Effects of dietary L-carnitine supplementation on growth performance and some biochemical parameters in Japanese quails (Coturnix

Effects of dietary L-carnitine supplementation on growth performance and some biochemical parameters in Japanese quails (Coturnix coturnix japonica) S. YALÇIN 1, B. ÖZSOY 2, Ö. CENGİZ* 3, T. BÜLBÜL 4 1 Department of Animal Nutrition, Faculty of Veterinary Medicine, Ankara University, 06110 Ankara, TURKEY. 2 Department of Animal Nutrition, Faculty of Veterinary Medicine, Mustafa Kemal University, 31040 Hatay, TURKEY. 3 Department of Animal Nutrition, Faculty of Veterinary Medicine, Adnan Menderes University, 09016 Aydın, TURKEY. 4 Department of Animal Nutrition, Faculty of Veterinary Medicine, Afyon Kocatepe University, 03200 Afyon, TURKEY. *Corresponding author: ozcancen@gmail.com SUMMARY This experiment was carried out to determine the effects of different contents of L-carnitine added to quail diets on some growth characteristics and blood biochemical parameters. For that, 345 one week old Japanese quail chickens (Coturnix coturnix japonica) were divided into 5 equal groups according to the L carnitine contents added to the diet for 4 weeks (0 mg/kg of diet in the control group, 50, 100, 150 and 200 mg/kg in the treatment groups 1, 2, 3 and 4, respectively). Body weights, body weight gains, food intake and food efficiency were determined weekly. At the 4 th week of treatment, carcass traits and blood biochemical parameters (serum total protein, cholesterol and triglyceride concentrations as well as serum ALP, AST and ALT activities) were analyzed from 12 randomly selected males in each group. With the highest L carnitine dosage, body weights significantly increased at the 2 nd week of treatment and the cumulative body weight gains on the whole period were significantly higher than in the other groups. In parallel, the total food intake tended to slightly decrease whereas food efficiency and weights and yields of carcasses tended to be improved in the group treatment 4 but not significantly. By contrast, lower dosages of L carnitine failed to induce changes in weight gain, food consumption or in carcass traits. Furthermore, biochemical parameters were not affected by L carnitine supplementation whatever the dose used. These results suggest that higher dosages of L carnitine (above 200 mg/kg) would be necessary for improving lipid metabolism and growth in quails. Keywords: Carnitine, supplementation, Japanese quail, growth, carcass, cholesterol, triglyceride, hepatic enzyme. RÉSUMÉ Effets d un apport alimentaire en L-carnitine sur la croissance et sur quelques paramètres biochimiques chez la caille japonaise (Coturnix coturnix japonica) Les objectifs de cette étude ont été de déterminer les effets d une supplémentation alimentaire en L carnitine à des doses variables sur la croissance et les paramètres biochimiques sanguins chez la caille japonaise (Coturnix coturnix japonica). Au total, 345 poussins âgés d une semaine ont été répartis en 5 groupes égaux en fonction de la quantité de L carnitine ajoutée à la ration pendant 4 semaines (0 mg/kg/jour : groupe contrôle, 50, 100, 150 et 200 mg/kg/jour : respectivement groupes 1, 2, 3 et 4). Les poids vifs, les gains pondéraux, la prise alimentaire et l efficacité de la ration ont été déterminés hebdomadairement. A l issue de la 4 ième semaine de traitement, les caractéristiques des carcasses de 12 mâles pris au hasard dans chaque groupe ont été appréciées et les paramètres biochimiques sériques (protéinémie, cholestérolémie, triglycéridémie, activités sériques des phosphatases alcalines, de l AST et de l ALT) ont été mesurés chez tous les animaux. Lorsque les cailles ont été traitées par la plus forte dose de L carnitine, les poids vifs ont significativement augmenté dès la 2 ième semaine de traitement et les gains pondéraux cumulés sur l ensemble de la période expérimentale ont été significativement plus élevés que dans les autres groupes. Parallèlement, la prise alimentaire totale a semblé diminuer alors que l efficacité alimentaire ainsi que les poids et les rendements des carcasses sont apparus améliorés dans le groupe 4 mais de façon non significative. En revanche, une supplémentation en L carnitine réalisée à de plus faibles doses n a induit aucun effet sur la croissance pondérale, la consommation alimentaire ou les caractéristiques des carcasses. En outre, quelque soit le dosage de L carnitine, les paramètres biochimiques explorés n ont pas été modifiés. Ces résultats suggèrent que des doses plus élevées de L carnitine (supérieures à 200 mg/kg) seraient nécessaires pour pouvoir améliorer le métabolisme lipidique et la croissance des cailles. Mots-clés : Carnitine, apport alimentaire, caille japonaise, croissance, carcasse, cholestérol, triglycéride, enzyme hépatique. Introduction Carnitine is an alternative food additive in poultry diets because of its beneficial effects on enhancing resistance to metabolic diseases, preventing some diseases, strengthening immune system, improving poultry performance and playing role in metabolic and physiological processes [10, 17, 21]. Carnitine is an endogenous betain derivate of β- hydroxy butyrate, found in the structure of human, animal, plant and some micro organisms in various levels [8]. Carnitine having

L-CARNITINE SUPPLEMENTATION AND GROWTH IN QUAILS 503 similar effects of vitamins takes part in many metabolic functions in organism, such as lipid catabolism and energy production [10, 28]. Broiler and quail diets contain a high percentage of cereal grains, which supply low L-carnitine contents [5]. Therefore, in case of increased metabolic demands, insufficient endogenous L-carnitine synthesis coupled to a low dietary L-carnitine supply could become limiting for metabolic requirements [9]. Several studies on broilers have shown that growth performance and food efficiency were significantly improved by dietary supplementation with L-carnitine [24-26]. In contrast some researchers failed to observe any beneficial effects of L-carnitine on broiler performance [9, 19, 20, 22]. Only scarce information on the effects of the dietary L carnitine supplementation in quails is available [4, 31]. Therefore the present study was undertaken to evaluate the effects of different contents of dietary carnitine supplementation on some productive characteristics and blood parameters of one-week-old quails. Measurement of blood parameters could be useful to detect some metabolic effects of L-carnitine as already shown in broilers [19, 20, 29]. Materials and Methods ANIMALS, DIETS AND EXPERIMENTAL PROTOCOL A total of 345 Japanese quail chicks (Coturnix coturnix japonica), one week old, with uniform body weight (30.3 ± 0.3 g) were used in this study. They were randomly divided into 5 equal groups (each group contained 69 quails) according to the dietary L carnitine (50% L-carnitine, Lohmann Animal Health, Cuxhaven, Holland) supplementation for four weeks (0 mg/kg diet (control group), 50 mg/kg diet (treatment group 1), 100 mg/kg diet (treatment group 2), 150 mg/kg diet (treatment group 3) and 200 mg/kg diet (treatment group 4)). Each group was divided into three replicate groups of 23 quails each. A photoperiod of 24 h was maintained. Food and water were provided ad libitum and the diets were presented in mash form. They were formulated to be isocaloric and isonitrogenous and their composition determined according to the AOAC [2] and chemical analyses are presented in Table I. The analyses of calcium [16] and phosphorus [1] were made after dry combustion of diets. Dietary metabolisable energy levels were estimated using the following prediction equation [18]: ME (kcal/kg) = 53 + 38 (% crude protein + 2.25 x % ether extract + 1.1 x % starch + % sugar). The dietary L-carnitine contents were calculated based on the L-carnitine supply in each ingredient reported by BAUMGARTNER and BLUM [5]. Quails were weighed individually at the beginning of the experimental period and weekly for calculating body weight gains. Mortality was recorded when it occurred. Food consumption was recorded weekly and expressed as g per quail per week and the food efficiency ratio was calculated as kg food per kg body weight gain. At the end of the experimental period, the sex ratio was established in each group and 12 male quails of each group (4 male quails from each replicate) were randomly selected and weighed to determine the carcass yield. Quails were slaughtered by severing the jugular vein, inert organs, head and foot were removed manually after wet defeathering. Chilled carcass weights were determined after allocation 18 hours at 4 C and carcass yield was calculated. Blood samples were collected from male quails during slaughtering into sterile tubes without anticoagulant and after clotting at room temperature for 4 hours, they were centrifuged at 3000 g for 10 min at room temperature. Thereafter, sera were carefully harvested and stored at -20 C until determination of biochemical parameters. Serum total protein (g/l; kit n : 11929991), triglyceride (g/l; kit n : 11730711) and cholesterol (mmol/l; kit n : 11491458) concentrations as well as serum activities of a lanine aminotransferase (ALT; kit n : 11876805), aspartate amino transferase Ingredients % Chemical composition (analyzed) Corn 46.05 Dry matter 92.41% Soybean meal 43.00 Crude protein 24.83% Meat and bone meal 4.00 Ether extract 7.05% Vegetable fat 4.50 Crude fibre 4.02% Limestone 1.00 Crude ash 6.81% Dicalcium phosphate 0.80 Nitrogen free extract 49.70% Salt 0.25 Calcium 1.19% Vitamin premix 1 0.15 Phosphorus 0.75% Mineral premix 2 0.10 Metabolisable energy 3 3 126 kcal/kg DL methionine 0.10 L carnitine 4 12.75 mg/kg Lysine 0.05 1 Vitamin premix composition (per 1.5 kg): 15 500 000 IU vitamin A, 2 500 000 IU vitamin D 3, 15 500 IU vitamin E, 500 mg vitamin B 1, 6 g vitamin B 2, 2 g vitamin B 6, 15 mg vitamin B 12, 2 g vitamin K 3, 1.5 g folic acid, 30 g niacin, 8 g calcium D-pantothenate. 2 Mineral premix composition (per 1 kg): 80 g Mn, 50 g Zn, 7 g Cu, 60 g Fe, 0.3 g I, 0.15 g Se, 400 g choline chloride. 3 The metabolisable energy of diets was estimated according to the prediction equation given in LEESON and SUMMERS [18]. 4 Carnitine content of diets was calculated using the values of BAUMGARTNER and BLUM [5]. TABLE I : Ingredients and chemical composition of the basal diet.

504 CENGIZ (Ö.) AND COLLABORATORS (AST; kit n : 11876848) and alkaline phosphatase (ALP; kit n : 12173107) were determined with a Hitachi auto analyser (Hitachi ltd, Tokyo Serial n : 1238-23) using their accompanying commercial kits (Roche Diagnostics, Tokyo, Japan). STATISTICAL ANALYSIS Statistical analysis was done using the SPSS software package for Windows (SPSS Inc., Chicago, IL, USA). One way ANOVA was used to evaluate the effects of L carnitine on performance and blood biochemical parameters according to the quail groups and the significance of mean differences was tested by the Duncan's test. The effect of L carnitine supplementation on the mortality and on the sex ratio in quails was evaluated by the X 2 test [14]. The level of significance used in all results was p< 0.05. Results A total of 20 quails (5.8%) (3 quails in the control group and 3 to 5 quails in the different treatment groups) died during the whole experimental period (Table II). The mortality rate was not significantly affected by the dietary L supplementation (p > 0.05). In the same way, the respective numbers of males and females did not significantly differ between the different assay groups (p > 0.05). The effects of L carnitine on body weight, body weight gains, food intake and food efficiency are shown in Table II. Only the highest L carnitine dosage (200 mg/kg diet) has induced significant increases of body weights after 2, 3 and 4 weeks of treatment (p < 0.05). Although intermediate body weight gains calculated weekly were similar whatever the experimental groups and were ranged from 4.3 to 5.4 g/day, Parameters Experimental groups 0 mg/kg 50 mg/kg 100 mg/kg 150 mg/kg 200 mg/kg (Control) (Treat. 1) (Treat. 2) (Treat. 3) (Treat. 4) Mortality (%) 4.35 5.80 4.35 7.25 7.25 (number of death) (3) (4) (3) (5) (5) Sex ratio (M / F) 34/32 30/35 31/35 35/29 34/30 Weight Initial BW (g) 30.2 ± 0.5 29.9 ± 0.6 30.4 ± 0.6 30.7 ± 0.5 30.5 ± 0.6 BW on week 1 (g) 60.3 ± 0.9 60.7 ± 1.0 61.3 ± 1.0 62.6 ± 1.1 62.8 ± 1.1 BWG0-1 (g) 30.1 ± 1.3 30.8 ± 0.7 30.9 ± 0.5 31.9 ± 1.9 32.3 ± 0.6 BW on week 2 (g) 91.9 ± 1.4 b 92.3 ± 1.5 b 94.9 ± 1.3 ab 96.0 ± 1.5 ab 99.1 ± 1.6 a BWG1-2 (g) 31.6 ± 1.3 31.6 ± 2.9 33.6 ± 0.9 33.4 ± 2.8 36.3 ± 1.8 BW on week 3 (g) 129.4 ± 1.5 b 128.1 ± 1.7 b 131.4 ± 1.6 ab 131.9 ± 1.7 ab 136.1 ± 1.7 a BWG2-3 (g) 37.5 ± 2.3 35.8 ± 2.0 36.5 ± 1.9 35.9 ± 2.2 37.0 ± 0.4 Final BW (g) 162.2 ± 1.8 b 163.4 ± 2.0 b 164.1 ± 1.5 b 166.3 ± 1.6 ab 169.7 ± 1.4 a BWG3-4 (g) 32.8 ± 1.1 35.3 ± 1.9 32.7 ± 0.5 34.4 ± 0.5 33.6 ± 0.9 BWG0-4 (g) 132.0 ± 1.6 b 133.5 ± 0.3 b 133.7 ± 0.6 b 135.6 ± 1.9 ab 139.2 ± 0.9 a Food Intake (g) FI0-1 74.4 ± 2.2 75.2 ± 4.3 79.5 ± 1.0 75.6 ± 0.6 76.5 ± 1.2 FI1-2 101.9 ± 0.3 104.5 ± 1.3 102.5 ± 1.6 106.3 ± 1.1 102.3 ± 4.0 FI2-3 129.0 ± 3.4 131.8 ± 3.1 132.9 ± 0.8 130.3 ± 4.8 128.9 ± 5.3 FI3-4 147.7 ± 7.7 149.1 ± 4.4 146.8 ± 7.2 143.1 ± 11.3 147.8 ± 6.4 FI0-4 452.0 ± 11.6 460.5 ± 4.0 461.6 ± 10.1 455.3 ± 11.6 455.4 ± 9.7 Food Efficiency (g/g) FE0-1 2.44 ± 0.19 2.44 ± 0.16 2.57 ± 0.02 2.37 ± 0.15 2.37 ± 0.02 FE1-2 3.22 ± 0.13 3.31 ± 0.33 3.05 ± 0.04 3.18 ± 0.25 2.82 ± 0.06 FE2-3 3.44 ± 0.12 3.68 ± 0.29 3.64 ± 0.22 3.63 ± 0.09 3.48 ± 0.14 FE3-4 4.50 ± 0.28 4.22 ± 0.30 4.49 ± 0.16 4.16 ± 0.38 4.40 ± 0.29 FE0-4 3.42 ± 0.05 3.45 ± 0.03 3.45 ± 0.09 3.36 ± 0.06 3.27 ± 0.05 Treat.: Treatment; M: male; F: female; BW: body weight; BWG: body weight gain: BWGi-j: body weight gain calculated between the ith and the jth weeks of treatment; FI: food intake; FIi-j: food intake calculated between the ith and the jth weeks of treatment; FE: food efficiency; FEi-j: food efficiency calculated between the ith and the jth weeks of treatment. Different superscripts a,b in the same line indicated significant differences (p < 0.05). TABLE II : Effects of dietary L carnitine supplementation for 4 weeks on mortality and growth parameters of one week old Japanese quails. Quails (n = 69 in each group) received 0 (control group), 50, 100, 150 and 200 mg/kg of L carnitine into diets. Results are expressed as mean ± standard error.

L-CARNITINE SUPPLEMENTATION AND GROWTH IN QUAILS 505 the overall mean body weight gain obtained for the whole experimental period (4 weeks) was significantly enhanced in quails receiving the highest dosage (p < 0.05). In contrast, food intake and food efficiency were not significantly modified by L carnitine supplementation compared to controls albeit the cumulative food efficiency ratio (calculated throughout the 4 weeks of treatment) tended to be improved when 200 mg/kg of L carnitine was added to diets. In addition, the carcass mean weight and the carcass mean yield obtained from male quails at the end of the experiment tended to increase in groups supplemented with 150 and 200 mg/kg diet L carnitine (treatment groups 3 and 4) (Table III) but the differences with the other treated groups and with the control group were not statistically significant. As shown in Table IV, the L carnitine supplementation even with the highest dosage did not induce any significant effect on blood biochemical parameters investigated in the present study (serum total protein, cholesterol and triglyceride concentrations and ALP, AST and ALT activities). Parameters Experimental groups 0 mg/kg 50 mg/kg 100 mg/kg 150 mg/kg 200 mg/kg (Control) (Treat. 1) (Treat. 2) (Treat. 3) (Treat. 4) Body weight (g) 165.8 ± 5.4 160.6 ± 4.2 163.7 ± 4.8 168.9 ± 4.8 169.0 ± 5.5 Carcass weight (g) 116.7 ± 3.7 113.8 ± 3.4 115.2 ± 2.6 120.2 ± 2.9 124.0 ± 4.7 Carcass yield (%) 70.4 ± 0.8 70.9 ± 1.3 70.4 ± 1.0 71.2 ± 1.1 73.4 ± 1.1 Treat.: Treatment. TABLE III : Effects of dietary L carnitine supplementation for 4 weeks on carcass weight and carcass yield determined on 12 randomly selected male quails in each group at the end of treatment. Quails received 0 (control group), 50, 100, 150 and 200 mg/kg of L carnitine into diets. Results are expressed as mean ± standard error. Biochemical Experimental groups parameters 0 mg/kg 50 mg/kg 100 mg/kg 150 mg/kg 200 mg/kg (Control) (Treat. 1) (Treat. 2) (Treat. 3) (Treat. 4) Total protein (g/l) 28.3 ± 1.1 28.7 ± 1.0 30.0 ± 0.9 29.6 ± 1.3 28.0 ± 1.2 Cholesterol (mmol/l) 5.11 ± 0.27 5.51 ± 0.33 4.81 ± 0.41 5.00 ± 0.34 5.66 ± 0.40 Triglyceride (g/l) 2.76 ± 0.37 3.22 ± 0.45 3.00 ± 0.35 2.90 ± 0.36 2.83 ± 0.33 ALP (U/L) 462.42 ± 55.39 480.83 ± 57.35 376.67 ± 50.10 412.83 ± 28.36 472.33 ± 61.06 AST (U/L) 272.17 ± 13.17 282.17 ± 24.46 252.50 ± 13.55 259.08 ± 10.75 271.92 ± 15.15 ALT (U/L) 8.33 ± 0.59 9.33 ± 0.94 8.75 ± 0.91 8.42 ± 0.50 9.17 ± 0.81 Treat.: Treatment; ALP: alkaline phosphatase; AST: aspartate amino transferase; ALT: alanine amino transferase. TABLE IV : Effects of dietary L carnitine supplementation on blood biochemical parameters (serum total protein, cholesterol and triglyceride concentrations and ALP, AST and ALT activities) determined on 12 randomly selected male quails in each group at the end of the treatment. Quails received 0 (control group), 50, 100, 150 and 200 mg/kg of L carnitine into diets. Results are expressed as mean ± standard error. Discussion In the present study, a dietary treatment of one week old Japanese quails with L carnitine for 4 weeks did not reduce the mortality of quails whatever the dosage used. Similarly, some researchers found that mortality of broilers [9, 22] or of quails [31] was not affected by carnitine supplementation. By contrast, DAȘKIRAN and TEETER [13] reported that carnitine supplementation at the doses of 40, 80, 120, 160 and 200 mg/kg diet improved the liveability by promoting a more efficient utilization of fatty acids in heart muscle. Moreover, the weight gain of quails was not significantly increased by the oral L carnitine treatment except when birds received the highest dosage of the present study (200 mg/kg diet). In agreement with these findings, dietary carnitine supplementation with dosages of 50 mg/kg diet [26], 100 mg/kg diet [9, 22] or 160 mg/kg diet [13, 20] did not alter the body weight of broilers. On the other hand, when 200 mg/kg of L carnitine was added to the basal diets, a positive effect on body weight was observed since the second week of treatment until the 4 th week whereas the intermediate body weight gains calculated weekly were not significantly modified. Only the cumulative body weight gain was significantly enhanced in the group treatment 4. In disagreement with the present results, DAȘKIRAN and TEETER [13] have not observed any significant positive effect of 200 mg/kg carnitine supplementation on weight gain in broilers. BAYRAM et al. [6] have also reported that supplementation with L carnitine at 500 mg/kg diet had no significant effect on body weight of quails. In parallel, mean carcass weights and carcass yields in male quails were not significantly increased by L carnitine supplementation as already observed in male quails [6] and in broilers [9, 13, 22], even if they tended to be

506 CENGIZ (Ö.) AND COLLABORATORS augmented in the groups treatment 3 and 4 (150 and 200 mg/kg of L carnitine, respectively). Although the highest L carnitine dosage (200 mg/kg) tended to positively modified, but not significantly, the cumulative food intake and food efficiency obtained in quails for 4 weeks of treatment, the L carnitine supplementation did not globally significantly affect these 2 parameters in the present study. These observations are in agreement with previous studies conducted in laying hens [12, 19, 23], in laying quails [19, 30] and in broilers [9, 11, 20, 22]. Similarly, food efficiency ratio was not significantly altered in broilers dietary supplemented with L-carnitine at 50 mg/kg diet [29], 100 mg/kg diet [9, 22], 160 mg/kg diet [20] and 200 mg/kg diet [13]. In the present study, L carnitine supplementation did not induce any significant change of blood serum biochemical parameters (serum total protein, cholesterol and triglyceride concentrations and ALP, AST and ALT activities) whatever the carnitine dosage. Previous studies in laying quails [30] have also failed to demonstrate significant effects of L carnitine administration on blood serum parameters. In agreement with these findings, carnitine supplementation with dosage of 100 mg carnitine chlorhydrate / L of water did not alter the serum cholesterol, triglyceride and total protein concentrations in 6 week old quails [4]. UYSAL et al. [27] detected in male 42 day old quails that 500 mg/kg diet L-carnitine caused no significant change in serum total protein and cholesterol concentrations as well as in ALT and ALP activities but they observed a significant decrease of serum triglyceride concentration. In the same way, serum cholesterol concentrations of broilers [20] and laying hens [19] were not significantly affected by a dietary L-carnitine supplementation. However, contradictory results have been obtained by EDER [15] who reported marked increase of cholesterolemia in L carnitine treated rats whereas BELL et al. [7] observed significant reduction of serum cholesterol and triglyceride concentrations in L carnitine supplemented rabbits. XU et al. [29] concluded that L-carnitine supplementation reduced the concentration of triglyceride in serum of male broilers. The eventual discrepancies between results of the present study and those of previous reports would be linked to the particular sensitivity of birds (influence of species, age, sex and environmental conditions) and to the experimental protocol used (dietary nutrient composition, modalities of diet distribution, dosages of L carnitine and/or its precursors in the diet and the treatment duration) [3, 30]. 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