- Email: [email protected]
Effects of in ovo injection of L-carnitine on hatchability and subsequent broiler performance and slaughter yield1, 2
Effects of in ovo injection of L-carnitine on hatchability and subsequent broiler performance and slaughter yield1,2 M. M. Keralapurath, A. Corzo, R. Pulikanti, W. Zhai, and E. D. Peebles3 Department of Poultry Science, Mississippi State University, Mississippi State 39762 ABSTRACT Effects of in ovo injection of l-carnitine on the hatchability, grow-out performance, and slaughter yield of Ross × Ross 308 broilers from a young breeder flock were determined through 48 d of age. Fertilized eggs 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. Hatchability and hatch rate of fertilized eggs were assessed. Furthermore, subsequent mortality, BW gain, feed intake per bird, and feed conversion were determined through 46 d posthatch. On d 47, live body, carcass, and abdominal fat pad weights, along with the weights of all major commercial cuts including the thigh, drumstick, wings, and breast muscles, were determined. Individual doses
of supplemental l-carnitine had no significant effect on the hatchability or rate of hatch of fertilized eggs; however, significant trends were noted for increased hatchability and length of egg incubation in conjunction with increases in l-carnitine dose. Nevertheless, there were no significant treatment effects on any of the grow-out performance or slaughter yield parameters investigated. In conclusion, although increasing the levels of lcarnitine added to commercial vaccine diluent between 0.5 and 8.0 mg/100 µL for commercial in ovo injection did not significantly influence subsequent broiler growout performance or slaughter yield, l-carnitine dosages above those used in this study have the potential for significantly increasing incubation length and hatchability of broiler hatching eggs.
Key words: broiler, hatchability, in ovo injection, l-carnitine, yield 2010 Poultry Science 89:1497–1501 doi:10.3382/ps.2009-00551
INTRODUCTION Fatty acid β-oxidation is established as a primary source of energy and as a foundation for developmental processes in the avian embryo necessary for its ultimate emergence from the eggshell (Rinaudo et al., 1991; Sato et al., 2006; Moran, 2007). Casillas and Newburgh (1969) have also shown that carnitine specifically facilitates the transfer of fatty acyl groups from yolk into tissues of embryonic chicks via the yolk sac membrane. However, chicken embryos have a limited capacity to synthesize l-carnitine during incubation (Casillas and Newburgh, 1969). Furthermore, freshly laid eggs from hens fed diets of plant origin possess low concentrations of l-carnitine (Chiodi et al., 1994). ©2010 Poultry Science Association Inc. Received November 9, 2009. Accepted March 18, 2010. 1 This is journal no. J-11683 from the Mississippi Agricultural and Forestry Experiment Station supported by MIS-322210. 2 Use of trade names in this publication does not imply endorsement by the Mississippi Agricultural and Forestry Experiment Station of these products, nor similar ones not mentioned. 3 Corresponding author: [email protected]
Dietary l-carnitine at 40 mg/kg has been shown to alter the metabolism of dark meat in commercial broilers, which resulted in an increase in relative thigh tissue accretion and a subsequently higher thigh muscle yield in broilers fed a 20% CP diet (Kidd et al., 2009). Supplementation of the diets of young broiler breeder hens with 25 mg/kg of l-carnitine has been shown to increase l-carnitine concentrations in the yolk sacs and livers of 18-d broiler embryos and to subsequently influence yolk sac fatty acid β-oxidation (Peebles et al., 2007). In a study companion to Peebles et al. (2007), Kidd et al. (2005) also reported that the supplemental l-carnitine in the diets of the same broiler breeder hens led to a decrease in abdominal fat in the progeny. Furthermore, the l-carnitine decreased carcass fat and increased breast meat in those progeny fed high nutrient density diets (Kidd et al., 2005). When l-carnitine was injected in eggs from Single Comb White Leghorn hens, in a 0.05 to 10 µmol/egg (0.008 to 1.612 mg/egg) dose range on d 17 or 18 of incubation, it was shown to have no effect on hatchability or subsequent hatching chick BW (Zhai et al., 2008a). Nevertheless, Zhai et al. (2008a) considered l-carnitine as a potential candidate for improving hatchability and
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grow-out performance of commercial layers when injected in ovo at concentrations higher than 10 µmol/ egg (1.612 mg/egg). In a recent study companion to the current one, Keralapurath et al. (2010) reported that 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 decreased liver glucose and increased pipping muscle moisture concentrations of chicks on d 0 and 3 posthatch, respectively. The changes in liver glucose and pipping muscle moisture in response to the supplemental l-carnitine caused their levels to return to those that were commensurate with noninjected controls. The aforementioned reports suggest that the in ovo administration of l-carnitine to broiler hatching eggs may influence hatchability and posthatch performance. Therefore, in this study, l-carnitine was injected into the amnion of broiler breeder eggs on d 18 of incubation, at concentrations similar to and higher than those used by Zhai et al. (2008a), to determine if exogenous supplementation of l-carnitine to the embryo would affect hatchability and the subsequent grow-out performance and slaughter yield of broilers through 47 d of posthatch age.
MATERIALS AND METHODS Incubation, Treatment, and Brooding 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 on the middle 4 of 8 tray levels) in an individual incubator. Descriptions of the incubator, incubational conditions, and egg candling procedures were provided by Keralapurath et al. (2010). On d 18 of incubation, all of the eggs were weighed and then injected into the amnion according to the following treatment descriptions: 1) noninjected control, 2) sham-injected control (dry punch), 3) sham-injected 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). The eggs on each replicate tray level were randomly assigned to the 6 treatment groups so that 28 eggs belonged to each treatment group on each replicate tray. Egg treatment groups were randomly arranged on each replicate tray level so that treatment effects would not be influenced by their position within
the incubator. Egg injection materials and methods and the preparation, osmolality, and pH of each of the treatment solutions have been described in detail by Keralapurath et al. (2010). Pilot tests with visible dye confirmed the safe delivery of solutions into the amnion (Keralapurath et al., 2010). After the injection, 16 randomly selected embryonated eggs per treatment replicate group were transferred to respective individual hatching baskets in the same incubator. Embryonated noninjected control eggs were likewise randomly selected and transferred to respective individual hatching baskets in the same incubator. 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 every tray level. On d 0 (21.5 d of incubation), chicks (12 per incubational replicate treatment group) were randomly selected, tagged, and transferred to pens in a brooder battery. Chicks from each incubational replicate treatment group were assigned a single pen in the battery so that there were a total of 24 pens accommodating chicks from each of the 6 treatment groups on all 4 replicate tray levels. On d 20 posthatch, 6 birds from each brooder battery pen were randomly selected and transferred to larger grow-out battery pens. Descriptions of the brooder, grow-out batteries, brooding, grow-out conditions, and procedures were as described by Keralapurath et al. (2010).
Data Collection Time of hatch for each chick was monitored every 6 h from 19.5 to 21.5 d. The mean length of incubation was expressed as the time after 20.5 d of incubation. Hatchability was calculated and expressed as a percentage of fertilized eggs. In each pen, mortality was recorded daily and total bird BW, bird numbers, and the weight of unconsumed and added feed were recorded on d 0, 3, 10, 28, and 46 posthatch. Mean BW gain, feed consumption, and feed conversion were calculated for each replicate pen between 0 and 3, 0 and 10, 0 and 28, and 0 and 46 d. In each time period, BW gain was calculated and expressed as grams per bird. Feed consumption (g of feed intake/bird) over the entire grow-out period was calculated by totaling feed consumption in each time interval between each bird sampling. Feed conversion (g of feed consumed/g of BW gain) was calculated by dividing total feed consumption by total weight gain in each pen. Treatment replicate groups were represented by brooder battery pens on d 0, 3, and 10 and by growout battery pens on d 28 and 48. On d 47 of posthatch grow-out, approximately 5 birds from each pen that already had their feed withdrawn for 12 h were weighed, slaughtered, and processed. Weights of the carcass, abdominal fat pad, and the major commercial cuts, including drumsticks, thighs, wings, and breasts, were recorded. Carcass, abdominal fat pad, and commercial cut weights were expressed as percentages of live BW.
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EFFECTS OF l-CARNITINE ON BROILER PERFORMANCE
Statistical Analysis 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 components of the study. Individual birds were considered as subsamples within each replicate pen. A 1-way ANOVA was used to test for the effect of treatment on all parameters. Using SAS software (SAS Institute, 2003), the REG procedure was used to regress the means for time of hatch and hatchability on l-carnitine concentration, the GLIMMIX procedure was used for the analysis of mortality data, and the MIXED procedure was used for the analysis of all other data. Fisher’s protected least significant difference test was used to compare means. Comparisons between means were made when there were significant global effects when P ≤ 0.05 (Steel and Torrie, 1980).
RESULTS AND DISCUSSION Mean length of incubation or time of hatch after 20.5 d (492 h) and hatchability of fertilized eggs for each treatment group are provided in Table 1. Length of incubation and hatchability of fertilized eggs were not significantly different between individual treatments. However, regression analysis performed on the replicate means of the 2 parameters for the 0 (sham), 0.5, 2.0, and 8.0 mg of supplemental l-carnitine treatments revealed significant positive trends for each in association with increasing l-carnitine concentration. There was a significant (P ≤ 0.03) positive relationship between time of hatch and increasing l-carnitine concentration in the diluent (R2 = 0.97). Furthermore, there was a significant (P ≤ 0.03) positive relationship between hatchability and increasing l-carnitine
(R2
= 0.93). Water is a concentration in the diluent metabolic by-product of fat catabolism (Berg et al., 2002). It is also well recognized that sufficient amounts of water must be lost from the avian egg before hatch to accommodate the production of metabolic water and that an insufficient loss of water will prolong incubation length (Rahn and Ar, 1974; Ar and Rahn, 1980). Zhai et al. (2008b) reported that higher concentrations of l-carnitine in the yolk of hatching eggs were associated with decreased hatchling yolk sac fat content, indicating a stimulatory effect of l-carnitine on fat utilization by the developing embryos. In the current study, the positive relationship that l-carnitine concentration had with time of hatch and hatchability indicates that the increase in l-carnitine dose may have stimulated embryonic metabolism, increased yolk fat utilization, and subsequently increased internal egg water content. The possibility exists that the use of an increased number of eggs or higher doses of l-carnitine may lead to significant increases in length of incubation and hatchability. In a companion study conducted by Keralapurath et al. (2010), in which the same birds and treatments were examined on d 0, 3, 10, 28, and 48 posthatch, it was shown that the addition of supplemental l-carnitine to commercial diluent caused chick liver glucose at hatch and pipping muscle moisture on d 3 to return to concentrations that were similar to those of chicks from noninjected control eggs. These results suggested that l-carnitine affected the metabolism of those tissues. However, because Keralapurath et al. (2010) noted no associated effects on liver or pipping muscle lipid concentrations throughout the posthatch period, the rates of fatty acid oxidation in those tissues appear to have not been affected by the in ovo administration of lcarnitine at the dosages described. These current data confirm that l-carnitine dosages as high as 8.0 mg/100 µL of commercial diluent (49.6 µmol/egg) are nontoxic to broiler embryos. Furthermore, the previously noted trends in the current study suggest that concentrations of in ovo supplemental lcarnitine in commercial diluent greater than 8.0 mg/100 µL (49.6 µmol/egg) may have potential for significantly lengthening the time of broiler hatching egg incubation and increasing subsequent hatchability. Because the
Table 1. Broiler hatching egg time of hatch after 20.5 d (492 h) and hatchability of fertilized eggs in noninjected control, dry punch, sham-injected with commercial diluent, injected with diluent containing 0.5 mg of l-carnitine, injected with diluent containing 2.0 mg of l-carnitine, and injected with diluent containing 8.0 mg of l-carnitine treatments1 Item Noninjected control Dry punch Diluent 0.5 mg of carnitine in diluent 2.0 mg of carnitine in diluent 8.0 mg of carnitine in diluent Pooled SEM 1n
Time of hatch after 20.5 d (h:min)
Hatch of fertilized eggs (%)
2:42 3:12 0:36 1:30 2:12 5:06 1:57
93.8 79.7 84.4 82.8 84.4 90.6 4.07
= 4 replicate groups used for calculation of each treatment mean for each parameter.
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Table 2. Body weight gain, feed consumption, feed conversion, and mortality in noninjected control, dry punch, sham-injected with commercial diluent, injected with diluent containing 0.5 mg of l-carnitine, injected with diluent containing 2.0 mg of l-carnitine, and injected with diluent containing 8.0 mg of l-carnitine treatments between 0 and 46 d of posthatch grow-out1
Item Noninjected control Dry punch Diluent 0.5 mg of carnitine in diluent 2.0 mg of carnitine in diluent 8.0 mg of carnitine in diluent Pooled SEM 1n
BW gain (g/bird)
Feed consumption (g/bird)
Feed conversion (g of feed consumption/g of BW gain)
Mortality (%)
2,177 2,118 2,168 2,159 2,244 2,275 73.9
4,114 3,911 4,137 4,164 4,142 4,251 120
1.80 1.81 1.85 1.83 1.80 1.80 0.035
0 4.55 4.35 4.55 0 6.25 2.143
= 4 replicate groups used for calculation of each treatment mean for each parameter.
rates of lipid metabolism vary between egg- and meattype strains of poultry (Sato et al., 2006), embryos in these 2 types of poultry may differ in their response to supplemental l-carnitine. Nevertheless, in the current study, the results were similar to those of Zhai et al. (2008a), who used Single Comb White Leghorns. It was shown in that study that the in ovo administration of up to 10 µmol of l-carnitine (1.612 mg/egg) was nontoxic, and it was suggested that l-carnitine levels higher than 10 µmol (1.612 mg/egg) may improve hatchability and hatchling BW. There were no significant treatment effects on mortality, BW gain, feed consumption, or feed conversion between 0 and 3, 0 and 10, 0 and 28, or 0 and 46 d posthatch. The treatment means for BW gain, feed consumption, feed conversion, and mortality over the entire grow-out period (0 to 46 d) are provided in Table 2 for observation. There were also no significant treatment effects on live BW or on relative carcass, abdominal fat pad, drumstick, thigh, wing, or breast weights on d 47 of posthatch grow-out (Table 3). An increased efficiency in fatty acid oxidation may reduce the embryo’s dependency upon gluconeogenic pathways and spare muscle tissue protein in the posthatch chick, which could subsequently lead to an increase in muscle yield during grow-out. Because skeletal muscle is a major site for fatty acid oxidation (Klasing, 1998), the effects of exogenous l-carnitine on lipid utilization may become most evident in various muscle groups. Kidd et al. (2009) reported that 40 mg/kg of supplemental dietary l-carnitine heightened dark meat
yield in commercial broilers, resulting in a relative elevation in thigh tissue accretion without influencing BW gain, feed consumption, feed conversion, or mortality, and without having any compromising effect on breast tissue accretion. Further, Xu et al. (2003) reported that when fed at a dose of 25 mg/kg to male broilers, lcarnitine increased breast fat concentration. The use of L-carnitine at a concentration of 25 mg/kg in the diets of broiler breeder hens showed no gross evidence of having any influence on subsequent broiler embryo growth and metabolism (Peebles et al., 2007). Furthermore, in a companion study by Kidd et al. (2005), in which the same breeder hens and diets were used, no effects on posthatch growth performance measurements due to l-carnitine supplementation were reported. Nevertheless, despite the lack of any noted effects in the embryonic and grow-out periods due to l-carnitine supplementation, later effects on carcass fat deposition in broilers at slaughter age were found by Kidd et al. (2005). Kidd et al. (2005) showed that maternally fed l-carnitine decreased abdominal fat and more specifically decreased carcass fat and increased breast meat yield in those progeny that had been fed high nutrient density diets. In other studies, when lcarnitine 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) and 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. However,
Table 3. Live BW and relative carcass, abdominal fat pad, drumstick, thigh, wing, and breast weights in noninjected control, dry punch, sham-injected with commercial diluent, injected with diluent containing 0.5 mg of l-carnitine, injected with diluent containing 2.0 mg of l-carnitine, and injected with diluent containing 8.0 mg of l-carnitine treatments on d 47 of posthatch grow-out1 Item Noninjected control Dry punch Diluent 0.5 mg of carnitine in diluent 2.0 mg of carnitine in diluent 8.0 mg of carnitine in diluent Pooled SEM 1n
Live BW (g)
Carcass (%)
Abdominal fat pad (%)
Drumstick (%)
Thigh (%)
Wing (%)
Breast (%)
2,233 2,255 2,113 2,298 2,314 2,350 89.7
67.2 66.7 66.5 66.9 66.8 66.0 0.57
1.40 1.44 1.45 1.41 1.31 1.31 0.129
9.13 9.68 9.53 9.50 9.45 9.21 0.177
12.1 11.8 12.0 11.8 12.3 11.8 0.38
7.84 7.97 8.15 7.94 8.00 7.90 0.127
20.6 19.8 20.1 20.1 19.5 19.6 0.443
= 4 replicate groups used for calculation of each treatment mean for each parameter.
EFFECTS OF l-CARNITINE ON BROILER PERFORMANCE
in contrast to the above statement on the role of lcarnitine 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. In conclusion, l-carnitine added to commercial diluent at levels between 0.5 and 8.0 mg/100 µL for the commercial injection of broiler hatching eggs did not significantly influence subsequent grow-out performance or slaughter yield. However, significant positive trends in length of incubation and hatchability of fertilized eggs with increased dosage of l-carnitine suggest that in ovo l-carnitine dosages greater than 49.6 µmol/ egg (8.0 mg/egg) have the potential for significantly increasing incubation length and hatchability of 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 Ar, M., and H. Rahn. 1980. Water in the avian egg: Overall budget of incubation. Am. Zool. 20:373–384. Berg, J. M., J. L. Tymoczko, and L. Styer. 2002. Fatty acid metabolism. Pages 601–632 in Biochemistry. 5th ed. W. H. Freeman and Company, New York, NY. Casillas, E. R., and R. W. Newburgh. 1969. l-Carnitine and derivatives in embryonic chick tissue. Biochim. Biophys. Acta 184:566–577. Chiodi, P., B. Ciani, S. Kentroti, F. Maccari, A. Vernadakis, L. Angelucci, and M. T. Ramacci. 1994. Carnitine and derivatives in the central nervous system of chick embryos. Int. J. Biochem. 26:711–720. Keralapurath, M. M., R. W. Keirs, A. Corzo, L. W. Bennett, R. Pulikanti, and E. D. Peebles. 2010. Effects of in ovo injection of l-carnitine on subsequent broiler chick tissue nutrient profiles. Poult. Sci. 89:335–341. Kidd, M. T., J. Gilbert, A. Corzo, C. Page, W. S. Virden, and J. C. Woodworth. 2009. Dietary l-carnitine influences broiler thigh yield. Asian-australas. J. Anim. Sci. 22:681–685.
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Kidd, M. T., C. D. McDaniel, E. D. Peebles, S. J. Barber, A. Corzo, S. L. Branton, and J. C. Woodworth. 2005. Breeder hen dietary l-carnitine affects progeny carcass traits. Br. Poult. Sci. 46:91– 103. Klasing, K. C. 1998. Lipids. Pages 171–200 in Comparative Avian Nutrition. CAB International, New York, NY. Moran, E. T. Jr. 2007. Nutrition of the developing embryo and hatchling. Poult. Sci. 86:1043–1049. Peebles, E. D., M. T. Kidd, C. D. McDaniel, J. P. Tanksley, H. M. Parker, A. Corzo, and J. C. Woodworth. 2007. Effects of breeder hen age and dietary l-carnitine on progeny embryogenesis. Br. Poult. Sci. 48:299–307. Rabie, M. H., and M. Szilagyi. 1998. 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. 80:391–400. Rabie, M. H., M. Szilagyi, and T. Gippert. 1997. Effects of dietary l-carnitine supplementation and protein level on performance and degree of meatness and fatness of broilers. Acta Biol. Hung. 48:221–239. Rahn, H., and A. Ar. 1974. The avian egg: Incubation time and water loss. Condor 76:147–152. Rinaudo, M. T., M. Curto, R. Bruno, M. Piccinini, and C. Marino. 1991. 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. Rodehutscord, M., R. Timmler, and A. Dieckmann. 2002. Effect of l-carnitine supplementation on utilization of energy and protein in broiler chicken fed different dietary fat levels. Arch. Tierernahr. 56:431–441. SAS Institute. 2003. SAS Proprietary Software Release 9.1. SAS Institute Inc., Cary, NC. Sato, M., T. Tachibana, and M. Furuse. 2006. Heat production and lipid metabolism in broiler and layer chickens during embryonic development. Comp. Biochem. Physiol. A 143:382–388. Steel, R. G. D., and J. H. Torrie. 1980. Principles and Procedures of Statistics. A Biometrical Approach. 2nd ed. McGraw-Hill, New York, NY. Xu, Z. R., M. Q. Wang, H. X. Mao, X. A. Zhan, and C. H. Hu. 2003. Effects of l-carnitine on growth performance, carcass composition, and metabolism of lipids in male broilers. Poult. Sci. 82:408–413. Zhai, W., S. Neuman, M. A. Latour, and P. Y. Hester. 2008a. The effect of in ovo injection of l-carnitine on hatchability of White Leghorns. Poult. Sci. 87:569–572. Zhai, W., S. Neuman, M. A. Latour, and P. Y. Hester. 2008b. The effect of male and female supplementation of l-carnitine on reproductive traits of White Leghorns. Poult. Sci. 87:1171–1181.
of supplemental l-carnitine had no significant effect on the hatchability or rate of hatch of fertilized eggs; however, significant trends were noted for increased hatchability and length of egg incubation in conjunction with increases in l-carnitine dose. Nevertheless, there were no significant treatment effects on any of the grow-out performance or slaughter yield parameters investigated. In conclusion, although increasing the levels of lcarnitine added to commercial vaccine diluent between 0.5 and 8.0 mg/100 µL for commercial in ovo injection did not significantly influence subsequent broiler growout performance or slaughter yield, l-carnitine dosages above those used in this study have the potential for significantly increasing incubation length and hatchability of broiler hatching eggs.
Key words: broiler, hatchability, in ovo injection, l-carnitine, yield 2010 Poultry Science 89:1497–1501 doi:10.3382/ps.2009-00551
INTRODUCTION Fatty acid β-oxidation is established as a primary source of energy and as a foundation for developmental processes in the avian embryo necessary for its ultimate emergence from the eggshell (Rinaudo et al., 1991; Sato et al., 2006; Moran, 2007). Casillas and Newburgh (1969) have also shown that carnitine specifically facilitates the transfer of fatty acyl groups from yolk into tissues of embryonic chicks via the yolk sac membrane. However, chicken embryos have a limited capacity to synthesize l-carnitine during incubation (Casillas and Newburgh, 1969). Furthermore, freshly laid eggs from hens fed diets of plant origin possess low concentrations of l-carnitine (Chiodi et al., 1994). ©2010 Poultry Science Association Inc. Received November 9, 2009. Accepted March 18, 2010. 1 This is journal no. J-11683 from the Mississippi Agricultural and Forestry Experiment Station supported by MIS-322210. 2 Use of trade names in this publication does not imply endorsement by the Mississippi Agricultural and Forestry Experiment Station of these products, nor similar ones not mentioned. 3 Corresponding author: [email protected]
Dietary l-carnitine at 40 mg/kg has been shown to alter the metabolism of dark meat in commercial broilers, which resulted in an increase in relative thigh tissue accretion and a subsequently higher thigh muscle yield in broilers fed a 20% CP diet (Kidd et al., 2009). Supplementation of the diets of young broiler breeder hens with 25 mg/kg of l-carnitine has been shown to increase l-carnitine concentrations in the yolk sacs and livers of 18-d broiler embryos and to subsequently influence yolk sac fatty acid β-oxidation (Peebles et al., 2007). In a study companion to Peebles et al. (2007), Kidd et al. (2005) also reported that the supplemental l-carnitine in the diets of the same broiler breeder hens led to a decrease in abdominal fat in the progeny. Furthermore, the l-carnitine decreased carcass fat and increased breast meat in those progeny fed high nutrient density diets (Kidd et al., 2005). When l-carnitine was injected in eggs from Single Comb White Leghorn hens, in a 0.05 to 10 µmol/egg (0.008 to 1.612 mg/egg) dose range on d 17 or 18 of incubation, it was shown to have no effect on hatchability or subsequent hatching chick BW (Zhai et al., 2008a). Nevertheless, Zhai et al. (2008a) considered l-carnitine as a potential candidate for improving hatchability and
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grow-out performance of commercial layers when injected in ovo at concentrations higher than 10 µmol/ egg (1.612 mg/egg). In a recent study companion to the current one, Keralapurath et al. (2010) reported that 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 decreased liver glucose and increased pipping muscle moisture concentrations of chicks on d 0 and 3 posthatch, respectively. The changes in liver glucose and pipping muscle moisture in response to the supplemental l-carnitine caused their levels to return to those that were commensurate with noninjected controls. The aforementioned reports suggest that the in ovo administration of l-carnitine to broiler hatching eggs may influence hatchability and posthatch performance. Therefore, in this study, l-carnitine was injected into the amnion of broiler breeder eggs on d 18 of incubation, at concentrations similar to and higher than those used by Zhai et al. (2008a), to determine if exogenous supplementation of l-carnitine to the embryo would affect hatchability and the subsequent grow-out performance and slaughter yield of broilers through 47 d of posthatch age.
MATERIALS AND METHODS Incubation, Treatment, and Brooding 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 on the middle 4 of 8 tray levels) in an individual incubator. Descriptions of the incubator, incubational conditions, and egg candling procedures were provided by Keralapurath et al. (2010). On d 18 of incubation, all of the eggs were weighed and then injected into the amnion according to the following treatment descriptions: 1) noninjected control, 2) sham-injected control (dry punch), 3) sham-injected 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). The eggs on each replicate tray level were randomly assigned to the 6 treatment groups so that 28 eggs belonged to each treatment group on each replicate tray. Egg treatment groups were randomly arranged on each replicate tray level so that treatment effects would not be influenced by their position within
the incubator. Egg injection materials and methods and the preparation, osmolality, and pH of each of the treatment solutions have been described in detail by Keralapurath et al. (2010). Pilot tests with visible dye confirmed the safe delivery of solutions into the amnion (Keralapurath et al., 2010). After the injection, 16 randomly selected embryonated eggs per treatment replicate group were transferred to respective individual hatching baskets in the same incubator. Embryonated noninjected control eggs were likewise randomly selected and transferred to respective individual hatching baskets in the same incubator. 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 every tray level. On d 0 (21.5 d of incubation), chicks (12 per incubational replicate treatment group) were randomly selected, tagged, and transferred to pens in a brooder battery. Chicks from each incubational replicate treatment group were assigned a single pen in the battery so that there were a total of 24 pens accommodating chicks from each of the 6 treatment groups on all 4 replicate tray levels. On d 20 posthatch, 6 birds from each brooder battery pen were randomly selected and transferred to larger grow-out battery pens. Descriptions of the brooder, grow-out batteries, brooding, grow-out conditions, and procedures were as described by Keralapurath et al. (2010).
Data Collection Time of hatch for each chick was monitored every 6 h from 19.5 to 21.5 d. The mean length of incubation was expressed as the time after 20.5 d of incubation. Hatchability was calculated and expressed as a percentage of fertilized eggs. In each pen, mortality was recorded daily and total bird BW, bird numbers, and the weight of unconsumed and added feed were recorded on d 0, 3, 10, 28, and 46 posthatch. Mean BW gain, feed consumption, and feed conversion were calculated for each replicate pen between 0 and 3, 0 and 10, 0 and 28, and 0 and 46 d. In each time period, BW gain was calculated and expressed as grams per bird. Feed consumption (g of feed intake/bird) over the entire grow-out period was calculated by totaling feed consumption in each time interval between each bird sampling. Feed conversion (g of feed consumed/g of BW gain) was calculated by dividing total feed consumption by total weight gain in each pen. Treatment replicate groups were represented by brooder battery pens on d 0, 3, and 10 and by growout battery pens on d 28 and 48. On d 47 of posthatch grow-out, approximately 5 birds from each pen that already had their feed withdrawn for 12 h were weighed, slaughtered, and processed. Weights of the carcass, abdominal fat pad, and the major commercial cuts, including drumsticks, thighs, wings, and breasts, were recorded. Carcass, abdominal fat pad, and commercial cut weights were expressed as percentages of live BW.
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EFFECTS OF l-CARNITINE ON BROILER PERFORMANCE
Statistical Analysis 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 components of the study. Individual birds were considered as subsamples within each replicate pen. A 1-way ANOVA was used to test for the effect of treatment on all parameters. Using SAS software (SAS Institute, 2003), the REG procedure was used to regress the means for time of hatch and hatchability on l-carnitine concentration, the GLIMMIX procedure was used for the analysis of mortality data, and the MIXED procedure was used for the analysis of all other data. Fisher’s protected least significant difference test was used to compare means. Comparisons between means were made when there were significant global effects when P ≤ 0.05 (Steel and Torrie, 1980).
RESULTS AND DISCUSSION Mean length of incubation or time of hatch after 20.5 d (492 h) and hatchability of fertilized eggs for each treatment group are provided in Table 1. Length of incubation and hatchability of fertilized eggs were not significantly different between individual treatments. However, regression analysis performed on the replicate means of the 2 parameters for the 0 (sham), 0.5, 2.0, and 8.0 mg of supplemental l-carnitine treatments revealed significant positive trends for each in association with increasing l-carnitine concentration. There was a significant (P ≤ 0.03) positive relationship between time of hatch and increasing l-carnitine concentration in the diluent (R2 = 0.97). Furthermore, there was a significant (P ≤ 0.03) positive relationship between hatchability and increasing l-carnitine
(R2
= 0.93). Water is a concentration in the diluent metabolic by-product of fat catabolism (Berg et al., 2002). It is also well recognized that sufficient amounts of water must be lost from the avian egg before hatch to accommodate the production of metabolic water and that an insufficient loss of water will prolong incubation length (Rahn and Ar, 1974; Ar and Rahn, 1980). Zhai et al. (2008b) reported that higher concentrations of l-carnitine in the yolk of hatching eggs were associated with decreased hatchling yolk sac fat content, indicating a stimulatory effect of l-carnitine on fat utilization by the developing embryos. In the current study, the positive relationship that l-carnitine concentration had with time of hatch and hatchability indicates that the increase in l-carnitine dose may have stimulated embryonic metabolism, increased yolk fat utilization, and subsequently increased internal egg water content. The possibility exists that the use of an increased number of eggs or higher doses of l-carnitine may lead to significant increases in length of incubation and hatchability. In a companion study conducted by Keralapurath et al. (2010), in which the same birds and treatments were examined on d 0, 3, 10, 28, and 48 posthatch, it was shown that the addition of supplemental l-carnitine to commercial diluent caused chick liver glucose at hatch and pipping muscle moisture on d 3 to return to concentrations that were similar to those of chicks from noninjected control eggs. These results suggested that l-carnitine affected the metabolism of those tissues. However, because Keralapurath et al. (2010) noted no associated effects on liver or pipping muscle lipid concentrations throughout the posthatch period, the rates of fatty acid oxidation in those tissues appear to have not been affected by the in ovo administration of lcarnitine at the dosages described. These current data confirm that l-carnitine dosages as high as 8.0 mg/100 µL of commercial diluent (49.6 µmol/egg) are nontoxic to broiler embryos. Furthermore, the previously noted trends in the current study suggest that concentrations of in ovo supplemental lcarnitine in commercial diluent greater than 8.0 mg/100 µL (49.6 µmol/egg) may have potential for significantly lengthening the time of broiler hatching egg incubation and increasing subsequent hatchability. Because the
Table 1. Broiler hatching egg time of hatch after 20.5 d (492 h) and hatchability of fertilized eggs in noninjected control, dry punch, sham-injected with commercial diluent, injected with diluent containing 0.5 mg of l-carnitine, injected with diluent containing 2.0 mg of l-carnitine, and injected with diluent containing 8.0 mg of l-carnitine treatments1 Item Noninjected control Dry punch Diluent 0.5 mg of carnitine in diluent 2.0 mg of carnitine in diluent 8.0 mg of carnitine in diluent Pooled SEM 1n
Time of hatch after 20.5 d (h:min)
Hatch of fertilized eggs (%)
2:42 3:12 0:36 1:30 2:12 5:06 1:57
93.8 79.7 84.4 82.8 84.4 90.6 4.07
= 4 replicate groups used for calculation of each treatment mean for each parameter.
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Table 2. Body weight gain, feed consumption, feed conversion, and mortality in noninjected control, dry punch, sham-injected with commercial diluent, injected with diluent containing 0.5 mg of l-carnitine, injected with diluent containing 2.0 mg of l-carnitine, and injected with diluent containing 8.0 mg of l-carnitine treatments between 0 and 46 d of posthatch grow-out1
Item Noninjected control Dry punch Diluent 0.5 mg of carnitine in diluent 2.0 mg of carnitine in diluent 8.0 mg of carnitine in diluent Pooled SEM 1n
BW gain (g/bird)
Feed consumption (g/bird)
Feed conversion (g of feed consumption/g of BW gain)
Mortality (%)
2,177 2,118 2,168 2,159 2,244 2,275 73.9
4,114 3,911 4,137 4,164 4,142 4,251 120
1.80 1.81 1.85 1.83 1.80 1.80 0.035
0 4.55 4.35 4.55 0 6.25 2.143
= 4 replicate groups used for calculation of each treatment mean for each parameter.
rates of lipid metabolism vary between egg- and meattype strains of poultry (Sato et al., 2006), embryos in these 2 types of poultry may differ in their response to supplemental l-carnitine. Nevertheless, in the current study, the results were similar to those of Zhai et al. (2008a), who used Single Comb White Leghorns. It was shown in that study that the in ovo administration of up to 10 µmol of l-carnitine (1.612 mg/egg) was nontoxic, and it was suggested that l-carnitine levels higher than 10 µmol (1.612 mg/egg) may improve hatchability and hatchling BW. There were no significant treatment effects on mortality, BW gain, feed consumption, or feed conversion between 0 and 3, 0 and 10, 0 and 28, or 0 and 46 d posthatch. The treatment means for BW gain, feed consumption, feed conversion, and mortality over the entire grow-out period (0 to 46 d) are provided in Table 2 for observation. There were also no significant treatment effects on live BW or on relative carcass, abdominal fat pad, drumstick, thigh, wing, or breast weights on d 47 of posthatch grow-out (Table 3). An increased efficiency in fatty acid oxidation may reduce the embryo’s dependency upon gluconeogenic pathways and spare muscle tissue protein in the posthatch chick, which could subsequently lead to an increase in muscle yield during grow-out. Because skeletal muscle is a major site for fatty acid oxidation (Klasing, 1998), the effects of exogenous l-carnitine on lipid utilization may become most evident in various muscle groups. Kidd et al. (2009) reported that 40 mg/kg of supplemental dietary l-carnitine heightened dark meat
yield in commercial broilers, resulting in a relative elevation in thigh tissue accretion without influencing BW gain, feed consumption, feed conversion, or mortality, and without having any compromising effect on breast tissue accretion. Further, Xu et al. (2003) reported that when fed at a dose of 25 mg/kg to male broilers, lcarnitine increased breast fat concentration. The use of L-carnitine at a concentration of 25 mg/kg in the diets of broiler breeder hens showed no gross evidence of having any influence on subsequent broiler embryo growth and metabolism (Peebles et al., 2007). Furthermore, in a companion study by Kidd et al. (2005), in which the same breeder hens and diets were used, no effects on posthatch growth performance measurements due to l-carnitine supplementation were reported. Nevertheless, despite the lack of any noted effects in the embryonic and grow-out periods due to l-carnitine supplementation, later effects on carcass fat deposition in broilers at slaughter age were found by Kidd et al. (2005). Kidd et al. (2005) showed that maternally fed l-carnitine decreased abdominal fat and more specifically decreased carcass fat and increased breast meat yield in those progeny that had been fed high nutrient density diets. In other studies, when lcarnitine 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) and 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. However,
Table 3. Live BW and relative carcass, abdominal fat pad, drumstick, thigh, wing, and breast weights in noninjected control, dry punch, sham-injected with commercial diluent, injected with diluent containing 0.5 mg of l-carnitine, injected with diluent containing 2.0 mg of l-carnitine, and injected with diluent containing 8.0 mg of l-carnitine treatments on d 47 of posthatch grow-out1 Item Noninjected control Dry punch Diluent 0.5 mg of carnitine in diluent 2.0 mg of carnitine in diluent 8.0 mg of carnitine in diluent Pooled SEM 1n
Live BW (g)
Carcass (%)
Abdominal fat pad (%)
Drumstick (%)
Thigh (%)
Wing (%)
Breast (%)
2,233 2,255 2,113 2,298 2,314 2,350 89.7
67.2 66.7 66.5 66.9 66.8 66.0 0.57
1.40 1.44 1.45 1.41 1.31 1.31 0.129
9.13 9.68 9.53 9.50 9.45 9.21 0.177
12.1 11.8 12.0 11.8 12.3 11.8 0.38
7.84 7.97 8.15 7.94 8.00 7.90 0.127
20.6 19.8 20.1 20.1 19.5 19.6 0.443
= 4 replicate groups used for calculation of each treatment mean for each parameter.
EFFECTS OF l-CARNITINE ON BROILER PERFORMANCE
in contrast to the above statement on the role of lcarnitine 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. In conclusion, l-carnitine added to commercial diluent at levels between 0.5 and 8.0 mg/100 µL for the commercial injection of broiler hatching eggs did not significantly influence subsequent grow-out performance or slaughter yield. However, significant positive trends in length of incubation and hatchability of fertilized eggs with increased dosage of l-carnitine suggest that in ovo l-carnitine dosages greater than 49.6 µmol/ egg (8.0 mg/egg) have the potential for significantly increasing incubation length and hatchability of broiler hatching eggs.
ACKNOWLEDGMENTS We express appreciation for the expert technical assistance of Sharon K. Womack of the Mississippi State University Poultry Science Department.
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