Effects of in ovo administration of L-carnitine on hatchability, subsequent performance, carcass traits and blood cholesterol of turkey poults

Effects of in ovo administration of L-carnitine on hatchability, subsequent performance, carcass traits and blood cholesterol of turkey poults M. SALMANZADEH*, Y. EBRAHIMNEZHAD, H. AGHDAM SHAHRYAR Department of Animal Science, Shabestar branch, Islamic Azad University, 53815-159 Shabestar, IRAN. *Corresponding author: salmanzadeh_mehdi@yahoo.com SUMMARY The present study aims to investigate the effects of in ovo administration of L-carnitine on hatchability as well as on subsequent performance, carcass traits and cholesterolemia of turkey poults 42 days after hatching. Fertilized eggs (n = 126 per group) were injected into the yolk sac with L-carnitine (10, 20, or 30 mg dissolved in 0.5 ml of deionized water) at the 6 th day of incubation. Two control egg groups (not injected and injected with 0.5 ml of deionized water) were also included. Hatchability and subsequent weight growth, performances, carcass traits (carcass weight and relative weights of liver, breast and gizzard) and cholesterolemia on day 42 post-hatching were assessed. The hatchability was slightly but significantly depressed in all injected eggs compared to the not injected ones, but significantly increased in eggs treated with 20 and 30 mg L-carnitine compared to the sham egg controls. Furthermore, the weights at hatching, weight gains and food efficiency were significantly increased in chickens from L-carnitine treated eggs compared to the control chickens. In addition, carcass weights and relative weights of breast and gizzard were also markedly increased in chickens treated in ovo with L-carnitine whereas cholesterolemia was not significantly altered throughout the whole experimental period. These data suggest that the in ovo injection of L-carnitine may improve growth performance in turkey chickens. Keywords: L-carnitine, in ovo injection, turkey, hatchability, growth performance, carcass, cholesterol. RÉSUMÉ Effets de l administration in ovo de L-carnitine sur l éclosabilité ainsi que sur la croissance, les caractéristiques de la carcasse et la cholestérolémie des dindonneaux éclos L objectif de cette étude a été de déterminer les effets de l administration in ovo de L-carnitine sur l éclosabilité ainsi que sur les performances consécutives de croissance, les caractéristiques des carcasses et la cholestérolémie des dindonneaux 42 jours après l éclosion. Les œufs fertilisés (126 par groupe) ont reçu dans le jaune une injection de L-carnitine (10, 20 ou 30 mg dissoute dans 0.5 ml d ion désionisée) au 6 ème jour d incubation. Deux groupes témoins (œufs non injectés et œufs injectés par 0.5 ml d eau désionisée) ont aussi été inclus dans l étude. L éclosabilité ainsi que les performances correspondantes de croissance, les caractéristiques des carcasses (poids des carcasses et poids relatifs des blancs, foies et gésiers) et la cholestérolémie ont été mesurées sur une période de 42 jours. L éclosabilité a été faiblement mais significativement diminuée pour tous les groupes d œufs injectés par rapport à ceux non injectés mais elle a été significativement augmentée lorsque les œufs ont été traités par 20 ou 30 mg de L-carnitine par rapport aux œufs injectés témoins. De plus, les poids à l éclosion, les gains de poids et l efficacité alimentaire ont été significativement augmentés chez les poussins issus des œufs traités par la L-carnitine par rapport aux poussins témoins. Les poids des carcasses ainsi que les poids relatifs des blancs et des gésiers ont également été nettement augmentés chez les oiseaux traités in ovo par la L-carnitine alors qu aucune variation significative de la cholestérolémie n a été mise en évidence sur toute la durée de l expérimentation. Ces résultats suggèrent qu un traitement in ovo par la L-carnitine améliore les performances de croissance chez les dindonneaux. Mots clés : L-carnitine, injection in ovo, dinde, éclosabilité, croissance, carcasse, cholestérol. Introduction All nutrients needed for embryogenesis are provided by the hen by the time the fertile egg is laid [18]. If nutritional deficiencies occur during the formation of the egg, it can have significant repercussions on the developing embryo [24]. Hen diets are composed mainly of corn and soy, which contain low amounts of L-carnitine [8]. Therefore, eggs contain little or no L-carnitine [12]. Also, the chick embryo may have limited capability to synthesize L-carnitine during incubation [10]. The γ-butyrobetaine, an intermediate substance required for L-carnitine biosynthesis, is limited in embryos and young animals due to the low activity of γ-butyrobetaine hydroxylase [6, 29]. Low levels of L-carnitine synthesis may make supplementation of L-carnitine beneficial to chick embryos. As an example, hatchability of eggs from broiler breeder hens consuming diets supplemented with 50 or 100 mg of L-carnitine for 3 weeks as compared with controls increased from 83 to 87% and from 82.4 to 85.3%, respectively [22]. Moreover, a novel method of supplementing the in ovo (IO) nutrition of oviparous species, described as in ovo feeding (IOF) within the US Patent (6592878) of UNI and FERKET [34], was demonstrated to be an effective way to administer exogenous nutrient to support the development of the embryos and neonates in broiler hens [35]. In ovo feeding of supplemental nutrients may help to overcome the constraint of limited egg nutrients [15]. In such

IN OVO L-CARNITINE INJECTION AND HATCHABILITY AND PERFORMANCES IN TURKEY CHICKENS 449 situations, exogenous supplementation of L-carnitine could be advantageous [8] and could in turn be used by the chick during hatching. L-carnitine is a water-soluble quaternary amine that exists naturally in micro-organisms, plants and animals and is required for the long chain fatty acid transfer from cytoplasm to mitochondrial matrix for subsequent β-oxidation and energy production [7]. Under circumstances of increased metabolism, when the demand for energy escalates, L-carnitine availability could become a limiting factor for β-oxidation of fatty acids. L-carnitine also possesses some antioxidant properties [16, 40, 41]. Chick embryonic tissues contain high amounts of polyunsaturated fatty acids, which are essential components of cell membrane phospholipids [25, 32]. Polyunsaturated fatty acids are susceptible to lipid peroxidation caused by free radicals, which are produced by mitochondria because of the high metabolic rate of rapidly developing embryos [33]. Moreover, L-carnitine may work as an antioxidant to scavenge free radicals [1, 2, 37]. Thus, the presence of L-carnitine in the fertile egg may decrease embryonic mortality by reducing oxidative stress during the hatch process, thereby increasing hatch rate. Because chick embryos show a high requirement for L-carnitine, whereas the L-carnitine amount is low in egg, this study aims to determine the effects of L-carnitine injection into the yolk sac of turkey breeder eggs on day 6 of incubation on hatchability, subsequent performance, carcass traits and blood cholesterol of turkey poults. Material and Methods INCUBATION AND IN OVO INJECTIONS A total of 630 fertile eggs were obtained from 32 weeks old hens of Nicholas turkey breeder strain. All eggs were collected from the same breeder flock and weighed on a balance with 0.1 g precision and eggs with a weight of 80 ± 1 g were incubated at 37.8 C and 63% RH (relative humidity). At the 5 th day of incubation, the eggs were candled, and the infertile ones or those containing only dead embryos were removed. At the 6 th day, fertile eggs were randomly divided into 5 treatments with 3 replicates per treatment and 42 eggs per replicate according to in ovo injection: in the 3 assay-groups, eggs were injected with L-carnitine (10, 20 and 30 mg, respectively) dissolved in 0.5 ml of deionized water whereas in the sham group, eggs were only treated with deionized water (0.5 ml) and those of the control group received no injection. For that, each egg was candled to identify the location of the future injection. A hole was then punched using a 22- gauge needle and 0.5 ml of the administered solution was injected into the yolk sac using a 22-gauge needle to a depth of about 19 mm. The injection hole area was disinfected with an ethyl alcohol-laden swab, sealed with cellophane tape, and transferred to hatching baskets. Control eggs were removed from the incubator together with the treated groups, and kept in the same environment. The group of eggs designated as sham-injected controls were injected with 0.5 ml of deionized water. Deionized water injections were included as sham controls primarily to rule out a possible negative response caused by the stress of injection and handling. Injected solutions containing L-carnitine were prepared by directly dissolving L-carnitine hydrochloride (~98% purity; Sigma Chemical Co) in the deionized water. All of the treatment solutions were prepared in autoclaved water. BIRDS AND DATA COLLECTION After hatching, chickens were transferred to experimental house and reared for 42 days with same ration according to standard turkey ration (National Research Council, 1994) [26]. Each chick according to the treatment group was identified by neck tag and recorded. All chicks and treatments were randomly assigned to 1 of 15 pens. Each pen was bedded with soft pine wood shavings and equipped with automatic drinkers, and manual self-feeders. Ration and water were provided ad libitum. All animal experimentation was conducted in accordance with the regulations of Islamic Azad University, Animal Ethics Committee. Upon hatch, hatchability and weight of newly-hatched chickens were measured. Weight of newly-hatched chickens was determined by weighing all chicks hatched one by one. Hatchability was calculated by considering the ratio of salvable chicks hatched to the live embryos after the treatment and expressed as a percentage of fertilized eggs. In each pen, bird body weight and food intake were recorded on days 0, 21, and 42 post-hatching and thereafter mean body weight gain, food intake, and food conversion ratio were calculated for each pen (replicate) between 0 and 21, 22 or 42 days. In each time period, body weight gain was calculated and expressed as grams per bird. Food intake (g of food intake/bird) over the entire grow-out period was calculated by totalling food consumption in each time interval between each bird sampling. Food conversion ratio (g of food intake /g of body weight gain) was calculated by dividing total food intake by total weight gain in each pen. On days 0, 21 and 42, two bird selected from each replicate were subjected to blood sampling for determination of cholesterolemia. Blood samples were collected in non-heparinised blood sterile by cardiac puncture. After clotting for 4 hours at room temperature (18-24 C), samples were centrifuged at 3200 g for 5 minutes at room temperature and sera were carefully harvested, transferred into vials and stored at -20 C until used. The serum cholesterol concentrations were measured using a colorimetric commercial kit (Alpha diagnostic, Shabestar, Iran). On day 42, 6 birds per treatment were randomly chosen for the determination of carcass traits. Chickens were fasted for approximately 12 hours and then individually weighed, slaughtered (by severing the jugular vein), feathered and eviscerated. Weight of carcass, breast, liver, and gizzard were recorded and the corresponding percentages (% of live body weight) were calculated. STATISTICAL ANALYSIS Results were analyzed by ANOVA using the GLM procedure of SAS software (SAS institute, 2003) [30]. Differences

450 SALMANZADEH (M.) AND COLLABORATORS between treatments were compared by the Duncan s multiple range tests following ANOVA, and values were considered statistically different at P < 0.05. When data were percentages they were transformed by arc sin square root. Results As seen in Table I, the hatchability was significantly reduced for all groups of injected eggs, including sham eggs, compared to the control group (not injected eggs) (P < 0.001). Nevertheless, hatchability was partially and significantly restored with carnitine treatment at 20 and 30 mg compared to the sham eggs (P < 0.001). By contrast, the mean weights of newlyhatched chickens treated by carnitine whatever the dose were markedly higher than those of controls or sham eggs (P < 0.01). Groups Hatchability (%) Weight (g) Control 86 ± 1 a 52.86 ± 0.11 a Sham group 72 ± 1 c 52.94 ± 0.11 a Carnitine 10 mg 73 ± 1 c 53.29 ± 0.11 b Carnitine 20 mg 78 ± 1 b 53.56 ± 0.11 b Carnitine 30 mg 77 ± 1 b 53.51 ± 0.11 b Different superscripts a,b,c in the same column indicate significant differences between groups (P < 0.05 or more). TABLE I: Effects of in ovo administration of L-carnitine on weight and hatchability in newly-hatched chickens. Results are expressed as mean ± standard error of the mean (SEM). The effects of in ovo administration of L-carnitine on body weight gains, food intake and food conversion ratio are shown in Table II. There were no significant treatment effects on food intake during the starting (0-21 days) and the growing (22-42 days) periods. Body weight gains (P < 0.01 for the starting period, P < 0.001 for the growing period) and food efficiency (P < 0.05 for the starting period, P < 0.001 for the growing period) were significantly improved in chickens treated in ovo by L carnitine compared to the not injected and sham controls throughout the whole experimental period. Besides, chickens treated with 30 mg carnitine in ovo have exhibited a significantly greater weight gain for the growing period than others treated birds (P < 0.01). However, cholesterolemia has not significantly differed according to treatment and control groups on days 0, 21 and 42 post-hatching (Table III). As reported in Table IV, the carcass yield (P < 0.01) and the proportions of breast (P < 0.001) and gizzard (P < 0.001) were significantly augmented in chickens treated in ovo by L carnitine compared to the control (not injected and sham) groups whereas the liver proportions were not affected. Discussion ZHAI et al. [42] reported that in ovo injection of L-carnitine in a 0.05 to 10 µmol/egg dose range into fertile Single Comb White Leghorn eggs at the 17 th -18 th days of incubation did not affect hatchability and body weight gains. Moreover, KERA- LAPURATH et al. [17] demonstrate that in ovo injection of Groups Starting period Growing period (22-42 days) (0-21 days) BWG FI FCR BWG FI FCR Control 669.00 ± 2.68 a 915.33 ± 9.81 1.368 ± 0.016 a 1526.66 ± 3.77 a 3053.33 ± 11.57 1.99 ± 0.008 a Sham group 672.33 ± 2.68 a 919.67 ± 9.81 1.367 ± 0.016 a 1521.66 ± 3.77 a 3048.33 ± 11.57 2.00 ± 0.008 a Carnitine 10 mg 685.33 ± 2.68 b 896.33 ± 9.81 1.307 ± 0.016 b 1554.66 ± 3.77 b 3035.33 ± 11.57 1.65 ± 0.008 b Carnitine 20 mg 682.00 ± 2.68 b 891.67 ± 9.81 1.307 ± 0.016 b 1559.00 ± 3.77 b 3045.33 ± 11.57 1.95 ± 0.008 b Carnitine 30 mg 689.33 ± 2.68 b 888.67 ± 9.81 1.289 ± 0.016 b 1573.00 ± 3.77 c 3043.33 ± 11.57 1.93 ± 0.008 b BWG: body weight gain (g/bird); FI: Food intake (g/bird); FCR: Food Conversion ratio (FI/BWG). Different superscripts a,b,c in the same column indicate significant differences between groups (p < 0.05 or more). TABLE II: Effects of in ovo administration of L-carnitine on body weight gain (BWG), food intake (FI) and food conversion ratio (FCR) of turkey chickens in different growth periods. Groups Cholesterolemia (mmol/l) Day 0 Day 21 Day 42 Control 3.54 ± 0.48 2.53 ± 0.28 3.16 ± 0.39 Sham group 4.21 ± 0.48 3.18 ± 0.28 2.89 ± 0.39 Carnitine 10 mg 3.78 ± 0.48 2.36 ± 0.28 3.40 ± 0.39 Carnitine 20 mg 3.42 ± 0.48 2.79 ± 0.28 3.06 ± 0.39 Carnitine 30 mg 4.02 ± 0.48 2.94 ± 0.28 2.78 ± 0.39 TABLE III: Effects of in ovo administration of L-carnitine on cholesterolemia on days 0, 21 and 42 post-hatching in turkey chickens. Results are expressed as mean ± standard error of the mean (SEM).

IN OVO L-CARNITINE INJECTION AND HATCHABILITY AND PERFORMANCES IN TURKEY CHICKENS 451 Groups Carcass yield (%) Breast 1 Liver 1 Gizzard 1 Control 64.83 ± 0.151 a 29.27 ± 0.08 a 1.98 ± 0.12 1.72 ± 0.03 a Sham group 64.62 ± 0.151 a 29.41 ± 0.08 a 2.13 ± 0.12 1.74 ± 0.03 a Carnitine 10 mg 65.34 ± 0.151 b 29.85 ± 0.08 b 1.77 ± 0.12 2.07 ± 0.03 c Carnitine 20 mg 65.57 ± 0.151 b 29.68 ± 0.08 b 2.03 ± 0.12 1.92 ± 0.03 b Carnitine 30 mg 65.48 ± 0.151 b 29.88 ± 0.08 b 1.96 ± 0.12 2.02 ± 0.03 c 1 percentages of live weight (%). Different superscripts a,b,c in the same column indicate significant differences between groups (P < 0.05 or more). TABLE IV: Effects of in ovo administration of L-carnitine on carcass yield, breast and on some visceral organs (liver and gizzard) on day 42 post-hatching in turkey chickens. Results are expressed as mean ± standard error of the mean (SEM). L-carnitine (0.5, 2.0, or 8.0 mg dissolved in 100 μl of a commercial diluent) into broiler breeder eggs on day 18 of incubation had no significant effect on the hatchability. Nevertheless, these authors also suggest that L-carnitine dosages higher than 10 μmol (1.612 mg/egg) and 49.6 μmol/egg (8.0 mg/egg) may improve hatchability or body weight of newly hatched chickens. ARSLAN [3] reported that dietary L-carnitine supplementation in laying hens induced significant increases in hatchability, egg production and the quantity and quality of albumen. On the contrary, injection of L-carnitine (10, 20, or 30 mg dissolved in 0.5 ml of deionized water) on day 6 of incubation of turkey breeder eggs caused significant decrease of hatchability in the present study. Because the rates of lipid metabolism vary between egg-type strains [31], embryos in these 3 types (Leghorn breeder eggs, broiler breeder eggs, turkey breeder eggs) of poultry may differ in their response to supplemental L-carnitine. However it is also quite possible that the solution injection into the yolk sac has created a cavity that may interfere with embryo respiration leading to the embryo death and decrease in hatchability observed in the present study. The body weight gains and food efficiency of newly hatched chickens in the present study were markedly increased when eggs have been injected with 10, 20 or 30 mg/egg L-carnitine compared to the not injected and sham controls while food intake was not significantly modified. These results were in agreement with report of ZHAI et al. [42] that have suggested the use of L-carnitine at higher concentration than 10 µmol/egg for improving growth in chickens. By contrast, KERALAPURATH et al. [17] observed no significant effects of L-carnitine (0.5, 2.0, or 8.0 mg) injection in the amnion of fertilized eggs at the 18 th day of incubation on weight gains, food intake and food conversion ratio determined on days 3, 10, 28 or 46 posthatching. In other experiments conducted on broilers [9, 20, 21] dietary supplemented with L-carnitine (100 mg/kg of food/day), no direct effect on weight gain was evidenced whereas weight gain was improved in progeny from broiler breeders dietary supplemented with 50 mg/kg L- carnitine [27, 28]. YALCIN et al. [39] found that only the highest L-carnitine dosage (200 mg/kg diet) has induced significant increase in body weights after 2, 3 and 4 weeks of treatment in Japanese quails, but food intake and food conversion ratio were not significantly modified by L-carnitine supplementation compared to controls albeit the cumulative food efficiency (calculated throughout the 4 weeks of treatment) tended to be improved when 200 mg/kg of L-carnitine was added to diets of quails. Furthermore, DENIZ and TURKMEN [13] and DENIZ et al. [14] also demonstrated that the addition of Hepabial carnitine (combination of L-carnitine, artichoke leaf extract, sorbitol and choline) in drinking water has significantly increased the hot carcass weight and weight growth in broilers submitted to stressing conditions such as high stocking density and vaccinations, respectively. It is generally admitted that L-carnitine improves the peripheral utilisation of lipids throughout β-oxidation of fatty acids [3, 7]. For example, cholesterolemia and serum triglyceride concentrations were significantly depressed in lambs dietary treated with L-carnitine (200 mg/day) for 45 days [11]. However, the direct carnitine effects on lipid metabolism were not univocally evidenced in broilers and quails [9, 39] and in geese, ARSLAN et al. [5] reported that L-carnitine in drinking water induced a significant increase in saturated fatty acid content in abdominal fat whereas the content of polyunsaturated fatty acids was reduced. Indeed, YALCIN et al. [38, 39] failed to observe significant changes in serum cholesterol concentrations in quails dietary supplemented with L-carnitine at the doses of 50, 100, 150 and 200 mg/kg. In agreement with these findings, addition of L-carnitine chlorhydrate in drinking water at 100 mg / L of water did not alter cholesterolemia in 6 week old quails [4] and UYSAL et al. [36] did not observed significant change in serum cholesterol concentration in male 42 day old quails supplemented with 500 mg/kg L-carnitine in diet. In the same way, serum cholesterol concentrations of broilers [23] and laying hens [22] were not significantly affected by a dietary L-carnitine supplementation. In eggs, yolk lipids provide essential energy to growing embryos. In fact, approximately 90% of the total energy requirement of the developing embryo is derived from fatty acid oxidation of yolk lipids [25]. As chick embryos show a high requirement for L-carnitine whereas the L-carnitine content is low in yolk, the injection of L-carnitine into the yolk sac would promote lipid circulation from fat storage areas such as the yolk sac, leading to an increase in fatty acid catabolism. An increased efficiency in fatty acid oxidation may, likewise, reduce the dependency of the embryo upon gluconeogenesis, thereby sparing muscle tissue protein in the post-hatched chick. This could subsequently lead to an increase in muscle yield during grow-out. Because skeletal muscle is a major site for fatty acid oxidation [19], the effects of exogenous L-carnitine on lipid utilization may become most evident in various muscle

452 SALMANZADEH (M.) AND COLLABORATORS groups. In the present study, the in ovo administration of L- carnitine has significantly increased the breast muscle size. This result generally agrees with our hypothesis that exogenous nutrition provision can substitute for amino acids from the pectoral for glucose gluconeogenesis; i.e. exogenous nutrient supply increases protein deposition, probably by attenuating muscle wasting. In the present study, injection of L-carnitine in the yolk sac has also improved carcass, breast and gizzard weights compared to the control groups. In poultry, the main function of the gizzard is to grind and digest larger food particles. Therefore, the increase gizzard weight indicates a developing preparation for the normal storage and physical digestion of solid feed and improvement of performance in the turkey poults. KIDD et al. [18] showed that dietary L-carnitine supplementation in laying hens induced significant decreases in abdominal fat and more specifically in carcass fat deposits and significant increases in breast meat yield in the corresponding chickens fed with high nutrient density diets. In addition, YALCIN et al. [39] demonstrate that, 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. In contrast, KERALAPURATH et al. [17] stated that, there were also no significant treatment effects on live BW or on relative carcass, abdominal fat pad, drumstick, thigh, wing, or breast weights on day 47 of post-hatch grow-out. 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