Effect of an addition of Hepabial Carnitine in drinking water on the performance of broilers under stocking stress

Effect of an addition of Hepabial Carnitine in drinking water on the performance of broilers under stocking stress G. DENIZ* and I. I. TURKMEN Department of Animal Nutrition and Nutritional Diseases, Faculty of Veterinary Medicine, University of Uludag, 16059-Gorukle, Bursa, TURKEY * Corresponding author : Dr. Gulay DENIZ. Department of Animal Nutrition and Nutritional Diseases, Faculty of Veterinary Medicine, University of Uludag, 16059-Gorukle, Bursa, Turkey - E-mail: denizg@uludag.edu.tr SUMMARY The aim of this study was to investigate whether a L-carnitine, choline, artichoke leaf extract and sorbitol combination (Hepabial Carnitine ) supplied by drinking water would influence broiler performance under stress induced by high stocking density (18 chicks per square meter). A total of 270 1-dayold male broiler chicks (Avian Farm) were randomly distributed into 2 groups (Hepabial Carnitine treatment group and Control group), each with 5 replicate groups containing 27 chicks, and were fed with standard broiler diets. Hepabial Carnitine was added in drinking water of the treatment group at 1 ml/l beginning by day 35 (when the stocking density was over 30 kg total body weight/m 2 ) until the slaughter of the animals at the 42 nd day of the experiment. All chicks were individually weighed at hatching and thereafter on the 35 th and 42 nd days of the experiment. Body weight gain, feed intake and feed conversion ratio were determined on days 35 and 42. Carcass performance (hot carcass weight and hot carcass yield) was measured after slaughtering. Hepabial Carnitine supplied by drinking water had no significant effect on body weight, body weight gain, feed intake and hot carcass yield of broilers. However, birds receiving Hepabial Carnitine consumed less amount of feed (P=0.531) while gaining higher body weight (P=0.144) when compared with the control group. Feed conversion ratio was significantly improved (P<0.05) with a Hepabial Carnitine supplementation in drinking water. Similarly, hot carcass weight was found significantly higher (P<0.05) in birds receiving Hepabial Carnitine in comparison to the control group. It is concluded that L-carnitine, choline, artichoke leaf extract and sorbitol combination (Hepabial Carnitine ) added in drinking water could improve performance of broilers under stress induced by high stocking density. Keywords : L-carnitine, choline, artichoke leaf extract, sorbitol, broiler performance, high stocking density. RÉSUMÉ Effet d une addition d Hepabial Carnitine dans l eau de boisson sur les performances des poulets de chair lors d un stress induit par une hyperdensité. Cette expérience a été réalisée afin de déterminer l effet d un mélange de L-Carnitine, de choline, d'extrait de feuilles d'artichaut et de sorbitol (Hepabial Carnitine ), dilué dans l eau de boisson, sur les performances de poulets de chair soumis à un facteur de stress : une densité élevée d individus (18 poussins par mètre carré). Deux cents soixante dix poussins âgés de 1 jour, de sexe mâle (Avian Farm) ont été aléatoirement divisés en 2 groupes (le groupe traité recevant de l Hepabial Carnitine et le groupe témoin), chacun étant composé de 5 sous-groupes de 27 animaux. Tous les animaux étaient nourris avec un régime alimentaire standard préparé pour les poulets de chair. L Hepabial Carnitine a été ajouté dans l'eau de boisson du groupe traité à la dose de 1 ml/l à partir du 35 éme jour (la densité atteignait alors plus de 30 kg PV/m2) et ce jusqu'à l'abattage au 42 éme jour de l expérimentation. Tous les poussins ont été pesés individuellement à l éclosion, puis au 35 ème et au 42 ème jours. Le gain de poids vif, la quantité ingérée et l indice de consommation ont été déterminés au 35 ème et 42 ème jours. La qualité des carcasses (poids chaud et rendement à chaud de carcasse) a été estimée après l abattage. L Hepabial Carnitine n'avait pas d'effet significatif sur le poids vif, le gain de poids vif, l ingestion et le rendement à chaud des carcasses. Cependant, la quantité d aliments ingérés par le groupe supplémenté en Hepabial Carnitine était moins importante que celle du groupe témoin (P=0.144), alors que leur gain de poids vif était plus important (P=0.531). Aussi, l indice de consommation a été significativement amélioré par l addition d Hepabial Carnitine (P<0.05). De la même manière, le poids des carcasses à chaud était significativement plus important dans le groupe recevant l Hepabial Carnitine (P<0.05) par rapport au groupe témoin. Ces résultats suggèrent que lors de stress, la combinaison de L-carnitine, de choline, d'extrait de feuille d'artichaut et de sorbitol (Hepabial Carnitine ) peut améliorer les performances des poulets de chair. Mots-clés : L-carnitine, choline, extrait de feuille d'artichaut, sorbitol, performances des poulets de chair, densité élevée d individus. Introduction Maintaining a high stocking density of broilers is a common practice used for increasing economic returns per unit of floor space. However, crowding of broilers can lead to reductions in performance [37]. In most experiments with broilers under litter conditions a tendency of growth depression has been observed when the total body weight of broilers reached over 30 kg/m 2 [18,31,37,39]. The decrease of growth performances observed in high stocking density could be attributed to an increase of stress resulting from a competition for feed and water, a poor litter quality, a high ammonia level and heat. REITER and BESSEI [36] found a linear decrease in growth of broilers at 5 weeks of age with increasing stocking density and this was mainly due to increasing temperature between and underneath the birds. During stress,

14 DENIZ (G.) AND TURMEN (I.I.) nutrients in the feed are not efficiently digested and absorbed, leading to an increase of energy requirements and a decrease of growth performances in young birds [8]. There are possible ways to alleviate the stress response such as assuring adequate feeding and watering space, improving the ventilation and house environment, and covering increased energy needs [13,15,35]. L-carnitine (γ-trimethyl-amino-β-hydroxybutyrate) has been reported to play a significant role in energy metabolism as a carrier for the transport of the activated long chain fatty acid from the cytosol into the mitochondrial matrix for β- oxidation [20,35]. Carnitine can be biosynthesized endogenously from methionine and lysine [10,35]. Endogenous biosynthesis is sufficient to cover normal requirements. However, this is not the case in neonates (biosynthesis is not fully developed), conditions of stress, high performances and diets rich in fat [2, 7, 32, 35]. Choline and carnitine are composed of 3 methyl groups and both might have similar functions. Choline plays an essential role in fat metabolism in the liver, promoting its transport as lecithin or by increasing fatty acid catabolism in the liver [29]. Chick liver can synthesize choline, but at an insufficient rate to cover its needs [24]. Artichoke leaf extract is an herbal remedy made from the lower leaves of the artichoke plant (Cynara scolymus L.). The leaf has a variety of pharmacologic effects such as being choleretic (bile increasing) [23] and hepatoprotective [17]. Sorbitol is the alcohol form of sucrose and is naturally found in numerous berries and higher plants [27]. Dietary sorbitol increases both bile volume and bile salts excretion [38]. The aim of this study was to investigate whether L- carnitine, choline, artichoke leaf extract and sorbitol combination (Hepabial Carnitine ) supplied by drinking water would influence broiler performances under stress. Our first experiment [14] used feed change and vaccination as stress factor. In this second study we explored the effects of third kind of stress factors: high stocking density. Materials and Methods BIRDS, HOUSING AND MANAGEMENT The study was conducted with 270 1-day-old male broilers (Avian Farm). The chicks were individually weighed and randomly divided into 2 groups (Hepabial Carnitine treatment group and Control group), each group was divided in 5 pens containing 27 chicks, with a density of 18 chicks per square meter. All chicks were fed a standard broiler prestarter (0 to10 days-crumbles), a starter (10 to 21 days-crumbles), a grower (21 to 35 days-pellets) and a finisher (35 to 42 days-pellets) diet produced by a commercial feed company (Charoen Pokphand Foods Group, Bursa, TURKEY). The ingredients and nutrient composition of the broiler diets are shown in Table I. The prestarter, starter, grower and finisher diets contained 17.58, 17.60, 16.87 and 12.89 mg/kg of L-carnitine; 1783, 1731, 1638 and 1579 mg/kg of choline, respectively. The L-carnitine and choline content of the diets was calculated according to BAUMGARTNER and BLUM [4] and NRC [30], respectively. During the experimental period of 42 days, each group received feed and water ad libitum. Birds were exposed to 23 h of light and 1 h of dark per day and reared at a conventional ambient temperature (from 30 C reducing to 24 C by 3 C/week) with a relative humidity of 60-70 %. Wood shavings were used as litter. Mortality was recorded as it occurred. TREATMENT WITH HEPABIAL CARNITINE L-carnitine, choline, artichoke leaf extract and sorbitol combination (Hepabial Carnitine, Sogeval Laboratoires, Laval, France) were added in the drinking water of the treated group daily at 1 ml/l beginning by day 35 (when the stocking density reached over 30 kg total body weight/m 2 ) until the slaughter of the animals at day 42. Hepabial Carnitine contains carnitine hydrochloride 50 mg, sorbitol 400 mg, choline chloride 150 mg, artichoke extract (Cynara scolymus L.) 20 mg per ml. MEASUREMENTS AND ANALYSES All chicks were individually weighed at hatching and thereafter on the 35 th and 42 nd days of the experiment. Average feed intake, bird weight gain and feed conversion ratio adjusted for mortality were determined on a pen basis on days 35 and 42. On day 42, all birds were slaughtered and hot carcasses (without neck, giblets and feet) were weighed in order to determine hot carcass weight and yield. Experimental diets were chemically analyzed (Table I) for dry matter, crude protein, crude ash, ether extract, starch and saccharose according to the AOAC procedures [1]. Metabolisable energy of the experimental diets was calculated using the equation of HARTEL [21]: [Ether extract, % x 0.3431] + [Crude Protein, % x 0.1551] + [Saccharose, % x 0.1301] + [Starch, % x 0.1669] = MJ/Kg x 239 = Kcal/Kg. STATISTICAL ANALYSIS Body weight, body weight gain and hot carcass weight data were measured individually while feed intake, feed conversion ratio, hot carcass yield and mortality were determined on a pen basis. The statistical analyses for feed intake, feed conversion ratio and hot carcass yield were performed using the Mann-Whitney test. Body weight, body weight gain and hot carcass weight data were analysed by Student s t-test. Mortality rates were evaluated using the Chi-square test. Differences were considered significant at a probability level of P<0.05 in all analyses. All statistical analysis was performed with SPSS software (version 10.0, SPSS Inc, USA).

EFFECT OF HEPABIAL CARNITINE ON THE PERFORMANCE OF BROILERS UNDER STRESS 15 Prestarter Starter Grower Finisher Ingredients (% of diet) Ground yellow corn 58.55 60.15 63.50 64.32 Soybean meal (48 % CP) 11.48 7.95 3.39 7.14 Sunflower meal (38 % CP) 1.02 1.62 2.25 1.37 Full fat soybean 8.00 8.00 8.00 8.00 Corn gluten meal 7.00 7.00 7.00 7.00 Fish-meal (65 % CP) 5.73 6.00 5.69 - Poultry by-product meal 4.00 4.00 4.00 6.00 Vegetable oil 1.76 2.74 3.73 3.03 Limestone 0.74 0.65 0.66 0.89 Dicalcium phosphate 0.94 0.94 1.01 1.48 Salt 0.20 0.18 0.21 0.25 DL-Methionine 0.04 0.15-0.04 L-Lysine HCL 0.09 0.17 0.11 0.13 Vitamin & Mineral Premix 1 0.25 0.25 0.25 0.25 Anticoccidial 2 0.10 0.10 0.10 - Antioxidant 3 0.10 0.10 0.10 0.10 Total 100.0 100.0 100.0 100.0 Chemical composition Dry matter, % 89.45 89.20 89.89 89.60 Crude Protein, % 22.45 21.60 20.14 18.71 Ether extract, % 8.18 9.69 10.84 9.72 Crude ash, % 6.17 5.38 5.22 5.00 Starch, % 36.75 37.01 36.96 40.21 Saccharose, % 6.14 5.90 5.58 4.90 Calcium, % 1.16 1.01 1.22 1.19 Total phosphorus, % 0.73 0.72 0.67 0.62 L-carnitine 4, mg/kg 17.58 17.60 16.87 12.89 Choline 5, mg/kg 1783 1731 1638 1579 Metabolic energy 6, (kcal/kg) 3140 3236 3266 3230 TABLE 1 : Ingredients and Chemical Composition of the Broiler Diets. 1. Provided the following per kg of diet: 12000 IU Vitamin A, 1500 IU Vitamin D 3, 30 mg Vitamin E, 5 mg Vitamin K 3, 3 mg Vitamin B 1, 6 mg Vitamin B 2, 5 mg Vitamin B 6, 0.03 mg Vitamin B 12, 0.75 mg Folic acid, 10 mg Calcium-D-Pantothenate, 0.075 mg D-Biotin, 375 mg Choline chloride, 40 mg Nicotine amide, 80 mg Manganese, 40 mg Iron, 60 mg Zinc, 5 mg Copper, 0.4 mg Iode, 0.1 mg Cobalt, 0.15 mg selenium, 10 mg Antioxidant. 2. Provided the following per kg of diet: 70 mg Narasin. 3. Provided the following per kg of diet: 2.5 mg BHA, 3.125 mg Etoxyquine, 2.5 mg Citric acid, 2.5 mg Ortho-phosphoric acid, 2.5 mg Mono-diglyceride fatty acids. 4. Calculated according to Baumgartner and Blum [4]. 5. Calculated according to NRC [30]. 6. Calculated according to Hartel [21].

16 DENIZ (G.) AND TURMEN (I.I.) Results The results with regard to the growth performance (body weight, body weight gain, feed intake, feed conversion ratio), and carcass performance and mortality of broilers are summarized in Tables II and III, respectively. Our findings showed that Hepabial Carnitine supplied by drinking water had no significant effect on body weight, body weight gain and feed intake, but there were significant differences in feed conversion ratio (Table II) of broilers during the last week of the experiment when they received the treatment. Birds receiving Hepabial Carnitine consumed less feed (P=0.531) and gained higher body weight (P=0.144) when compared with the control group. Feed conversion ratio was significantly improved (P<0.05) after Hepabial Carnitine supplementation to drinking water. Similarly, hot carcass weight was found significantly higher (P<0.05) in birds receiving Hepabial Carnitine in comparison to the control group, but hot carcass yield and mortality were not affected by Hepabial Carnitine supplementation (Table III). Discussion These results indicated that broilers receiving L-carnitine, choline, artichoke leaf extract and sorbitol combination could cope with various stress factors better. No comparable document could be found related to the usage of L-carnitine, choline, artichoke leaf extract and sorbitol in broilers under stress caused by high stocking density. However, in a previous study we had carried out by using feed change and vaccination as stress factors, we noticed an increase of performances (body weight, body weight gain, hot carcass weight and feed efficiency) with the L-carnitine, choline, artichoke leaf extract and sorbitol combination (Hepabial Carnitine ) added in drinking water in broilers [14]. Dietary supplemental carnitine studies on poultry have mostly focused on broilers, due to the suggested beneficial effects of carnitine on broilers under stress. Several studies on broilers have shown that growth performance was significantly improved by feeding dietary L-carnitine [32-34]. In contrast, some investigators failed to observe any effects of dietary L-carnitine on broiler performance [3,9,25,26]. The inconsistent responses of broilers supplemented with L- carnitine may be related to differences in dosage of L- carnitine, period of supplementation, sex, broiler genotype, basal diet composition and/or environmental conditions. The calculated choline levels of diets in the present experiment were exceeding the requirements of broiler chickens according to the nutrient requirements outlined by the NRC [30]. The bioavailability of choline in feed ingredients is less than 100 %. Therefore, several researchers suggest that chicks fed with corn-soybean meal diets containing choline in excess of the NRC requirements still need supplemental choline to ensure optimal performance [5,28]. JUKES [22] observed sub-optimal performance and perosis in young chicks without a dietary supplement of choline. There has not been enough research related to the effect of sorbitol on the performance of broilers. In one study, the absence of an effect of sorbitol on performance was reported in broiler chicken by FRUSE et al. [16]. On the other hand, feed efficiency was improved in preruminant calf from birth to 3 weeks by sorbitol supplementation in the study of DANIELS et al. [12]. Also, studies on the effect of artichoke leaf extract on performance of broilers are limited. BONOMI [6] suggested that the replacement of dried lucerne meal with dried artichoke leaf meal in the diets of broiler, duck, guinea fowl, rabbits and pig at 4 to 10 % would improve performance parameters and reduce feed costs. In this study, feed efficiency and hot carcass weight were significantly improved in birds receiving Hepabial Carnitine when compared with the control group. However, we did not observe significant improvement in the body weight and body weight gain in contrast with our previous study [14]. In our first experiment feed change group had received Hepabial Carnitine at the beginning of the trial for three days, and then for two days in every feed change period while vaccination group had received Hepabial Carnitine for two days 6 hours after each vaccination. However, in the present study, Hepabial Carnitine was added to drinking water on day 35 when the total body weight of broilers was approximately 33 kg/m 2. Previous studies revealed that a tendency of growth depression occurs when the total body weight of broilers reaches over 30 kg/m 2 [18,31,37,39]. We think that the differences between the body weight and body weight gain parameters of broilers in these two experiments were probably related with the intensity of different stress factors, and period and duration of Hepabial Carnitine addition in drinking water. In the present study, the improvements noted on feed efficiency and hot carcass weight may be due to the combined positive effects of L-carnitine, choline, artichoke leaf extract and sorbitol on energy metabolism. L-carnitine is accepted as a protecting agent for broilers under heat stress [11] via balancing energy deficit by facilitating long-chain fatty acids oxidation [20,35]. As one of the major disadvantages of overcrowding is increased temperatures in the pen, it can be expected that supplemental L-carnitine will also be beneficial in cases of high stocking density. Dietary L-carnitine supplementation has been reported to improve fatty acid and energy utilization and, therefore, to improve the performance in poultry [19]. Choline is also known to increase fatty acid utilization in the liver [29]. Therefore, we suggest that choline may show a carnitine-like effect by increasing fatty acid catabolism in the liver. Moreover, lipid digestion may be improved by artichoke extract and sorbitol as both are considered to have choleretic effect [23,38]. It could be concluded that the L-carnitine, choline, artichoke leaf extract and sorbitol combination (Hepabial Carnitine ) added in drinking could improve the performances of broilers under stress induced by high stocking density. Further studies are required to explore the specific roles of L-carnitine, choline, and particularly artichoke leaf extract and sorbitol in broilers under stress.

EFFECT OF HEPABIAL CARNITINE ON THE PERFORMANCE OF BROILERS UNDER STRESS 17 Item Control group Hepabial Carnitine group P d 0 40.01 ± 0.29 39.74 ± 0.27 0.479 Body weight (g) d 35 1874.47 ± 12.87 1881.83 ± 13.51 0.688 d 42 2457.14 ± 15.83 2494.26 ± 14.32 0.984 Body weight gain (g) d 35-42 582.67 ± 13.01 612.43 ± 22.30 0.144 Feed intake (g) d 35-42 1281.69 ± 29.10 1240.60 ± 24.20 0.531 Feed/gain (g/g) d 35-42 2.20 ± 0.023 2.03 ± 0.028 0.012 TABLE II : Effect of Hepabial Carnitine on Growth Performance of Broilers under Stocking Stress (mean ± SEM, 5 pens of 27 chicks per treatment) Item Control group Hepabial Carnitine group P Pre-slaughter live weight (g) 2457.14 ± 15.83 2494.26 ± 14.32 0.984 Hot carcass weight (g) 1750.27 ± 9.39 1818.26 ± 9.85 0.018 Hot carcass yield (%) 71.34 ± 0.48 73.46 ± 0.40 0.245 Mortality (%) 2.96 ± 1.38 2.22 ± 0.91 0.702 TABLE III : Effect of Hepabial Carnitine on Carcass Performance and Mortality of Broilers under Stocking Stress (mean ± SEM, 5 pens of 27 chicks per treatment). 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