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Poult Sci 2008. 87:569-572. doi:10.3382/ps.2007-00348
© 2008 Poultry Science Association
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PHYSIOLOGY, ENDOCRINOLOGY, AND REPRODUCTION: Research Note

The Effect of In Ovo Injection of L-Carnitine on Hatchability of White Leghorns1

W. Zhai, S. Neuman, M. A. Latour and P. Y. Hester2

Department of Animal Sciences, Purdue University, West Lafayette, IN 47907

2 Corresponding author: phester{at}purdue.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Two experiments were conducted to determine if L-carnitine injected in ovo affected hatchability. Eggs of experiment 1 were injected with sterilized saline (0.85%) or L-carnitine (0.25, 0.50, 1.00, or 2.00 µmol dissolved in saline). An additional group of eggs served as noninjected controls. Hatchabilities were unaffected by treatment (94% for noninjected controls; 94% for saline injected eggs; and 87, 87, 88, and 88% for eggs injected with 0.25, 0.50, 1.00, or 2.00 µmol of L-carnitine, respectively; SEM = 1). Yolk sac weights retrieved from hatchings that were subjected to 0, 0.25, or 0.50 µmol of L-carnitine as embryos through in ovo injection were 3.9, 3.8, and 3.6 g, respectively (SEM = 0.1, P = 0.71). Eggs used in experiment 2 were injected with a wider dosimetry of L-carnitine. Fertile eggs were injected with sterilized saline (0.85%) or L-carnitine (0.05, 0.5, 5, or 10 µmol dissolved in saline). An additional group of eggs served as noninjected controls. Chick BW and % hatch were unaffected by treatment (76% for noninjected controls; 74% for saline injected eggs; and 77, 77, 68, and 76% for eggs injected with 0.05, 0.5, 5, or 10 µmol of L-carnitine, respectively; SEM = 3). In ovo injection of L-carnitine into fertile chicken eggs at 17 or 18 d of incubation did not affect hatchability, yolk sac weight, or BW.

Key Words: L-carnitine • in ovo injection • hatchability • embryo • White Leghorn


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
L-Carnitine acts as an antioxidant that ultimately results in a decrease in reactive oxygen species by removing excessive levels of intracellular acetyl-CoA that induces mitochondrial reactive oxygen species production (Vicari and Calogero, 2001; Agarwal and Said, 2004; Agarwal et al., 2005). Free radicals responsible for lipid peroxidation are scavenged by L-carnitine (Rebouche, 1992). Other anti-oxidants such as ascorbic acid and vitamin E, as well as antioxidant enzymes, are protected against potential peroxidation by L-carnitine (Kalaiselvi and Panneerselvam, 1998).

In addition to its role as an antioxidant, L-carnitine transports long chain fatty acids across mitochondrial membranes for β-oxidation of fatty acids. 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. In such situations, exogenous supplementation of L-carnitine could prove advantageous (Buyse et al., 2001) and could in turn be used by the chick during hatching.

L-Carnitine can be synthesized from lysine and methionine in animals. However, L-carnitine synthesis is limited in chicken embryos. Gamma-butyrobetaine, an intermediate substance required for L-carnitine biosynthesis, is limited in embryos and young animals due to low activity of {gamma}-butyrobetaine hydroxylase (Borum, 1983; Rebouche, 1992).

During late embryogenesis, feeding solutions administered into the amnionic fluid are consumed by the embryo, digested, and absorbed by the embryonic intestine prior to pipping (Uni et al., 2005). In ovo feeding of supplemental nutrients may help to overcome the constraint of limited egg nutrients (Foye et al., 2006).

Rapid development, a high energy requirement, and a low level of L-carnitine synthesis may make supplementation of L-carnitine beneficial to chicken embryos. We hypothesize that administration of L-carnitine to the amnion will improve fatty acid β-oxidation and suppress lipid peroxidation, thus increasing hatchability. Our hypothesis was tested by comparing hatchability, BW, and yolk sac weight of hatchlings administered exogenous L-carnitine in ovo at 17 or 18 d of incubation with controls.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Experiment 1

A preliminary study was done in which 20 fertile eggs were injected with violet dye at 17 d of incubation and then opened immediately following injection to verify if the injectable entered the amnion. Subsequently, 2,520 chicken eggs were collected from White Leghorns at 51 wk of age and stored at 10° C prior to initiation of incubation. The day of egg collection was recorded to allow for equal distribution of eggs to treatment groups relative to storage time. Eggs were incubated for 18 d using standard incubation conditions. Eggs were removed from the incubator at 18 d and candled individually for fertility. A total of 1,680 fertile eggs was available for the experiment. The eggs were injected in ovo at 18 d of incubation with sterilized saline (0.85%) or L-carnitine (0.25, 0.50, 1.00, 2.00 µmol dissolved in sterilized saline; Lonza Inc., Allendale, NJ) in 100-µL volume. In addition, a control group of eggs was used that received no injection. The saline or L-carnitine was injected into the large end of the egg using a 2.54-cm 21-gauge needle. The entire length of the needle was extended into the hatching egg to ensure that the needle punctured the amnion, allowing the embryo to swallow the amnion and the injectable. The injection site was not sealed.

Two-hundred eighty eggs were assigned to each treatment (6 treatments total) with 28 replicates per treatment. An experimental unit consisted of 10 fertile eggs. Ten eggs each were placed in their own individual hatching compartment following injection to keep experimental units separated. At 22 d of incubation, the number of chicks that hatched was counted in an experimental unit and group BW determined. All unhatched eggs were opened to determine stage of development to adjust hatchability for embryonic deaths that occurred prior to 18 d of incubation not identified via candling.

Hatchlings were euthanized, and yolk sacs were retrieved from chicks that hatched from eggs that served as noninjected controls and from hatchlings subjected to in ovo injections of 0.25 or 0.50 µmol of L-carnitine. Individual BW and yolk sac weights were collected.

Data were analyzed using ANOVA in a completely randomized block design (Steel et al., 1997) using the mixed model of the SAS system (SAS Institute, 2003). The in ovo injection treatment was considered a fixed effect. The incubator tray was the block.

Experiment 2

A total of 2,077 chicken eggs were collected from White Leghorns and stored at 10° C prior to initiation of incubation. The day of egg collection was recorded to allow for equal distribution of eggs to treatment groups relative to storage time. Eggs were incubated for 17 d using standard incubation conditions. Eggs were removed from the incubator and candled individually for fertility prior to injection. Prior to initiation of the in ovo injections of L-carnitine, 13 fertile eggs were injected with dye to verify that injections were conducted appropriately. A total of 1,680 fertile eggs were sanitized by spraying a bleach solution (0.12% citric acid, 0.01% NaBr, and 0.55% bleach) at the large end of the egg prior to injection at 17 d of incubation. Two-hundred eighty eggs were assigned to 1 of 6 treatments of control, saline, or 0.05, 0.5, 5, or 10 µmol of L-carnitine. Hatching eggs laid on different days were distributed evenly among experimental units that consisted of 10 hatching eggs. There were 28 replicates per treatment. With the exception of controls, all eggs were injected using a 3.81-cm 21-gauge needle with either sterilized saline or L-carnitine dissolved in saline using an injection volume of 100 µL. Eggs were not sealed at the injection site. Ten eggs each were placed in their own individual hatching compartment following injection so as to keep experimental units separated. At 22 d of incubation, the number of chicks that hatched was counted in an experimental unit and group BW determined. All unhatched eggs were opened to determine stage of development to adjust the hatchability for embryonic deaths that occurred before 17 d of incubation that were not detected through candling.

Data were analyzed using ANOVA in a completely randomized block design (Steel et al., 1997) using the mixed model of the SAS system (SAS Institute, 2003). The in ovo injection treatment was considered a fixed effect, and the incubator tray was the block.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Experiment 1

The preliminary study showed that each of the incubated embryos injected with dye had purple coloring in the amnionic fluid (20/20 or 100%). In ovo injection of L-carnitine into chicken eggs at 18 d of incubation did not affect hatchability, BW, or yolk sac weight (Tables 1Go and 2Go).


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  		Table 1. The effect of in ovo injection of L-carnitine on hatchability and hatchling BW determined at 22 d of incubation (experiment 1)
 

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  		Table 2. The effect of in ovo injection of L-carnitine on hatchling BW and yolk sac weight (experiment 1)
 
Experiment 2

Twelve of the 13 fertile eggs (92%) injected with dye had the injectable within the amnion of the 17-d-old embryo. Injecting L-carnitine into fertile eggs at 17 d of incubation at dosages that ranged from 0.05 to 10 µmol did not affect hatchability or hatchling BW compared with controls and eggs injected with saline (Table 3Go).


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  		Table 3. The effect of in ovo injection of L-carnitine on hatchability and hatchling BW determined at 22 d of incubation (experiment 2)
 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The avian embryo relies on the hen to provide nutrients needed for successful embryogenesis (Kidd et al., 2005). If nutritional deficiencies occur during the formation of the egg, it can have significant repercussions on the developing embryo (Moran, 2007). Most feed ingredients in the commercial diets of domesticated hens are from plants that are low in L-carnitine. For this reason, freshly laid eggs have low concentrations of free L-carnitine and acetyl L-carnitine (Chiodi et al., 1994). However, supplementing hen’s diets with L-carnitine increases yolk L-carnitine concentrations (Leibetseder, 1995; Peebles et al., 2007).

The chick embryo may have limited capability to synthesize L-carnitine during incubation (Casillas and Newburgh, 1969). Gamma-butyrobetaine, an intermediate substance required for L-carnitine biosynthesis, is limited in embryos and young animals due to the low activity of {gamma}-butyrobetaine hydroxylase (Borum, 1983; Rebouche, 1992). Low levels of L-carnitine synthesis may make supplementation of L-carnitine beneficial to chicken embryos. As an example, hatchability of eggs from broiler breeder hens consuming diets supplemented with 50 or 100 mg of L-carnitine for 3 wk as compared with controls increased from 83 to 87% and from 82.4 to 85.3%, respectively. No indication was given of statistical significance between treatments (Leibetseder, 1995). However, Peebles et al. (2007) showed no effect on hatchability of eggs from broiler breeders consuming 25 mg/kg of L-carnitine compared with controls.

Even though de novo synthesis of L-carnitine occurs in birds, exogenous L-carnitine may be beneficial, especially in embryos and hatchlings (Kidd et al., 2005). The conversion of fatty acids into esterified L-carnitine is an essential step for fatty acid oxidation (Rinaudo et al., 1991). Embryos and young chickens have much lower levels of free and total L-carnitine as well as short chain esterified L-carnitine in muscles, liver, and heart as compared with adult tissues (Rinaudo et al., 1991). The ratio of esterified short chain L-carnitine to free L-carnitine reaches the highest level on d 18 of incubation in all tissues. This ratio is even higher than in growing chicks, reflecting the high demand for fatty acid utilization for energy production in embryos.

In ovo feeding of supplemental nutrients may help overcome any constraint of limited egg nutrients (Foye et al., 2006). During late embryogenesis, providing exogenous feeding solutions into the amnionic fluid of the embryo allows for intestinal absorption of potentially beneficial nutrients prior to the initiation of pipping. For example, in ovo feeding of a solution containing carbohydrates, β-hydroxy-β-methylbutyrate (a leucine metabolite), or both at 17.5 d of incubation increased broiler hatching BW by reducing the need to produce glucose via gluconeogenesis from muscle protein (Uni et al., 2005).

Because chicken embryos show a high requirement for L-carnitine, yet contain low levels of L-carnitine, our hypothesis was that the injection of L-carnitine into the amniotic fluid of late-stage embryos would lead to its consumption, intestinal absorption, and circulation to fat storage areas such as the yolk sac to facilitate catabolism of fatty acids for energy. The L-carnitine-induced increase in ATP energy from fatty acid catabolism would in turn be used by the chick to facilitate hatching. Also, L-carnitine possesses antioxidant properties scavenging free radicals that cause lipid peroxidation. L-Carnitine could reduce the incidence of late dead embryos, in particular those chicks that die during the pipping process, perhaps leading to improved hatchabilities. Moreover, less glucose may be used as an energy source as more fatty acids are mobilized for energy production because of higher L-carnitine concentration in L-carnitine in ovo-injected embryos. Reduced utilization of glucose may spare muscle protein mobilization for gluconeogenesis during late-term embryonic development, thus increasing BW. However, under the conditions used in the current experiments, we were unable to show beneficial effects of injecting L-carnitine at dosages that ranged from 0.05 to 10 µmol on hatchability or hatchling BW.

It is concluded that under our experimental condition, the in ovo injection of dosimetric dosages of L-carnitine in the range of 0.05 to 10 µmol into fertile Leghorn eggs at 17 or 18 d of incubation did not affect hatchability, yolk sac weight, or BW. The possibility exists that dosages of L-carnitine greater than 10 µmol could improve hatchability and hatchling BW because there was no evidence of carnitine toxicity with the dosimetry utilized in the current study. Moreover, the response of broiler and turkey hatching eggs to in ovo carnitine injections may differ from Leghorn embryos. Differences in lipid metabolic rate between egg-laying and meat-type strains of poultry could influence response to in ovo injection of L-carnitine (Sato et al., 2006).


   ACKNOWLEDGMENTS
 
This study was supported by the US Poultry and Egg Association and Lonza Inc. The authors thank F. A. Haan and O. M. Van Dame for incubation management and M. E. Einstein for providing statistical advice (Purdue University).


   FOOTNOTES
 
1 Journal paper No. 2007-18170 of the Purdue University Agricultural Research Programs, West Lafayette, IN 47907. Back

Received for publication August 20, 2007. Accepted for publication November 18, 2007.


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 ABSTRACT
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 DISCUSSION
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Agarwal, A., and T. M. Said. 2004. Carnitines and male infertility. Reprod. Biomed. Online 8:376–384.[Web of Science][Medline]

Borum, P. R. 1983. Carnitine. Annu. Rev. Nutr. 3:233–259.[CrossRef][Web of Science][Medline]

Buyse, J., G. P. Janssens, and E. Decuypere. 2001. The effects of dietary L-carnitine supplementation on the performance, organ weights and circulating hormone and metabolite concentrations of broiler chickens reared under a normal or low temperature schedule. Br. Poult. Sci. 42:230–241.[CrossRef][Web of Science][Medline]

Casillas, E. R., and R. W. Newburgh. 1969. L-carnitine and derivatives in embryonic chick tissue. Biochim. Biophys. Acta 184:566–577.[Medline]

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 embryo. Int. J. Biochem. 26:711–720.[CrossRef][Web of Science][Medline]

Foye, O. T., Z. Uni, and P. R. Ferket. 2006. Effect of in ovo feeding egg white protein, beta-hydroxy-beta-methylbutyrate, and carbohydrates on glycogen status and neonatal growth of turkeys. Poult. Sci. 85:1185–1192.[Abstract/Free Full Text]

Kalaiselvi, C. J., and C. Panneerselvam. 1998. Effect of L-carnitine on the status of lipid peroxidation and antioxidants in aging rats. J. Nutr. Biochem. 9:575–581.[CrossRef][Web of Science]

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 carcase traits. Br. Poult. Sci. 46:91–103.

Leibetseder, J. 1995. Studies on the effects of L-carnitine in poultry. Arch. Ani. Nutr. 48:97–108.

Moran, E. T., Jr. 2007. Nutrition of the developing embryo and hatchling. Poult. Sci. 86:1043–1049.[Abstract/Free Full Text]

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.[CrossRef][Web of Science][Medline]

Rebouche, C. J. 1992. Carnitine function and requirements during the life cycle. FASEB J. 6:3379–3386.[Abstract]

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.[CrossRef][Web of Science][Medline]

SAS Institute. 2003. SAS/STAT User’s Guide. Version 9. SAS Inst. 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 Mol. Integr. Physiol. 143:382–388.[CrossRef][Medline]

Steel, R. G. D., J. H. Torrie, and D. A. Dickey. 1997. 3rd ed. Principles and procedures of statistics: A biometrical approach. McGraw Hill Book Co., New York, NY.

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