In Ovo Feeding Improves Energy Status of Late-Term Chicken Embryos1 Z. Uni,*,2 P. R. Ferket,† E. Tako,* and O. Kedar* *Faculty of Agricultural, Food and Environmental Quality Sciences, Department of Animal Sciences, Hebrew University of Jerusalem, PO Box 12, Rehovot, 76100, Isarel; and †North Carolina State University, Department of Poultry Science, College of Agriculture and Life Sciences, Raleigh, North Carolina 27695-7608 days of embryonic incubation until 25 d after hatching. We examined, using 600 birds from 2 different strains of commercial boilers, body and muscle weights and glycogen reserves following feeding embryos at d 17.5 of incubation with a solution containing maltose, sucrose, dextrin, and β-hydroxy-β-methylbutyrate (HMB). Providing carbohydrates and HMB to late-term embryos increased hatching weights by 5 to 6% over controls, improved liver glycogen by 2- to 5-fold, and elevated relative breast muscle size by 6 to 8%. These weight advantages were sustained through the end of the experiments at 25 d of age. It is reasonable to assume that the elevated glycogen levels in the in ovo treatment reduce the need to produce glucose via gluconeogenesis and, therefore, contribute to less use of muscle protein and hence a greater percentage of pectoral muscle weight in the in ovo birds.

ABSTRACT Maintenance of glucose homeostasis during late-term embryonic development is dependent upon the amount of glucose held in reserve primarily in the form of glycogen in the liver and upon the degree of glucose generated by gluconeogenesis from protein first mobilized from amnion albumen and then from muscle. Insufficient glycogen and albumen will force the embryo to mobilize more muscle protein toward gluconeogenesis, thus restricting growth of the late-term embryo and hatchling. We hypothesize that administration of available carbohydrates to the amnion will improve glycogen reserves and spare muscle protein mobilization for gluconeogenesis during late-term embryonic and posthatch neonatal development. Our hypothesis was tested by comparing BW gain, liver glycogen reserves, and muscle weight of in ovo fed and control embryos during last

(Key words: chicken, embryo, in ovo feeding, body weight, glycogen) 2005 Poultry Science 84:764–770

serves are withdrawn as embryos go through the hatching process (Christensen et al., 2001). The glycogen reserves begin to be replenished when the newly hatched chick has full access to feed and oxygen and can fully use the fat stored in the yolk sac (Rosebrough et al., 1978a; 1978b). Thus, the glucose level available to the late-term embryo for hatching and posthatch survival is dependent upon the glycogen reserve and gluconeogenesis, which is induced when glucose intake is insufficient to meet the metabolic glucose demands (Elwyn and Bursztein, 1993c). The late-term embryo and neonatal chick depend on gluconeogenesis from amino acids (Dickson and Langslow, 1978; John et al., 1988; Hamer and Dickson, 1989), resulting in the depletion of muscle protein reserves and reduced early growth and development (Vieira and Moran, 1999a,b). To reduce the use of liver glycogen reserves and the depletion of muscle protein we hypothesized that administration of carbohydrate and β-hydroxy-β-methylbutyrate (HMB) into the embryonic amnion fluid prior to hatch would support the energy status of the hatchling

INTRODUCTION Establishment of a stable and sufficient glucose status is critical for the late-term embryonic developmental hatching process and posthatch development of poultry until feed consumption is initiated. Toward the end of incubation embryos use their energy reserves to meet the high demand for glucose to fuel hatching activities (Freeman, 1965; John et al., 1987, 1988; Christensen et al., 2001). Although glucose can be synthesized from fat and protein (Elwyn and Bursztein, 1993a,b,c), glucose is primarily generated from protein by gluconeogenesis or by glycolysis of glycogen reserves because oxygen is limited during the last quarter of incubation (Bjonnes et al., 1987; John et al., 1987). In birds, the major glycogen reserves are the liver and glycolytic muscles (John et al., 1988). These glycogen re-

2005 Poultry Science Association, Inc. Received for publication April 18, 2004. Accepted for publication January 25, 2005. 1 This research was supported by Research Grant Award Number IS3311-02 from BARD, United States-Israel Binational Agriculture Research and Development. 2 To whom correspondence should be addressed: [email protected].

Abbreviation Key: HMB = hydroxy-β-methylbutyrate.

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by elevating the glycogen reserves, moderating the use of muscle proteins, and thus contributing to enhanced posthatch performance. In addition to the carbohydrate source, which is included in the in ovo feeding solution, HMB was chosen because it was reported by Nissen et al. (1994) that this leucine metabolite decreases chicken mortality, increases carcass yield, and plays an important role in protein metabolism in muscle, mainly in attenuating proteolysis (Nissen et al., 1996). Moreover, HMB has been shown to prevent excessive muscle proteolysis during metabolic stress (reviewed by Nissen and Abumrad, 1997; Slater and Jenkins, 2000) and has also been reported to stimulate protein synthesis in rat skeletal muscle (Anthony et al., 2000). The objective of this study was to measure effects of in ovo feeding solution, containing highly digestible carbohydrates and HMB, on body and muscle weights and glycogen reserves of broiler chicks from 3 d prehatch until 25 d posthatch.

MATERIALS AND METHODS Birds Two experiments of similar treatment design and protocol were conducted at 2 different locations with 2 different strains of commercial broilers. In experiment 1, fertile eggs from a flock of Cobb 500 breeders, 39 wk in lay, were obtained from Brown Hatchery.3 The eggs were incubated under optimal conditions at the Department of Animal Sciences of the Faculty of Agriculture, Food and Environmental Sciences, Rehovot, Israel. In experiment 2, fertile eggs from a flock of Ross 308 breeders, 43 wk in lay, were obtained from Townsend Poultry4 and incubated to hatch at the Department of Poultry Science, North Carolina State University, Raleigh, NC.

In Ovo Feeding Procedure In each experiment, at 17.5 d of incubation, 300 eggs containing viable embryos were weighed and divided into 2 groups of 150 eggs with equal weight frequency distribution. The amnion in the in ovo feeding group was identified by candling and was injected with 1 mL of in ovo feeding solution using a 21-gauge needle. The in ovo feeding solution contained 25 g/L maltose,5 25 g/L sucrose,5 200 g/L dextrin,5 1 g/L HMB6 all dissolved in 5 g/L NaCl. After the eggs were injected, the injection holes were sealed with cellophane tape. The other group of 150 eggs was not injected and served as the control treatment because preliminary experiments conducted in our laboratory indicated that injection of 1 mL of 5 g/L

TABLE 1. Hatchability of Cobb (experiment 1) and Ross (experiment 2) fertile eggs following conventional and in ovo feeding procedures Treatment

Experiment

Stock

n

Hatchability

In ovo fed Control In ovo fed Control

1 1 2 2

Cobb Cobb Ross Ross

150 150 150 150

88% 89% 87% 86%

NaCl did not affect (P < 0.05) embryo or chick BW, intestinal development, or brush border membrane enzymatic activity. Finally, all eggs were placed in hatching trays such that each treatment was equally represented in each location of the incubator.

Tissue Sampling Within each experiment, chicks were sampled randomly from each treatment group to evaluate the proportional changes in the mass of glycogen content in pectoral muscle and liver. Ten random chicks from each of the in ovo and control treatments were sampled for analysis at 18, 19, and 20 d of incubation (18E, 19E, and 20E, respectively); on the day of hatch; and at d 10 and 25 posthatch. Body, liver, and pectoral (pectoralis major and pectoralis minor) weights were recorded for each bird sampled. Both liver and pectoral muscle samples were frozen in liquid nitrogen immediately after collection and stored at −80°C for glycogen determination.

Housing In experiment 1, completed in Rehovot, Israel, 1 building was devided into 4 pens. One hundred control chicks were randomly assigned to 2 pens, 50 chicks per pen. One hundred in ovo chicks were randomly assigned to the other 2 pens. The pens were 2.5 × 4.0 m, and the stocking density was 0.2 m2 per broiler chick. Each pen was equipped with an automatic drinker and a self feeder. Bedding was wood shavings, and incandescent heat was provided to 32°C for wk 1 and to 27°C for wk 2. Individual chick BW were recorded upon hatch and at d 10 and 25. In experiment 2, each treatment group of chicks was randomly assigned on the day of hatch to 10 replicate pens (1 m2) of 10 chicks and was housed in a total confinement building at North Carolina State University. Each concrete floor pen was equipped with an automatic nipple drinker, manual self feeder, soft pine wood shavings litter, and supplemental incandescent heat. Heat was provided to 32°C for wk 1 and to 27°C for wk 2. Individual chick BW were recorded upon hatch and at d 10 and 25.

Animal Care 3

Hod Hasharon, Israel. 4 Siler City, NC. 5 Sigma Diagnostics, Sigma, St. Louis, MO. 6 Metabolic Technologies Inc., Ames, IA.

All experimental protocols were approved by the Institutional Animal Care and Use Committees at Hebrew University of Jerusalem and North Carolina State University. All birds were given ad libitum access to water and

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UNI ET AL. TABLE 2. Experiment 1: egg, body, and pectoral muscle weights (g) of embryos (Cobb) from controls and in ovo feeding treatments at 19 and 20 d of incubation Days of incubation 1

202

19 Control 3

Egg weight (g) BW3 (g) BW per egg weight (%) Pectoral muscle weight3 (g) Pectoral muscle weight per BW (%)

57.82 48.31 83.7 0.76 1.58

± ± ± ± ±

NS

0.8 0.5NS 0.5NS 0.10NS 0.03*

In ovo

Control

± ± ± ± ±

± ± ± ± ±

57.84 49.02 84.7 0.82 1.66

1.1 0.9 0.5 0.03 0.04

58.63 50.54 86.2 0.79 1.57

In ovo NS

1.2 1.1NS 0.2NS 0.03* 0.05*

57.72 49.55 85.8 0.86 1.74

± ± ± ± ±

0.8 0.9 0.6 0.03 0.07

1

Thirty-six hours after in ovo feeding procedure. Sixty hours after in ovo feeding procedure. 3 Weight values are means ± SE of 10 embryos. *Treatment means within a sample time are significantly different (P < 0.05). NS P > 0.05. 2

diet formulated to meet NRC (1994) recommendations in all experiments.

Data was analyzed with the SAS software7 separating for each sampling time (embryonic age or chick age) within each breed, using Student’s t-test. Values were considered statistically different at P < 0.05. When data were percentages they were transformed by arc sin square root.

than the controls (Table 2); however, there were no treatment effects observed in relative pectoral muscle weight among the Ross embryos in experiment 2 (Table 3). On the day of hatch, the in ovo fed hatchlings in the Ross and Cobb experiments were 5 to 6% heavier than the control hatchlings, and these BW differences continued until the end of the experiment (d 25; Tables 4 and 5). In addition, the relative weights of pectoral muscles of the in ovo fed chickens were 5 to 8% higher than controls, regardless of experiment (strain). Liver glycogen levels in the Cobb chicks in experiment 1 increased by 8-fold at 36 h after in ovo feeding in comparison with the controls (Figure 1A). The same response of higher liver glycogen content after in ovo feeding was observed in the Ross embryos in experiment 2 (Figure 2A).Both strains exhibited a reduction in liver glycogen levels as the embryos approached hatch. At day of hatch, in ovo fed and control Cobb hatchlings exhibited very low liver glycogen levels, whereas in ovo fed Ross hatchlings had higher liver glycogen levels than the control hatchlings (Figures 1A and 2A). Regardless of strain (experiment), the glycogen content in the pectoral muscle was approximately 5% of that observed in the liver glycogen, and it did not change significantly after in ovo feeding (Figures 1B and 2B). On the day of hatch, however, in ovo fed Cobb hatchlings had significantly higher glycogen in their pectoral muscle than the respective control group (Figure 2B).

RESULTS

DISCUSSION

The percentage hatch of fertile eggs in the 2 experiments was similar, and there were no significant treatment effects observed (Table 1). Body weights among the Cobb (experiment 1) and Ross (experiment 2) embryos during the last days of incubation were similar, regardless of the experimental treatments (Tables 2 and 3). At 36 h after in ovo feeding, the Cobb embryos in experiment 1 had greater pectoral muscle weight relative to embryonic BW

The present study shows that providing the in ovo feeding solution to the late-term embryo increased hatching weights by 5 to 6% over those of controls, improved liver glycogen by 2- to 5-fold, and increased relative breast muscle size (% of broiler BW) by 6 to 8%. These weight advantages were sustained through to the end of the experiments at 25 d of age. Previous studies have indicated that hatching weight is a major predictor of marketing weight in chickens. Wilson (1991) reported that each 1 g of increase in BW at hatch leads to 8 to 13 g of increase in BW at marketing.

Glycogen Determination Liver and muscle tissue glycogen contents were determined by a colorimetric method based on the reduction of iodine as described by Dreiling et al (1991). From each bird, 0.2-g samples of liver and muscle were added to 0.8 mL of 8% HClO4, homogenized (in ice) for 45 s, and centrifuged at 700 × g and 4°C for 20 min. The supernatant was removed, and 1 mL of petroleum ether (60 to 80°C) was added to each tube. After being mixed, the petroleum ether fraction was removed, and samples from the bottom layer (10 µL for the liver and 100 µL for the pectoralis muscles) were transferred to a new tube, and then 650 µL of color reagent (Dreiling et al., 1991) was added. After incubation for 10 min at room temperature, the absorbance at 450 nm was determined.

Statistical Analysis

7

SAS User’s Guide, 1986, Version 6, SAS Institute Inc., Cary, NC.

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IN OVO FEEDING OF LATE-TERM CHICKEN EMBRYOS TABLE 3. Experiment 2: Egg, body, and pectoral muscle weights (g) of embryos (Ross) from controls and in ovo feeding treatments at 19 and 20 d of incubation Days of incubation 1

202

19 Control 3

Egg weights (g) BW3 (g) Egg weight per BW (%) Pectoral muscle weight3 (g) Pectoral muscle weight per BW (%)

56.8 43.9 77.3 0.65 1.48

± ± ± ± ±

NS

0.8 0.4NS 0.8NS 0.04NS 0.08NS

In ovo 58.7 44.04 75.1 0.71 1.61

± ± ± ± ±

Control

0.6 0.5 0.7 0.03 0.07

55.1 46.4 84.2 0.77 1.66

± ± ± ± ±

NS

0.9 0.7NS 0.5NS 0.05NS 0.10NS

In ovo 56.4 47.7 84.5 0.81 1.70

± ± ± ± ±

0.8 0.6 0.8 0.02 0.06

1

Thirty-six hours after in ovo feeding procedure. Sixty hours after in ovo feeding procedure. 3 Weight values are means ± SE of 10 embryos. NS P > 0.05. 2

Although this correlation between hatch weight and market weight may differ among strains, the influence of hatch weight on market weight is apparently increasing as broiler breeding companies continue to select for everincreasing growth rate (Wilson, 1991; Vieira and Moran, 1999a,b; Havenstein et al., 2003). In this study we showed that a 2-g difference in BW at hatch due to in ovo feeding resulted in 50 to 60 g of increase in BW at d 25. The positive association between BW at hatch and body glycogen status level has been demonstrated in several studies (Christensen et al., 1991, 1999, 2000, 2001, 2003; John et al., 1988; Warriss et al., 1992). All of these studies clearly demonstrate that depletion of glycogen reserves may occur due to lack of maternal nutrients, withdrawal of external food, or prolonged egg storage. In both experiments of our study, the levels and depletion of hepatic and pectoral muscle glycogen upon hatching of the control group were similar to those observed by Rosebrough et al. (1978a, 1979), John et al. (1987), and Christensen et al. (2001). In both experiments using different strains, in ovo feeding resulted in an increase of approximately 6 to 12 mg of glycogen per gram of embryonic liver tissue. This additional energy source probably supported the late-term development of the embryo, resulting in a significant increase in BW and pectoral muscle weight at hatch. In addition, it is reasonable to conclude that the elevated glycogen levels in the in ovo

treatment also spared the utilization of muscle protein to produce glucose via gluconeogenesis, resulting in higher pectoral muscle weight per BW. The in ovo feeding procedure did not have a major effect on pectoral muscle glycogen reserves, and significant differences were observed at hatch only among the Cobb chicks in experiment 1. Edwards et al. (1999) concluded that avian skeletal muscle glycogen is relatively insensitive to nutritive status after observing that muscle glycogen levels were not significantly increased over control levels after refeeding, even though liver glycogen was increased by 380%. Evidently, muscle glycogen level is regulated by metabolic homeostatic mechanisms that are not influenced by nutritional status before or after hatch. Several researchers have tested various treatments during the incubation period and first days posthatch in an attempt to stimulate the early growth and increase BW. In ovo photostimulation of embryos with artificial light accelerates embryonic development and early hatching (Siegel et al., 1969; Fairchild and Christensen, 2000). Green light photostimulation results in increased BW in broilers and turkey poults ( Rozenboim et al., 1999; Halevy et al., 2003) and affects skeletal muscle satellite cell proliferation (Halevy et al., 1998) and breast muscle weight relative to BW (Halevy et al., 2003). Ad libitum access to feed immediately after hatch has been demonstrated to in-

TABLE 4. Experiment 1: body and pectoral muscle weight (g) of chickens (Cobb) from controls and in ovo feeding treatments at day of hatch and at 10 and 25 d posthatch Age Day of hatch

BW1 (g) BW difference (%) Pectoral muscle weight2 (g) Pectoral difference (%) Pectoral muscle weight per BW (%) Pectoral muscle weight difference (%)

Control

In ovo

46.1 ± 0.7*

48.7 ± 0.9 +5.6 0.97 ± 0.05 +19.7 1.99 ± 0.06 +7.5

0.81 ± 0.04* 1.85 ± 0.05*

Day 10

Day 25

Control 240 ± 3* 23.2 ± 0.8* 9.6 ± 0.25*

In ovo

Control

In ovo

257 ± 5 +7 27.5 ± 0.8 +18.5 10.3 ± 0.1 +7.2

941 ± 22*

1,003 ± 20 +6.5 128 ± 6.1 +18.5 12.4 ± 0.2 +8.7

The BW values are means ± SE of 80 to 100 birds (equal numbers of males and females). Pectoral muscle weights are means ± SE of 10 birds (equal numbers of males and females). *The difference between the 2 treatment means within an age sample time is significant (P < 0.05).

1 2

108 ± 3.0* 11.4 ± 0.4*

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UNI ET AL.

FIGURE 1. Glycogen concentration (mg/g of wet tissue) in the liver (A) and in the pectoral muscle (B) of late term embryos (Cobb) from controls (Con) and in ovo feeding treatment (IOvo) shown according to d 18, 19, and 20 of embryonic incubation (18E, 19E, 20E, respectively) and day of hatch. A,BDifferent letters, within days of embryonic incubation, indicate a significant difference between the 2 treatments (P < 0.05).

FIGURE 2. Glycogen concentration (mg/g of wet tissue) in the liver (A) and in the pectoral muscle (B) of late-term embryos (Ross) from controls (Con) and in ovo feeding treatment (IOvo). Shown according to d 18, 19, and 20 of embryonic incubation (18E, 19E, 20E, respectively) and day of hatch. A,BDifferent letters are different within day of embryonic incubation indicate a significant difference between the 3 treatments (P < 0.05).

crease BW gain of neonatal chicks and poults, enhance the size of pectoralis major and minor muscle, and improve the development of intestine (Halevy et al., 1998, 2003; Noy and Sklan, 1998, 1999). In contrast, poor nutritional status posthatch inhibits mitotic activity of muscle satellite cells and consequently reduces breast muscle size (Mozdziak et al., 1997; Halevy et al., 2000) and depresses the early expansion of the intestinal villus surface area, including the depression of villi crypt development and maturation of enterocytes in the small intestine (Geyra et al., 2001).

Our data show that the amnionic administration of an in ovo feeding solution, containing carbohydrates and HMB, just before internal piping likely provides the embryo the caloric resources to fuel the hatching process and early development. Few studies have been conducted to test the addition of nutrients into hatching eggs to improve the energy status and growth of late-term embryos and hatchlings. By using pressure differentials, John et al. (1988) dipped fertile turkey eggs into a solution containing 10% glucose and antibiotics before they were set in an incubator and found it increased glycogen con-

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IN OVO FEEDING OF LATE-TERM CHICKEN EMBRYOS TABLE 5. Experiment 2: body and pectoral muscle weights (g) of chickens (Ross) from controls and in ovo feeding treatments at day of hatch and at d 10 and 25 posthatch Age Day of hatch

1

BW (g) BW difference (%) Pectoral muscle weight2 (g) Pectoral difference (%) Pectoral muscle weight per BW (%) Difference (%) in pectoral muscle weight

Day 10

Day 25

Control

In ovo

Control

In ovo

Control

In ovo

45.3 ± 0.3*

47.02 ± 0.3 +3.7 0.95 ± 0.06 +10.4 2.05 ± 0.05 +6.2

243 ± 4 *

254 ± 3 +4.2 30.3 ± 0.8 +8.6 12.3 ± 0.3 +5.2

943 ± 21*

997 ± 20 +5.7 130 ± 6 +14.1 13.0 ± 0.2 +8.3

0.86 ± 0.03* 1.93 ± 0.06*

27.9 ± 0.8* 11.4 ± 0.3*

114 ± 3* 12.0 ± 0.4*

The BW values are means ± SE of 80 to 100 birds (equal numbers of males and females). Pectoral muscle weights are means ± SE of 10 birds (equal numbers of males and females). *The difference between the 2 treatment means within an age sample time is significant (P < 0.05).

1 2

tent of liver and pectoral muscle of late-term embryos. Al-Murrani (1982) and Ohta et al. (2001) found that injection of amino acids into the air cell of fertile chicken eggs during the first week of incubation increased amino acid contents of embryo, yolk albumen, and allantoic and amnionic fluids on d 19 of incubation and elevated embryonic BW. These experiments demonstrate the benefits of adding external nutrients to hatching eggs and clearly illustrate the limitations of avian species, which, unlike mammals, do not have a continuous energy supply from a maternal source to support embryonic and neonatal growth. However, the research of Al-Murrani (1982), John et al. (1988), and Ohta et al. (2001) differed considerably from the technology presented in our study because the nutrients were not directly exposed to embryonic intestine when it is most needed near the end of incubation. Our technology is novel in feeding the embryo before hatch by administering a feeding solution into the amnionic fluid of the embryo during last period of incubation (Uni and Ferket, 2003). The added nutrients are subsequently exposed to the tissues of the gastrointestinal tract after the embryo naturally consumes the amniotic fluid prior to piping and are then subjected to digestion and absorption by the embryonic intestine. Based on positive preliminary experimentation in our laboratories, the in ovo feeding solution formulation was developed to contain disaccharides (maltose and sucrose), readily digested polysaccharides (dextrin), and the leucine metabolite, HMB. Carbohydrates were chosen to be an important component of the in ovo feed solution because of their importance for the final stage of embryonic development prior to emergence from the shell (Christensen et al., 1993). HMB was added to the feeding solution, because this leucine catabolite has been reported to stimulate protein synthesis in rat skeletal muscle (Anthony et al., 2000) and prevent protein proteolysis (Nissen et al., 1996). Furthermore, HMB supplementation to the poultry diet decreases mortality and increases carcass yield in broilers, and addition of 0.01% HMB to broiler feed increases BW at marketing (42 d) by 1.4% when compared with control broilers (Nissen et al., 1994). However, the mechanism by which HMB affects muscle protein accretion is presently unclear. It has been claimed

that it acts as an anticatabolic agent, perhaps by being a source for cholesterol synthesis, which is required for the synthesis of new membranes or regenerating damaged ones (Nissen and Abumrad, 1997). In addition, it has been suggested that HMB may affect muscle growth via a direct effect on cell proliferation because it increases protein synthesis and decreases protein degradation (Peterson et al., 1999; Siwicki et al., 2000). Several studies have demonstrated that heavier chicks yield the highest body and muscle weight at market age. The enhanced muscle growth is due, in part, to higher proliferation of myoblasts during embryonic development and to the presence of more satellite cells in early posthatch development (Halevy et al., 2003). The in ovo feeding formulation we used might have enhanced the proliferation of embryonic and neonatal myoblasts and satellite cells, thereby contributing to the 7 to 8% increase in relative pectoral muscle mass among the in ovo fed chicks. Our results further demonstrated that in ovo feeding of carbohydrates and HMB at 17.5 d of incubation can improve the energy status of late-term broiler embryos and improve early growth to enhance the genetic potential for late embryonic and early posthatch growth.

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