Effects of in ovo injection of L-carnitine on subsequent broiler chick tissue nutrient profiles 1,2

Transcription

1 Effects of in ovo injection of L-carnitine on subsequent broiler chick tissue nutrient profiles 1,2 M. M. Keralapurath,* R. W. Keirs, A. Corzo,* L. W. Bennett, R. Pulikanti,* and E. D. Peebles * 3 * Department of Poultry Science, Department of Basic Sciences, and Department of Pathobiology and Population Medicine, Mississippi State University, Mississippi State, MS ABSTRACT Effects of in ovo injection of l-carnitine on BW and the moisture and nutrient biochemical concentrations of various organs and muscles of Ross Ross 308 broiler chicks, hatched from eggs laid by a 28-wk-old breeder flock, were determined through 48 d posthatch. Eggs containing live embryos were injected in the amnion with l-carnitine (0.5, 2.0, or 8.0 mg dissolved in 100 μl of a commercial diluent) on d 18 of incubation using an automated egg injector. Three control groups (noninjected and injected with or without diluent) were also included. On d 0, 3, 10, 28, and 48 posthatch, bird BW and the proportional weights and moisture concentrations of various organs and muscles were determined. Glycogen, glucose, protein, and fat concentrations were also determined in certain tissue samples. Bird BW; proportional liver weight; breast, thigh, and gastrocnemius muscle moisture; liver glycogen, glucose, and protein concentrations; and breast and thigh muscle fat and protein concentrations changed with posthatch bird age. Liver glucose on d 0 and pipping muscle moisture on d 3 posthatch were significantly affected by treatment. In comparison to eggs injected with commercial diluent with no added l-carnitine, liver glucose was reduced by the injection of diluent containing either 0.5 or 8.0 mg of l-carnitine, and pipping muscle moisture was increased by the injection of commercial diluent containing either 0.5 or 2.0 mg of l-carnitine. The modified concentrations of the 2 parameters in response to these treatments were not different from those in noninjected control eggs. In conclusion, l-carnitine added to commercial vaccine diluent at levels between 0.5 and 8.0 mg/100 μl for the commercial injection of broiler hatching eggs may decrease liver glucose and increase pipping muscle moisture concentrations of chicks on d 0 and 3 posthatch, respectively, so that their levels are commensurate with noninjected controls. Key words: broiler chick, in ovo injection, l-carnitine, muscle, organ INTRODUCTION The physiological role of l-carnitine is to transport long-chain fatty acids across the mitochondrial membrane and to subsequently facilitate β-oxidation of long-chain fatty acids for energy production. Casillas and Newburgh (1969) have shown that l-carnitine specifically facilitates the transfer of fatty acyl groups from yolk into tissues of embryonic chicks via the yolk sac membrane. The ratio of esterified short-chain l-carnitine to free l-carnitine is highest in the tissues of avian 2010 Poultry Science Association Inc. Received July 9, Accepted October 26, This is journal no. J from the Mississippi Agricultural and Forestry Experiment Station supported by MIS Use of trade names in this publication does not imply endorsement by Mississippi Agricultural and Forestry Experiment Station of these products, nor similar ones not mentioned. 3 Corresponding author: dpeebles@poultry.msstate.edu 2010 Poultry Science 89 : doi: /ps embryos on d 18 of incubation, thus indicating the importance of fatty acid oxidation for energy production in embryos (Rinaudo et al., 1991). Freshly laid eggs, especially those from hens fed diets of plant origin, were found to have low concentrations of l-carnitine (Chiodi et al., 1994). It is also proven that chicken embryos have a limited capacity to synthesize l-carnitine during incubation (Casillas and Newburgh, 1969) due to the lower activity of the enzyme γ-butyrobetaine hydroxylase, which is essential for l- carnitine biosynthesis (Borum, 1983; Rebouche, 1992). During the later stages of incubation, especially during the pipping process, the embryo expends increased amounts of energy. Therefore, l-carnitine level could be a limiting factor for the β-oxidation of fatty acids during emergence from the eggshell. At such times, exogenous supplementation of l-carnitine could prove to be advantageous (Buyse et al., 2001). The injection of nutritional supplements into the amnion of avian embryos is analogous to in ovo feeding 335

2 336 because amniotic fluid is orally ingested by the embryo before it hatches (Uni and Ferket, 2004). In Single Comb White Leghorns, when l-carnitine was injected in ovo in a 0.05 to 10 µmol/egg dose range on d 17 or 18 of incubation, it was shown to have no effect on hatchability, yolk sac weight, or BW (Zhai et al., 2008). Nevertheless, considering its physiological benefits and the lack of any evidence for its toxicity in poultry when injected in doses up to 10 µmol/egg, Zhai et al. (2008) considered l-carnitine as a potential candidate for improving hatchability and grow-out performance of commercial layers when injected in ovo at concentrations higher than 10 µmol/egg. Sato et al. (2006) have suggested that the difference in lipid metabolic rate between meat-type strains and egg-type strains of poultry could influence their response toward in ovo administration of l-carnitine. Total (free and ester forms) l-carnitine concentrations in fresh 0-d yolks, 18-d yolk sacs, and 18-d embryo livers in eggs from broiler breeder hens that received no supplemental l-carnitine were determined to be 1.85, 4.15, and 28.9 mg/kg of tissue, respectively (Peebles et al., 2007). However, maternal dietary supplementation with 25 mg/kg of l-carnitine increased the l-carnitine concentrations of the 0-d yolk, 18-d yolk sac, and 18-d liver samples by 48.6, 21.7, and 10.0%, respectively, and influenced yolk sac fatty acid β-oxidation in embryos from young broiler breeder hens (Peebles et al., 2007). This same level of l-carnitine in the diet of the hen resulted in a later decrease in abdominal fat of the progeny (Kidd et al., 2005). These results suggest that the in ovo administration of l-carnitine to broiler hatching eggs may influence the nutrient biochemical levels in various organs and muscles of posthatch broilers throughout grow-out, which could subsequently affect performance. Therefore, in this study, l-carnitine was injected into the amnion of broiler breeder eggs on d 18 of incubation to determine if exogenous supplementation of l-carnitine to the embryo would affect the nutrient biochemical profiles of various organs and muscles of broiler progeny through 48 d of posthatch growth. MATERIALS AND METHODS Incubation and Treatments The current experimental protocol was approved by the Institutional Animal Care and Use Committee of Mississippi State University. Ross Ross 308 broiler hatching eggs from a young breeder flock (28 wk of age) were obtained from a commercial source. Within 3 d of egg collection, 168 eggs were weighed and set on each of 4 tray levels (672 total eggs were set). From top to bottom, the middle 4 tray levels (tray levels 3 to 6) of 8 tray levels were used in an individual AVN (1,344- egg capacity) single-stage incubator (Jamesway Incubator Company Inc., Cambridge, Ontario, Canada). The eggs on each replicate tray level were later randomly assigned to 6 treatment groups upon treatment Keralapurath et al. application on d 18 of incubation, so that 28 eggs belonged to each treatment group on each replicate tray. In conjunction with twice daily (a.m. and p.m.) wet and dry bulb recordings from the instrumentation of the machine, 2 data loggers were attached to tray levels 2 and 7 to record the RH and dry bulb temperature of the incubator microenvironment every 5 min. Averages of all dry bulb readings indicated that temperature in the incubator was maintained between 37.5 and 37.7 C. Also, incubator wet bulb temperatures were maintained between 26 and 30 C (RH between 53 and 56%). Eggs visually recognized as contaminated, odd-shaped, or with cracked or malformed shells were not set. On d 10 and 18 of incubation, all eggs were weighed and candled to identify and discard those that were contaminated, unfertilized, or with dead embryos. On d 18 of incubation, all of the eggs were weighed and then in ovo-injected into the amnion according to the following treatment descriptions: 1) noninjected control, 2) sham-injected control (dry punch), 3) shaminjected control [diluent punch; injected with 100 µl of commercial diluent (Merial Limited, Duluth, GA)], 4) injected with 100 µl of diluent containing 0.5 mg of l- carnitine (3.1 µmol of l-carnitine/egg), 5) injected with 100 µl of diluent containing 2.0 mg of l-carnitine (12.4 µmol of l-carnitine/egg), and 6) injected with 100 µl of diluent containing 8.0 mg of l-carnitine (49.6 µmol of l-carnitine/egg). Injected solutions containing l-carnitine were prepared by directly dissolving l-carnitine hydrochloride (~98% purity; Sigma Chemical Co., St. Louis, MO) in the commercial diluent. All of the treatment solutions were prepared in autoclaved water. The osmolality and ph of each of the treatment solutions are provided in Table 1. The groups of eggs belonging to each treatment were randomly arranged (front to back and side to side) on each replicate tray level so that treatment effects would not be influenced by their position within the incubator. Egg Injection and Hatching Sham and treatment solution in ovo injections were conducted on d 18 of incubation. An Intellilab automated single egg injector (AviTech LLC, Salisbury, MD) was used to inject eggs. The injector needle [18.4-cm length and 1.27-mm bore width (o.d.)] had a blunt end Table 1. Osmolality and ph of solutions in the following treatment groups: sham-injected with commercial diluent (diluent), injected with diluent containing 0.5 mg of l-carnitine (diluent l-carnitine), injected with diluent containing 2.0 mg of l-carnitine (diluent l-carnitine), and injected with diluent containing 8.0 mg of l-carnitine (diluent l-carnitine) Treatment Osmolality (mosm) ph Diluent Diluent l-carnitine Diluent l-carnitine Diluent l-carnitine 1,

3 EFFECTS OF l-carnitine ON BROILER CHICK TISSUES 337 and provided a 2.49-cm injection depth. The injector was equipped with an automated cleaning cycle, which ensured complete disinfection after every injection to prevent cross contamination between individual eggs. All treatment solutions were injected at the large end of the egg. Pilot tests with visible dye confirmed the safe delivery of the solutions into the amnion. Treatment solutions were drawn into a 60-mL syringe through a nanopore filter, and the syringe was attached directly to the injector machine. A cleaning cycle was run in the injector machine before injection, and the machine was primed with each of the treatment solutions before injecting respective treatment groups. A 100-μL volume of treatment solution was injected into each designated egg, with an SE of ± 0.1 μl/100 μl. To avoid cross contamination between treatment solutions, eggs belonging to all of the replicate groups within a particular treatment were completely injected before changing the treatment solutions. Furthermore, each tray of eggs designated for treatment remained out of the incubator at room temperature for a maximum of 15 min before being returned to the incubator. After the injection of 16 randomly selected embryonated eggs per treatment replicate group, they were transferred to respective individual hatching baskets in the same incubator. Embryonated noninjected control eggs were likewise randomly selected and transferred. On each of the 4 tray levels, each hatching basket contained 16 eggs from 1 of the 6 treatment groups, so that all 6 treatment groups were again represented on each tray level (96 eggs on each of the 4 tray levels; 384 total eggs transferred). Chick Brooding and Grow-Out On d 0 posthatch (21.5 d of incubation), 12 chicks belonging to the same hatching basket (treatment replicate group) were randomly selected and tagged and then randomly assigned and transferred as a group to a pen in a brooder battery (Petersime Incubator Co., Gettysburg, OH). In the battery, there were a total of 24 pens accommodating chicks from each of the 6 treatment groups on all 4 replicate tray levels. Heat lamps were used to maintain standard commercial brooding temperatures in each pen. On d 20 posthatch, 6 birds from the same brooder battery pen were randomly selected and then randomly assigned and transferred as a group to a larger grow-out battery pen (Petersime Incubator Co.), so that a total of 24 grow-out battery pens were occupied. The temperatures of all 24 pens in the brooder and grow-out batteries were recorded once daily throughout the 48-d posthatch period. Birds were maintained on 24 h of light per day through d 5 and then on 20 h of light per day thereafter. Chicks were provided ad libitum access to feed and water, and diets were formulated to meet or exceed NRC (1994) recommendations throughout the grow-out period. Ingredient percentages and calculated analysis of the broiler starter, grower, and finisher diets were provided by Peebles et al. (2002). Percentage CP in the starter, grower, and finisher diets was 23, 20, and 18, respectively, and ME (kcal/kg) in all 3 diets was 3,194. Data Collection On d 0, 3, 10, 28, and 48 of the posthatch period, chicks were weighed, killed, and necropsied for determination of BW and the weights, moisture concentrations, and nutrient biochemical constituents of various organs and muscles. From each of 4 treatment replicate groups, 2 chicks were sampled on d 0, 3, and 10 posthatch, and 1 chick was sampled on d 28 and 48 posthatch for determination of the specific parameters indicated below. Samples collected included the liver, yolk sac, pipping muscle, and left breast, thigh, and gastrocnemius muscles on d 0 and 3; the liver and left breast, thigh, and gastrocnemius muscles on d 10 and 48; and the liver and left breast and gastrocnemius muscles on d 28. Bird BW was determined on all sampling days. Proportional weights (percentages of live chick BW) of the liver and yolk sac and moisture concentrations of the liver, yolk sac, and 4 muscle groups were determined. In addition, approximately 0.25 g of tissue samples of the liver, left breast muscle, and left thigh muscle collected on d 0, 10, and 48, and of the pipping muscle collected on d 0, were preserved in 10% perchloric acid at 20 C for further biochemical analysis. Tissue samples used for moisture analyses were dried according to the procedure described by Peebles et al. (1998). Tissue moisture concentration was calculated as a percentage of fresh tissue sample weight (Peebles et al., 1999). Tissue nutrient parameters, including glycogen, glucose, protein, and fat concentrations, were determined quantitatively from a single fresh tissue sample using colorimetric methods. Tissue glucose and glycogen concentrations were determined using the phenol-sulfuric acid method as described by Bennett et al. (2007). Colorimetric protein estimation was done according to Lowry et al. (1951), and colorimetric fat estimation was performed according to the methodology devised by Van Handel (1985). Glycogen, glucose, protein, and fat concentrations were reported on a DM basis, adjusting for the calculated moisture contents of the tissue. Statistical Analysis In the current study, a randomized complete block experimental design was employed for the incubational component of the study (d 0). Incubator tray levels were treated as blocks, with all 6 treatments equally represented on each of the 4 tray levels. When moved to the brooder battery, chicks from the same treatment replicate group were assigned to a single pen, whereas the pens were allocated at random to each of the treatment replicate groups. Because of pen number limitations on each level of both the brooder and grow-out batteries, a completely randomized experimental design was employed in the brooding and further grow-out com-

4 338 ponents of the study. Due to differences in experimental design, the data from d 0 were analyzed separately from that on d 3, 10, 28, and 48. Treatment replicate groups were represented by incubator tray level on d 0, by brooder battery pen on d 3 and 10, and by grow-out battery pen on d 28 and 48. Individual chicks were considered as subsamples within each replicate tray or pen. Both 1-way (d 0) and split-plot (d 3, 10, 28, and 48) analyses were used, depending on posthatch age and the parameters analyzed. The effect of treatment was tested on d 0, and for d 3, 10, 28, and 48, the main effects and interactions of age and treatment were tested. The MIXED procedure of SAS software (SAS Institute, 2003) was employed for the analysis of the data, and Fisher s protected least significant difference test was used to compare means. Comparisons between means were made when there were significant global effects (Steel and Torrie, 1980). Global effects and differences among least squares means were considered significant at P RESULTS AND DISCUSSION Age Effects on Parameters Significant bird age (d 3, 10, 28, and 48 posthatch) main effects were noted for BW (P ), proportional liver weight (P ), and breast (P ), thigh (P 0.002), and gastrocnemius muscle (P 0.02) moisture concentrations (data not shown). The age-related changes in broiler BW and proportional liver weight in this study were comparable with those in earlier reported studies by Latour et al. (1994) and Peebles et al. (1997, 2002, 2006). The current data further confirm that the moisture concentrations of the breast, thigh, and gastrocnemius muscles are subject to fluctuations with broiler age throughout the grow-out period. There were also significant age (d 10 and 48 posthatch) main effects for liver glycogen (P ), glucose (P 0.001), and protein (P ) concentrations, as well as for breast muscle fat (P 0.02) and protein (P 0.003) and thigh muscle fat (P ) and protein (P 0.04) concentrations (data not shown). These data indicated that like the moisture concentrations of the muscles, the nutrient biochemical profiles of the livers and muscles of broilers are subject to change with posthatch broiler age. Age-related changes in tissue moisture and nutrient concentrations during embryonic (Murray, 1926) and posthatch (Marion and Edwards, 1962; Marion, 1965; Edwards et al., 1973; MacDonald and Swick, 1981; Pulliainen and Tunkkari, 1984) developmental stages of birds have likewise been previously reported. Ontogenetic changes in broiler tissue contents during the posthatch period may be initiated by the nutritional transition and subsequent serum constituent changes experienced by young chicks (Latour et al., 1995) in association with the loss of the yolk sac by 3 to Keralapurath et al. 4 d of age and the sole consumption of a carbohydraterich exogenous diet. Treatment Effects on Parameters The growing embryo derives most of its energy by the β-oxidation of fatty acid reserves within the egg (Rinaudo et al., 1991). This is particularly important during the pipping process, when the embryo expends extra energy. l-carnitine plays a major role in transferring long-chain fatty acids across the mitochondrial membrane and thus facilitates fatty acid oxidation for energy production (Friedman and Fraenkel, 1955; Fritz, 1955). However, l-carnitine biosynthesis is limited in chick embryos (Casillas and Newburgh, 1969; Borum, 1983; Bremer, 1983; Rebouche, 1992). Consequently, l- carnitine supplementation may increase the efficiency of yolk utilization by growing embryos and of yolk and body tissue lipid reserves in posthatch chicks. An increased efficiency in fatty acid oxidation may, likewise, reduce the dependency of the embryo upon gluconeogenesis, thereby sparing muscle tissue protein in the posthatch chick. This could subsequently lead to an increase in muscle yield during grow-out. Skeletal muscle is a major site for fatty acid oxidation (Klasing, 1998); therefore, the effects of exogenous l-carnitine on lipid utilization may become most evident in various muscle groups. Based on the above information, changes would be expected for proportional yolk sac weight and for the nutrient biochemical profiles of the liver and muscle tissue of birds provided supplemental l-carnitine. Supplemental dietary l-carnitine at a dose of 25 mg/ kg increased ovarian follicle yolk deposition in 27-, 32-, and 38-wk-old broiler breeder hens and altered fatty acid β-oxidation in the yolk sacs of embryos from 27-wk-old breeder hens, causing yolk palmitoleic acid concentrations to be reduced on d 18 of incubation (Peebles et al., 2007). In a companion study, Kidd et al. (2005) reported that the supplemental l-carnitine in the diets of the same breeder hens led to a decrease in abdominal fat in the progeny. Furthermore, the l- carnitine decreased carcass fat and increased breast meat in progeny fed high nutrient density diets (Kidd et al., 2005). Arslan et al. (2004) also found that 100 mg/l of l-carnitine administered to goslings via their drinking water caused total saturated and unsaturated fatty acid concentrations to increase and decrease, respectively, in their abdominal fat stores. When fed at a dose of 25 mg/kg to male broilers, l-carnitine has been shown to increase breast fat concentration (Xu et al., 2003). Clark et al. (2007) also observed that dietary supplementation with l-carnitine caused liver triglyceride levels in aging ovariectomized rats to be reduced, suggesting an increased utilization of fatty acids. However, in the current study, the in ovo administration of l-carnitine did not affect proportional yolk sac weight or yolk sac moisture between d 0 and 3 posthatch (data not shown). Furthermore, liver or muscle tissue fat con-

5 EFFECTS OF l-carnitine ON BROILER CHICK TISSUES 339 Table 2. Liver glucose concentration on d 0 (LGD0) and pipping muscle moisture concentration on d 3 (PMD3) of posthatch growout in noninjected control (NI control), dry punch (dry), sham-injected with commercial diluent (diluent), injected with diluent containing 0.5 mg of l-carnitine (diluent l-carnitine), injected with diluent containing 2.0 mg of l-carnitine (diluent l- carnitine), and injected with diluent containing 8.0 mg of l-carnitine (diluent l-carnitine) treatments 1 Treatment Item NI control Dry Diluent Diluent l-carnitine Diluent l-carnitine Diluent l-carnitine Pooled SEM LGD0 (mg/kg) 26,564 bc 41,623 a 36,923 ab 24,421 c 25,286 bc 24,158 c 4,281 PMD3 (%) 82.1 a 81.7 a 78.5 b 83.2 a 82.3 a 80.7 ab 0.94 a c Treatment means within a row (parameter) with no common superscript differ significantly (P 0.05). 1 For the calculation of means, 2 birds were sampled from each of 4 replicate groups on d 0 and 3. centration was not affected by the l-carnitine concentrations used (data not shown). l-carnitine, therefore, did not appear to exhibit a definitive role in the oxidation of the yolk sac lipid reserves of the chick or in the oxidation of fatty acids in the liver and muscles. Arslan et al. (2004) observed that the proportional liver weight of goslings at 12 wk of age was increased by the provision of l-carnitine in the drinking water at a dose of 100 mg/l. Despite the lack of any treatment effects on proportional liver weight or fat concentration in the current study, liver glucose concentration on d 0 was significantly affected by treatment (P 0.02; Table 2). Liver glucose concentration was higher in the dry punch treatment in comparison to the noninjected controls and those injected with diluent containing the 3 levels of l-carnitine. The liver glucose concentrations of chicks from the diluent punch eggs were also greater than those from eggs that received 0.5 or 8.0 mg of l-carnitine. However, all l-carnitine treatment groups were not different from the noninjected controls. l-carnitine has a significant role in preventing the negative feedback of high acetyl-coenzyme A levels on pyruvate dehydrogenase, due to its buffering action on the acetylcoenzyme A:coenzyme A ratio. This, in turn, causes an increased oxidation of glucose, leading to a better utilization of glucose in tissues, which results in lower plasma glucose concentrations (Feller and Rudman, 1988). A similar result was obtained in the present study, in which the 0.5- and 8.0-mg doses of l-carnitine resulted in a significant decrease in liver glucose concentration on d 0 when compared with chicks hatched from eggs that were sham-injected with commercial diluent. These results suggest that the supplemental l-carnitine may have caused increased glucose oxidation in the livers of the newly hatched chicks. Nevertheless, the lack of any effects of l-carnitine on glycogen and glucose content in the pipping muscle as well as the other muscles examined indicates that the effects of l-carnitine on carbohydrate metabolism occurred solely in the liver and not in the muscular tissue. Although researchers have not identified the role of l-carnitine in increasing the BW in birds, when l-carnitine was supplemented in broiler breeder hen diets at a dose of 50 mg/kg, a significant improvement in BW gain of the progeny chicks resulted (Rabie et al., 1997; Rabie and Szilagyi, 1998). This improvement in BW was explained to be the result of an increase in the efficiency of fatty acid oxidation caused by l-carnitine, which subsequently led to an improved utilization of dietary nitrogen. Dietary l-carnitine at 40 mg/kg has been shown to increase the metabolism of dark meat in commercial broilers, which resulted in an increase in proportional thigh tissue accretion and a subsequently higher thigh muscle yield in broilers fed 200 g of CP/ kg of diet (Kidd et al., 2009). In contrast to the above statements on the role of l-carnitine in dietary nitrogen utilization, Rodehutscord et al. (2002) found no evidence that dietary l-carnitine had any effect on protein utilization and nitrogen balance of tissues. This was similarly evidenced in the current study, in which none of the l-carnitine concentrations were able to cause any significant responses in the protein concentrations of the liver or muscle samples tested (data not shown). On d 3 posthatch, there was a significant effect due to treatment for pipping muscle moisture concentration (P 0.04; Table 2). Pipping muscle moisture concentration on d 3 in the birds from the diluent punch treatment was significantly lower than all other treatments including noninjected controls, except for the diluent and 8.0 mg of l-carnitine treatment, which was intermediate. The highest level of l-carnitine (8.0 mg/100 µl), therefore, had no effect. However, all l-carnitine treatment groups were not different from the noninjected controls. Despite the effect of l-carnitine at the 0.5 and 2.0 mg/100 µl doses on pipping muscle moisture content, the lack of effect of l-carnitine at any level on its fat content suggests that lipid metabolism in the muscle was not influenced by the supplemental l-carnitine. Nevertheless, the observed effect on the pipping muscle occurred after hatch and regression of the pipping muscle starts soon after hatching and continues up to 8 d posthatch (Pohlman, 1919). Therefore, the observed increase in pipping muscle moisture on d 3 posthatch in response to the addition of 0.5 or 2.0 mg of l-carnitine to the commercial diluent may lack relevance for the hatching process. Furthermore, despite the effect on pipping muscle moisture, l-carnitine concentrations did not produce any significant effect on the moisture concentrations of other tissues including the liver and muscles.

6 340 In conclusion, l-carnitine added to commercial diluent, for the commercial injection of broiler hatching eggs, at the 0.5 and 8.0 mg/100 µl levels decreased liver glucose and at the 0.5 and 2.0 mg/100 µl levels increased pipping muscle moisture concentrations of chicks on d 0 and 3 posthatch, respectively. Because liver glucose and pipping muscle moisture concentrations returned to those commensurate with noninjected controls after treatment, it is suggested that l-carnitine may allow liver glucose and pipping muscle moisture concentrations to be normalized in subsequent early posthatch chicks that were previously subjected to the stress of an in ovo injection of commercial vaccine diluent on d 18 of incubation. These effects warrant further investigations into the use of injected supplemental l-carnitine in broiler hatching eggs. ACKNOWLEDGMENTS We express appreciation for the expert technical assistance of Sharon K. Womack of the Mississippi State University Poultry Science Department. REFERENCES Arslan, C., M. Citil, and M. Saatci Effects of l-carnitine administration on growth performance, carcass traits, serum lipids and abdominal fatty acid compositions of geese. Rev. Med. Vet. 155: Bennett, L., R. W. Keirs, E. D. Peebles, and P. D. Gerard Methodologies of tissue preservation and analysis of the glycogen content of the broiler chick liver. Poult. Sci. 83: Borum, P. R Carnitine. Annu. Rev. Nutr. 3: Bremer, F Carnitine Metabolism and functions. Physiol. Rev. 63: Buyse, J., G. P. Janssens, and E. Decuypere The effects of dietary l-carnitine supplementation on the performance, organ weights and circulating hormone and metabolite concentration of broiler chickens reared under a normal or low temperature schedule. Br. Poult. Sci. 42: Casillas, E. R., and R. W. Newburgh l-carnitine and derivatives in embryonic chick tissue. Biochim. Biophys. Acta 184: Chiodi, P., B. Ciani, S. Kentroti, F. Maccari, A. Vernadakis, L. Angelucci, and M. T. Ramacci Carnitine and derivatives in the central nervous system of chick embryos. Int. J. Biochem. 26: Clark, R., A. Balakrishnan, D. Waters, D. Aggarwal, K. Owen, and S. Koo l-carnitine increases liver α-tocopherol and lowers liver and plasma triglycerides in aging ovariectomized rats. J. Nutr. Biochem. 18: Edwards, H. M. Jr., F. Denman, A. Abou-Ashour, and D. Nugara Carcass composition studies. 1. Influences of age, sex and type of dietary fat supplementation on total carcass and fatty acid composition. Poult. Sci. 52: Feller, A. G., and D. Rudman Role of carnitine in human nutrition. J. Nutr. 118: Friedman, S., and G. Fraenkel Reversible enzymatic acetylation of carnitine. Arch. Biochem. Biophys. 59: Fritz, I. B The effect of muscle extracts on the oxidation of palmitic acid by liver slices and homogenates. Acta Physiol. Scand. 34: Kidd, M. T., J. Gilbert, A. Corzo, C. Page, W. S. Virden, and J. C. Woodworth Dietary l-carnitine influences broiler thigh yield. Asian-australas. J. Anim. Sci. 22: Kidd, M. T., C. D. McDaniel, E. D. Peebles, S. J. Barber, A. Corzo, S. L. Branton, and J. C. Woodworth Breeder hen dietary Keralapurath et al. l-carnitine affects progeny carcass traits. Br. Poult. Sci. 46: Klasing, K. C Lipids. Pages in Comparative Avian Nutrition. CAB International, New York, NY. Latour, M. A., E. D. Peebles, C. R. Boyle, and J. D. Brake The effects of dietary fat on growth performance, carcass composition, and feed efficiency in the broiler chick. Poult. Sci. 73: Latour, M. A., E. D. Peebles, C. R. Boyle, J. D. Brake, and T. F. Kellogg Changes in serum lipid, lipoprotein and corticosterone concentrations during neonatal chick development. Biol. Neonate 67: Lowry, O. H., N. J. Rosenbrough, A. L. Farr, and R. J. Randall Protein measurement with the folin phenol reagent. J. Biol. Chem. 193: MacDonald, M. L., and R. W. Swick The effect of protein depletion and repletion on muscle-protein turnover in the chick. Biochem. J. 194: Marion, J. E Effect of age and dietary fat on the lipids of chicken muscle. J. Nutr. 85: Marion, J. E., and H. M. Edwards Jr Observations on the influence of diet and age upon liver lipid changes in the chick. J. Nutr. 77: Murray, H. A. Jr Physiological ontogeny. A. Chicken embryos. VII. The concentration of the organic constituents and calorific value as functions of age. J. Gen. Physiol. 9: NRC Nutrient Requirements of Poultry. 9th rev. ed. Natl. Acad. Press, Washington, DC. Peebles, E. D., W. D. Berry, R. W. Keirs, L. W. Bennett, and P. D. Gerard Effects of injected gluconeogenic supplementation on the performance of broilers from young breeders. Poult. Sci. 85: Peebles, E. D., J. D. Cheaney, J. D. Brake, C. R. Boyle, M. A. Latour, and C. D. McDaniel Effects of added lard fed to broiler chickens during the starter phase. 1. Body and selected organ weights, feed conversion, hematology, and serum glucose. Poult. Sci. 76: Peebles, E. D., M. T. Kidd, C. D. McDaniel, J. P. Tanksley, H. M. Parker, A. Corzo, and J. C. Woodworth Effects of breeder hen age and dietary l-carnitine on progeny embryogenesis. Br. Poult. Sci. 48: Peebles, E. D., L. Li, S. Miller, T. Pansky, S. Whitmarsh, M. A. Latour, and P. D. Gerard Embryo and yolk compositional relationships in broiler hatching eggs during incubation. Poult. Sci. 78: Peebles, E. D., T. Pansky, S. M. Doyle, C. R. Boyle, T. W. Smith, M. A. Latour, and P. D. Gerard Effects of dietary fat and eggshell cuticle removal on egg water loss and embryo growth in broiler hatching eggs. Poult. Sci. 77: Peebles, E. D., C. D. Zumwalt, P. D. Gerard, M. A. Latour, and T. W. Smith Market age live weight, carcass yield, and liver characteristics of broiler offspring from breeder hens fed diets differing in fat and energy contents. Poult. Sci. 81: Pohlman, A. G Concerning the causal factor in the hatching of the chick, with particular reference to the musculus complexus. Anat. Rec. 17: Pulliainen, E., and P. S. Tunkkari Seasonal changes in the weight and composition of the liver of the willow grouse (Lagopus lagopus) in the far north of Finland. Ann. Zool. Fenn. 21: Rabie, M. H., and M. Szilagyi Effects of l-carnitine supplementation of diets differing in energy levels on performance, abdominal fat content, and yield and composition of edible meat of broilers. Br. J. Nutr. 80: Rabie, M. H., M. Szilagyi, and T. Gippert Effects of dietary l-carnitine supplementation and protein level on performance and degree of meatness and fatness of broilers. Acta Biol. Hung. 48: Rebouche, C. J Carnitine function and requirements during the life cycle. FASEB J. 6: Rinaudo, M. T., M. Curto, R. Bruno, M. Piccinini, and C. Marino Acid soluble, short chain esterified and free carnitine in the liver, heart, muscle and brain of pre and post hatched chicks. Int. J. Biochem. 23:59 65.

7 EFFECTS OF l-carnitine ON BROILER CHICK TISSUES 341 Rodehutscord, M., R. Timmler, and A. Dieckmann Effect of l-carnitine supplementation on utilization of energy and protein in broiler chicken fed different dietary fat levels. Arch. Tierernahr. 56: SAS Institute SAS Proprietary Software. Release 9.1. SAS Inst. Inc., Cary, NC. Sato, M., T. Tachibana, and M. Furuse Heat production and lipid metabolism in broiler and layer chickens during embryonic development. Comp. Biochem. Physiol. A 143: Steel, R. G. D., and J. H. Torrie Principles and Procedures of Statistics. A Biometrical Approach. 2nd ed. McGraw-Hill, New York, NY. Uni, Z., and R. P. Ferket Methods for early nutrition and their potential. World s Poult. Sci. J. 60: Van Handel, E Rapid determination of total lipids in mosquitoes. J. Am. Mosq. Control Assoc. 1: Xu, Z. R., M. Q. Wang, H. X. Mao, X. A. Zhan, and C. H. Hu Effects of l-carnitine on growth performance, carcass composition, and metabolism of lipids in male broilers. Poult. Sci. 82: Zhai, W., S. Neuman, M. A. Latour, and P. Y. Hester The effect of in ovo injection of l-carnitine on hatchability of White Leghorns. Poult. Sci. 87: