, Brazil) solubilised in water and administered it by gavage in a volume of 0.3 ml per animal. The physicochemical properties of glycerol were determined by the company Mapric (Ipiranga, SP, Brazil): fatty acids and esters, 0.02%; heavy 266
metals, 0.05) of glycerol on water consumption or excretion, or crude protein in the carcass. However, there were significant differences (p < 0.05) in average daily feed intake and percentage of fat in the carcass. As shown in Table 1 and Fig. 2, the values of feed intake and carcass fat content decreased linearly according to the increase in the dose of glycerol (Fig. 2). The analysed plasma parameters showed that the use of glycerol as a supplement did not affect the levels of triglycerides. Regarding the variables, glucose, total cholesterol, HDLc, VLDLc + LDLc, ALT and AST, different glycerol levels in the diet had no effect (Table 2). As shown in Table 2, for the variables plasma glucose and total cholesterol levels, there was a significant quadratic with increasing values until the dose of 800 mg/kg, followed by a decrease with the higher dose. As for the levels of HDLc, there was an increase up to a dose of 800 mg/kg, with a declining trend with the dose of 1600 mg/kg, but levels were still within the standard for the species. For VLDLc + LDLc levels, there was a decrease with increasing glycerol doses, which remained within the standards. The liver enzymes AST and ALT showed a decreasing quadratic behaviour up to the dose of 800 mg/kg of glycerol
Table 1 Performance, water balance and carcass characteristics (crude protein, ether extract, ash and dry matter) of rats receiving different levels of glycerol by gavage for 28 days Glycerol, mg/kg Variable Performance Weight gain (g/day) Feed intake (g/day) Water consumption (ml/day) Urine volume (ml/day) Carcass characteristics Moisture (%) Crude protein (%) Ether extract (%) Ash (%)
p-Value
0
200
400
800
1600
SD
L
Q
RV
2.27 30.90 22.91 5.63
1.92 26.28 21.49 4.52
2.20 27.23 21.84 5.18
2.13 25.39 22.69 7.12
1.95 23.32 22.56 6.53
0.70 5.45 2.46 2.30
0.51 0.04 0.78 0.20
0.77 0.47 0.78 0.62
2.08 0.8* 17.9† 24.9† 7.8†
73.61 25.28 2.64 22.62
70.82 28.54 3.00 25.33
72.04 28.32 2.72 23.91
70.30 27.21 2.55 25.41
71.38 28.11 2.01 24.43
2.67 2.66 0.73 2.32
0.30 0.34 0.04 0.39
0.13 0.35 0.41 0.14
69.2 0.3* 21.4 0.2* 4.1 0.2* –
L, Linear regression; Q, quadratic regression; SD, standard deviation; VR, reference values. *Franco et al. (2007). †Carvalho et al. (2009).
Journal of Animal Physiology and Animal Nutrition © 2014 Blackwell Verlag GmbH
267
K. G. Lisenko et al.
Glycerol feeding and rats metabolism
(b) 3.5
y = –0.0038x + 28.915 R2 = 0.75
30 25 20 15 10
2.5 2.0 1.5 1.0 0.5
5 0
y = –0.0002x + 1.689 R2 = 0.76
3.0 Ether extract, %
Feed intake, g/day
(a) 35
0.0 0
400 800 1200 Glycerol, mg/kg
1600
0
400 800 1200 Glycerol, mg/kg
Fig. 2 Feed intake (a) and percentage of fat in the carcass (b) of rats receiving glycerol by gavage for 28 days.
1600
Table 2 Blood parameters of Wistar rats supplemented with different levels of glycerol by gavage for 28 days Glycerol, mg/kg
p-Value
Variable
0
200
400
800
1600
SD
L
Q
RV
Glucose (mg/dl) Triacylglycerols (mg/dl) Total cholesterol (mg/dl) HDLc (mg/dl) VLDLc + LDLc (mg/dl) ALT (U/L) AST (U/L)
91.00 28.26 95.52 39.44 56.08 47.83 109.9
89.83 27.46 91.00 44.65 46.36 46.30 101.5
95.67 30.12 96.47 55.45 40.47 36.48 75.3
99.00 24.68 107.15 59.53 37.62 40.69 66.7
84.50 27.22 85.29 48.22 37.32 40.44 126.4
9.85 7.67 11.73 10.77 11.91 7.20 33.61
0.28 0.65 0.25 0.08
ORIGINAL ARTICLE
Metabolic parameters in rats receiving different levels of oral glycerol supplementation K. G. Lisenko1, E. F. Andrade1, R. V. Lobato1, D. R. Orlando1, D. H. C. Damin2, A. C. Costa3, R. R. Lima4, R. R. Alvarenga3, M. G. Zangeronimo1, R. V. Sousa1 and L. J. Pereira1 1 2 3 4
Department of Veterinary Medicine, Federal University of Lavras Lavras, Brazil Department of Food Sciences, Federal University of Lavras Lavras, Brazil Department of Animal Sciences, Federal University of Lavras Lavras, Brazil, and Department of Exact Sciences, Federal University of Lavras Lavras, Brazil
Summary The use of glycerol in the diets for animals is of interest because it is a residue of biodiesel production and rich in energy. Thus, this study aimed to evaluate metabolic and physiological parameters of rats receiving supplemental pure glycerol by gavage. We used 30 Wistar rats (initial weight 202.7 29.98 g) receiving 0 (control/saline), 200, 400, 800 and 1600 mg glycerol/kg of body weight (bidistilled glycerine, 99.85% glycerol) beside food and water ad libitum for 28 days. We used a completely randomised design with five treatments and six replicates. At the end of the experiment, the animals were killed, and the results showed that there was no change (p > 0.05) in the intake and excretion of water, the average daily weight gain, dry matter, ash and crude protein in the carcass or plasma triacylglycerols. There was a beneficial effect (p < 0.05) up to a dose of 800 mg/kg glycerol on feed intake, percentage of carcass fat, plasma levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), high-density lipoprotein (HDLc) and low-/very low-density lipoprotein (LDLc + VLDLc). The levels of total cholesterol and glucose were increased with up to a dose of 800 mg/kg glycerol (but remained within the normal range); they were reduced with the dose of 1600 mg/kg. The total leucocyte count tended to be reduced, although it was within the reference values for rats. There were no renal or pancreatic lesions. In conclusion, glycerol presented as a safe supplement at the studied doses, even having some beneficial effects in a dose-dependent manner in rats. Keywords glycerine, metabolism, animal nutrition, supplementary feeding Correspondence Prof. Dr. L. J. Pereira, DMV – Department of Physiology and Pharmacology, University of Lavras – UFLA, PO Box 3037 – Campus, Lavras, Minas Gerais, Brazil. Tel: +55-35-38295211; Fax: +55-35-38291715; E-mail: [email protected] Received: 20 February 2014; accepted: 27 May 2014
Introduction The steady increase in the global consumption of fuel for engines in industrial, residential and automotive markets has generated a greater demand for energy production (Goldemberg, 2000). At present, the most used energy resources are derived from fossil oil compounds. However, the use of these products has become environmentally and economically unfavourable because they may cause major impacts that are difficult to predict (Crutzen et al., 2008). Thus, new sources of energy are required, creating conditions for biofuel development, production and research. Although biodiesel is considered an alternative form of energy, its production generates waste, such as glycerol, being necessary sustainable measures of reuse of these by-products. In recent years, several
studies have shown the effects of the use of residual glycerol as an energy source in diets of different animal species (Lammers et al., 2008; Berenchtein et al., 2010). Being a product that is rich in energy (approximately 4320 kcal of gross energy per kg of pure glycerol), the use of glycerol in the formulation of animal diets arouses immediate interest (Berenchtein et al., 2010) and has also been used as a regulator of body fluids during rehydration or exercise for horses (Schott et al., 2001) and even humans (Kavouras et al., 2006; Dini et al., 2007). According to the National Petroleum Agency (ANP, 2011) in Brazil, there are 66 plants used for the production of biodiesel, corresponding to a total production of 16 216 m3/day. In addition, pursuant to Resolution No. 6/2009 of the National Energy
Journal of Animal Physiology and Animal Nutrition 99 (2015) 265–272 © 2014 Blackwell Verlag GmbH
265
K. G. Lisenko et al.
Glycerol feeding and rats metabolism
Policy (CNPE), diesel oil sold from the year 2010 onwards throughout the Brazilian territory must contain 5% biodiesel (ANP, 2011). At the same time, Brazil has an annual production of 2.4 billion litres of biodiesel, with an installed capacity for the same year of approximately 5.8 billion litres (ANP, 2011). This production has placed the country among the largest producers and consumers of fuel in the world (ANP, 2011). As the by-products of biodiesel can be used in animal nutrition, this situation raises the need to check the toxicity of this substance, its optimal dose and its advantages as a substitute or supplement in food, as few studies have evaluated these variables. In this sense, the objective of this study was to evaluate the effect of pure glycerol as a feed supplement in order to find an optimal dose that is beneficial for the physiological and metabolic parameters of rats. Materials and methods Location of the experiment, animals and plants
The experiment was conducted in the period between July and August 2012, in the Department of Veterinary Medicine, Federal University of Lavras (UFLA), in Lavras, southern Minas Gerais State, Brazil. The experimental protocol was approved by the Ethics Committee on Animal Use (CEUA – protocol 022/12) of the Federal University of Lavras. We used 30 adult male healthy rats (Rattus norvegicus Albinus, Wistar), weighing 202.7 29.98 g. The animals were placed in individual metabolic cages and subjected to a period of 7 days of acclimatisation. The room was heated to a temperature of 22 2 °C and kept under 12-h/12-h light–dark cycles. A commercial diet and water were given ad libitum during the experimental period. Diets
The basal diet consisted of a commercial feed for rats (Nuvilabâ; Nuvital Feeding, Curitiba, PR, Brazil), containing by weight: 19% protein, 56% carbohydrate, 3.5% fat, 4.5% cellulose, 5.0% vitamins and minerals, 12% humidity and an energy content of 17.03 kJ/g. To supplement the diets, we used pure glycerol (glycerine with double distilled 99.85% of glycerol – ALMAD, Aracßatuba, SP, Brazil) solubilised in water and administered it by gavage in a volume of 0.3 ml per animal. The physicochemical properties of glycerol were determined by the company Mapric (Ipiranga, SP, Brazil): fatty acids and esters, 0.02%; heavy 266
metals, 0.05) of glycerol on water consumption or excretion, or crude protein in the carcass. However, there were significant differences (p < 0.05) in average daily feed intake and percentage of fat in the carcass. As shown in Table 1 and Fig. 2, the values of feed intake and carcass fat content decreased linearly according to the increase in the dose of glycerol (Fig. 2). The analysed plasma parameters showed that the use of glycerol as a supplement did not affect the levels of triglycerides. Regarding the variables, glucose, total cholesterol, HDLc, VLDLc + LDLc, ALT and AST, different glycerol levels in the diet had no effect (Table 2). As shown in Table 2, for the variables plasma glucose and total cholesterol levels, there was a significant quadratic with increasing values until the dose of 800 mg/kg, followed by a decrease with the higher dose. As for the levels of HDLc, there was an increase up to a dose of 800 mg/kg, with a declining trend with the dose of 1600 mg/kg, but levels were still within the standard for the species. For VLDLc + LDLc levels, there was a decrease with increasing glycerol doses, which remained within the standards. The liver enzymes AST and ALT showed a decreasing quadratic behaviour up to the dose of 800 mg/kg of glycerol
Table 1 Performance, water balance and carcass characteristics (crude protein, ether extract, ash and dry matter) of rats receiving different levels of glycerol by gavage for 28 days Glycerol, mg/kg Variable Performance Weight gain (g/day) Feed intake (g/day) Water consumption (ml/day) Urine volume (ml/day) Carcass characteristics Moisture (%) Crude protein (%) Ether extract (%) Ash (%)
p-Value
0
200
400
800
1600
SD
L
Q
RV
2.27 30.90 22.91 5.63
1.92 26.28 21.49 4.52
2.20 27.23 21.84 5.18
2.13 25.39 22.69 7.12
1.95 23.32 22.56 6.53
0.70 5.45 2.46 2.30
0.51 0.04 0.78 0.20
0.77 0.47 0.78 0.62
2.08 0.8* 17.9† 24.9† 7.8†
73.61 25.28 2.64 22.62
70.82 28.54 3.00 25.33
72.04 28.32 2.72 23.91
70.30 27.21 2.55 25.41
71.38 28.11 2.01 24.43
2.67 2.66 0.73 2.32
0.30 0.34 0.04 0.39
0.13 0.35 0.41 0.14
69.2 0.3* 21.4 0.2* 4.1 0.2* –
L, Linear regression; Q, quadratic regression; SD, standard deviation; VR, reference values. *Franco et al. (2007). †Carvalho et al. (2009).
Journal of Animal Physiology and Animal Nutrition © 2014 Blackwell Verlag GmbH
267
K. G. Lisenko et al.
Glycerol feeding and rats metabolism
(b) 3.5
y = –0.0038x + 28.915 R2 = 0.75
30 25 20 15 10
2.5 2.0 1.5 1.0 0.5
5 0
y = –0.0002x + 1.689 R2 = 0.76
3.0 Ether extract, %
Feed intake, g/day
(a) 35
0.0 0
400 800 1200 Glycerol, mg/kg
1600
0
400 800 1200 Glycerol, mg/kg
Fig. 2 Feed intake (a) and percentage of fat in the carcass (b) of rats receiving glycerol by gavage for 28 days.
1600
Table 2 Blood parameters of Wistar rats supplemented with different levels of glycerol by gavage for 28 days Glycerol, mg/kg
p-Value
Variable
0
200
400
800
1600
SD
L
Q
RV
Glucose (mg/dl) Triacylglycerols (mg/dl) Total cholesterol (mg/dl) HDLc (mg/dl) VLDLc + LDLc (mg/dl) ALT (U/L) AST (U/L)
91.00 28.26 95.52 39.44 56.08 47.83 109.9
89.83 27.46 91.00 44.65 46.36 46.30 101.5
95.67 30.12 96.47 55.45 40.47 36.48 75.3
99.00 24.68 107.15 59.53 37.62 40.69 66.7
84.50 27.22 85.29 48.22 37.32 40.44 126.4
9.85 7.67 11.73 10.77 11.91 7.20 33.61
0.28 0.65 0.25 0.08
