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Heavier chicks at hatch improves marketing body weight by enhancing skeletal muscle growth
Heavier Chicks at Hatch Improves Marketing Body Weight by Enhancing Skeletal Muscle Growth D. Sklan,1 S. Heifetz, and O. Halevy Faculty of Agriculture, Hebrew University of Jerusalem, Rehovot 76100, Israel smaller proportion of BW in large chicks, and its growth was not correlated with performance. Liver proportions were greater in heavier chicks. Pectoralis growth and satellite cell numbers and activity were greater in heavier chicks through 5 d posthatch, and pectoralis muscles were heavier at marketing. Examination of some of the growth factors involved suggested that in heavier chicks satellite cells underwent higher proliferation and earlier differentiation during their critical period of activity in the immediate posthatch days. To determine when these differences in activity were established, examination of 15-d embryonic myoblast activity indicated that at this stage activity was already greater in the heavier eggs. This finding suggests that programming of muscle growth may be completed in late embryonic stages. This study suggests that enhanced satellite cell activity is involved in increased growth of chicks hatching with higher BW.
(Key words: chick, gastrointestinal tract, growth, satellite cells, skeletal muscle) 2003 Poultry Science 82:1778–1786
INTRODUCTION Broiler performance is influenced by the age of the maternal flock and the weight of the hatching chick (Shanawany, 1984; Sinclair et al., 1990; Wilson, 1991; Vieira and Moran, 1999). Broiler weights at marketing were reported in the 1980s to increase by 8 to 13 g for each gram of increase in the hatching weight (Wilson, 1991), and, although these values differ between strains, this trend seems be increasing in modern birds (Wilson, 1991). Although egg composition changes with egg weight, these changes do not appear to have significant effects on broiler growth or processing yields (Vieira and Moran, 1999), suggesting that other systems are involved in these process. The digestive system of the chick is anatomically complete at hatching but undergoes major morphological and functional changes in the immediate posthatch period (Sklan, 2001). In the hatchling, uptake of glucose and amino acids is limited
2003 Poultry Science Association, Inc. Received for publication April 28, 2003. Accepted for publication July 1, 2003. 1 To whom correspondence should be addressed: sklan@agri. huji.ac.il.
and increases rapidly within 4 d posthatch (Noy and Sklan, 1999). Morphological and functional development of the small intestine as well as growth are stimulated by early access to feed (Noy and Sklan, 2001). Hence we hypothesized that development of the gastrointestinal tract (GIT) may be a limiting factor in broiler performance in the posthatch period. An additional system that is observed to be at a critical stage at hatch and affecting subsequent performance is the skeletal muscle satellite cell (Halevy et al., 2000). Skeletal muscle growth in posthatch birds is determined by hypertrophy and accumulation of nuclei in muscle fibers. The latter process is attributed to myogenic precursor cells, located beneath the basal lamina of the fibers, termed satellite cells (Mauro, 1961). In response to external signals, these cells are capable of entering the cell cycle and proliferating, differentiating, and either fusing into existing fibers or fusing with each other to form new fibers (Campion, 1984). Myogenic differentiation involves the coordination of muscle-specific gene expression and withdrawal from the cell cycle. The
Abbreviation Key: GIT = gastrointestinal tract; IGF-I = insulin-like growth factor-I.
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ABSTRACT This study examined some of the factors connected with the greater marketing weight observed in chicks hatching with higher BW. Examination of chicks hatching from maternal flocks of different ages indicated that BW at hatch increased quadratically and yolk sac weight linearly with age, whereas components of the gastrointestinal tract showed no significant trend. Growth of pectoralis muscles and gastrointestinal tract were compared in chicks hatching at the same weight from maternal flocks of 28 and 64 wk of age and in chicks from the same maternal flock (44 wk old) hatching at different weights. The results indicated that no differences were found among chicks hatching with the same weight from maternal flocks of different ages. In contrast, in chicks from the same maternal flock hatching at different weights the gastrointestinal tract tended to compose a
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MATERIALS AND METHODS Experimental Procedures Experiment 1. Chicks (Ross 308) were obtained from a commercial hatchery within 1 h of clearing the shell (n = 10 males and 10 females); birds were from a mother flock aged 26 through 68 wk, every 2 wk. BW of chicks were determined, and the yolk sac and small intestines were weighed, the latter after gently extruding contents. Experiment 2. Male chicks (Ross 308) were taken within 1 h of clearing the shell either from a flock at 44 wk of age with hatching weights of 43.5 ± 0.5 g and 53.1 ± 0.5 g or from flocks of 28 and 62 wk of age with weights of 49.3 ± 0.4 and 49.3 ± 0.4 g, respectively. Chicks were divided into groups on the basis of BW equalizing the variance between treatments (n = 80) and transported within 30 min to the facility. Chicks were provided with commercial feed according to the manufacturer’s protocol,2 and water was available ad libitum in floor pens in temperature-controlled rooms. 2
Matmor Feed Mill, M.P. Evtach, Israel. New England Nuclear, Boston, MA. 4 Packard, Downers Grove, IL. 5 Pierce, Rockford, IL. 6 Schleicher and Schuell, Dassel, Germany. 7 Zymed, San Francisco, CA. 3
For the first 5 d of age, 5 chicks per day were taken, and intestines and pectoral muscles were removed for further analyses. Chicks were weighed at 7, 14, 21, 35, and 41 d when chicks were marketed. All experimental procedures were approved by the Animal Welfare Committee of the Faculty of Agriculture, The Hebrew University of Jerusalem. Experiment 3. Eggs from a breeder flock (Ross 308) 46 wk of age at 15 d of incubation and weighing 68.2 ± 0.9 and 51.8 ± 0.9 were taken for examination of myoblast proliferation in the pectoral muscle.
Cell Cultures Skeletal muscle satellite cells were cultured from the pectoral muscle of poults at various ages as described by Halevy and Lerman (1993). Tissues were removed from the chicks under sterile conditions, chopped, and incubated with pronase (1.4 g/L) followed by trypsin (2.5 g/L) and DNAse I (0.5 g/L) for an additional 10 min. Enrichment of satellite cell culture was achieved with several centrifugations and tituration of the resuspended pellet. Cells were then filtered through a mesh (200 µm) and preplated for 2 h on a plastic Petri dish. Cells were counted using a hemocytometer and plated at 5 × 104 cells/cm2 in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 100 mL/L horse serum (HS) on gelatin-coated (1 g/L) dishes and maintained at 37°C in a humidified atmosphere containing 95% air and 5% CO2. On all days cells were prepared under similar conditions by using 6 g of pectoralis muscle that had been pooled from the experimental birds. An enriched population of myogenic cells was recovered, and less than 5% of the cells were nonmyogenic (Halevy et al., 1994).
Thymidine Incorporation DNA synthesis was assessed by [3H]-thymidine incorporation (Halevy and Lerman, 1993). [3H]-thymidine3 was added (74 kBq/well) for 2 h of incubation. Radioactivity in dissolved precipitates was counted using a TriCarb 1600CA scintillation counter.4 Equal plating efficiency was verified by determining cell numbers in parallel wells.
Western Blot Analysis Western blot analysis was performed as described by (Leshem et al., 2000). Muscle extracts were sonicated and normalized for protein content (BCA kit5), and equal amounts of protein were separated by SDS-PAGE and transferred to nitrocellulose filters.6 Membranes were incubated overnight at 4°C with the appropriate primary antibodies and then washed and incubated for 1 h with horseradish peroxidase-conjugated goat antimouse or goat anti-rabbit IgG.7 Proteins were visualized by enhanced chemiluminescence. The following primary antibodies were used: anti-IGF-I monoclonal anti-
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MyoD family of basic helix-loop-helix (bHLH) transcription factors (MyoD, myf5, myogenin, MRF4), form heterodimers with ubiquitous bHLH nuclear proteins (E proteins) and act in cooperation with the MEF2 family members to activate skeletal muscle structural genes (Naya and Olson, 1999; Weintraub, 1993). Proliferation of satellite cells is regulated by several growth factors and hormones, including fibroblast growth factors (Florini et al., 1996), platelet-derived growth factor (Yablonka-Reuveni et al., 1990), growth hormone (Halevy et al., 1994), and hepatocyte growth factor (Allen et al., 1995; Gal-Levi et al., 1998). Whereas these mitogens inhibit satellite cell differentiation, insulin-like growth factor-I (IGF-I) has been found to induce this process, as well as to improve muscle hypertrophy (Coleman et al., 1995). In broiler chicks, the process of proliferation and differentiation of satellite cells is transient but crucial, occurring in the first days posthatch, after which the satellite cell population declines dramatically (Moss et al., 1964; Halevy et al., 2000). Recent studies with birds have shown that early access to feed posthatch enhanced satellite cell proliferation and stimulated skeletal muscle growth in broilers, supporting the key role of these cells in dictating muscle size (Halevy et al., 2000). Another recent study has shown that the smaller muscle size found in chicks without access to feed following hatch is due to enhanced apoptosis of myonuclei (Mozdziak et al., 2002). This study examined changes in the GIT and pectoral muscle in chicks hatching from maternal flocks of different ages and of different BW and their correlation with subsequent development.
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body (1:3,000),8 polyclonal antibodies against chicken myogenin (1:6,000; a kind gift from Bruce Paterson, NIH, Bethesda, MD), and polyclonal anti-p21 (1:1,500).9 Densitometric analysis was performed on bands using GelPro software. Protein levels in each lane were normalized to the levels of protein loaded on that lane or to the levels of α-tubulin as an internal standard.
Histology Segments of approximately 2 cm were taken from the midpoint of the duodenum (duodenum), from the midpoint between the point of bile duct entry and Meckel’s diverticulum (jejunum), and midway between Meckel’s diverticulum and the ileocecal junction (ileum). Segments were gently flushed twice with 0.9% NaCl to remove the intestinal contents, fixed in fresh 4% buffered formaldehyde, and routinely processed into 5mm hematoxylin-eosin-stained sections. Morphometric indices were examined using computer-aided light microscope image analysis as previously described (Uni et al., 1999).
Statistical Analyses Data were analyzed by analysis of variance using the general linear models procedures of SAS software (SAS, 1986). Differences between means were examined using
8
Upstate Biotechnology, Lake Placid, NY. BD Biosciences, Lexington, KY.
9
FIGURE 2. Body weight (top panels) and pectoralis weight in chicks for 5 d posthatch (bottom panels). (A, C) Performance of chicks of the same BW at hatching from maternal flocks at 28 (filled circles) and 64 wk (filled triangles) of age (early-late). (B, D,) Performance of chicks hatching at 43 ± 0.5 g (open circles) and 53.1 ± 0.5 g (open triangles) from a maternal flock aged 44 wk (small-big). Results are means (n = 10), and bars are SD. BW were significantly different between chicks from big or small eggs from d 1 through 5, and pectoralis weight was different from d 3 to 5 (P < 0.05).
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FIGURE 1. Body weight (open triangles), weight of yolk sac or gastrointestinal track (GIT; circles), and weight of small and large intestines (filled triangles) at hatch from a maternal flock at different stages of lay. Results are means (n = 20), and bars are SD.
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the Tukey test, and significance was P < 0.05 unless otherwise stated.
RESULTS Experiment 1 Changes in BW of the hatchling and of some internal organs were examined with increasing age of the maternal flock. BW of chicks at hatching increased quadratically (r = 0.92, P < 0.001) and reached a plateau in the older flocks (Figure 1). In contrast, the weight of the yolk sac increased linearly (r = 0.78, P < 0.001) with age of the maternal flock. No correlations were observed between the age of the maternal flock and either small intestinal weight or the weight of any of the small intestinal segments. BW at hatch was correlated with the weight of the yolk sac (r = 0.216, P < 0.01) but not with the weight of the small intestines (r = 0.075, P > 0.05).
Experiment 2 This experiment was carried out to elucidate the connection between hatching weight and chick develop-
ment. Four groups of chicks were compared: chicks hatching with equal weights from maternal flocks in early (1) or late (2) stages of lay or large (3) and small (4) chicks from the same maternal flock at 44 wk of age. Chicks were examined at different ages through marketing to determine relative development. The parameters determined in these chicks are summarized in Figures 2 through 4. BW was not different between chicks from maternal flocks in early or late stages of lay either close to hatch (Figure 2A) or through 41 d (Figure 3A). Sizes of the pectoral muscles until d 5 (Figure 2C) and pectoral muscles and abdominal fat (Figure 3C) were not different at 41 d between these groups of chicks. In contrast, chicks hatching with different weights from the same maternal flock in midlay had different BW and pectoral muscle until 5 d and through 41 d (Figures 2B and D and 3B), whereas abdominal fat was not different at 41 d (Figure 3D). In the period until d 5, development of the GIT (expressed as a proportion of BW) did not change with stage of lay (Figure 4C). Relative sizes of the gizzard and duodenum were not different between treatments at 5 d of age; however, the jejunum, ileum, and cecum composed a smaller proportion of the BW in large chicks compared to small chicks.
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FIGURE 3. Body weight of chicks through marketing and pectoralis (pect) and abdominal fat (fat) weight from maternal flocks at 28 wk (filled circles, crosshatched bars) and 64 wk (filled triangles, gray bars) of age (A, C) and from chicks hatching at 43 ± 0.5 (open circles, open bars) and 53.1 ± 0.5 g (filled circles and black bars) from a maternal flock aged 44 wk (B, D). Results are expressed as a proportion of weights of chicks from the 28-wk-old maternal flock (C) and of chicks weighing 43 ± 0.5 g at hatch (D). BW differed significantly among chicks from big and small eggs through marketing, and at marketing pectoralis size was different among these chicks (P < 0.05).
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In contrast, liver development was affected by the difference in BW, and liver composed a greater proportion of BW in large chicks as compared to small chicks (Figure 4B). Morphometric measurements of villus height and width and crypt depth did not indicate significant differences between groups of chicks and the total number of enterocytes and absorptive areas reflected intestinal size (data not shown). Because development of the GIT was not correlated with BW, whereas pectoral muscle development was, the number and proliferation of satellite cells from pectoral muscles were determined in the early posthatch period. The temporal pattern of satellite cell proliferation per gram of muscle, as measured by thymidine
incorporation into DNA, and of changes in cell numbers were similar in large and small chicks; however, the magnitude was greater in the large chicks (Figure 5A, C). This difference in proliferation and number of satellite cells was even greater when calculated for whole muscle (Figure 5B, D). Protein levels of the muscle regulatory factor myogenin and the cyclin-dependent kinase inhibitor p21, both of which are involved in muscle differentiation, were examined in pectoral muscle tissues. Higher levels of these proteins were observed in the muscles of the large chicks compared to the small chicks from d 1 posthatch (Figure 6). In the large chicks, myogenin levels peaked on d 4, whereas in the small chicks these levels
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FIGURE 4. BW (A), liver weight as a percentage of BW (B) and gastrointestinal tract (GIT) as a percentage of BW (C) of chicks with equal weights from maternal flocks 28 wk of age (crosshatched bars) and 64 wk of age (diagonal lines) and in chicks from a maternal flock aged 44 wk hatching at 43 ± 0.5 g (open bars) and weighing 53.1 ± 0.5 g (filled bars) at 5 d of age. Results are means (n = 10), and bars are SD. BW and liver percentage were significantly greater, and jejunal, ileal, and cecal percentages were lower in chicks hatching at 53 g as compared with chicks hatching at 43 g (P < 0.05). GIZ = gizzard; Duo = duodenum; Jej = jejunum; Il = ileum; Cec = cecum.
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were reached only on d 5. IGF-I levels peaked in both groups on d 2 but were 2-fold higher in the large compared to the small chicks.
Experiment 3
FIGURE 5. Thymidine incorporation levels in satellite cells per gram of tissue (A) or per total pectoralis muscle (B) and number of satellite cells per gram of tissue (C) or per pectoralis muscle (D) during first 5 d of age in chicks, from a maternal flock aged 44 wk, weighing 43 ± 0.5 (circles) and 53.1 ± 0.5 g (triangles) at hatch. Results are means (n = 10), and bars are SD. Significant differences are marked with asterisks (P < 0.05).
DISCUSSION This study indicates that hatching weight of chicks is a major determinant of marketing weight and that this growth process is regulated, at least in part, by muscle growth and not by the development of the GIT. Previous studies have suggested that hatching weight is the major predictor of marketing weight in chickens
FIGURE 6. Protein levels of insulin-like growth factor-I (IGF-1), p21, and myogenin in pectoralis muscle of chicks weighing 43 ± 0.5 (circles) and 53.1 ± 0.5 g (triangles) at hatch from a maternal flock aged 44 wk from d 1 through 5. Densitometric analysis of protein levels relative to αtubulin protein levels in pectoralis muscles derived from experimental chicks at various days of age. Results are means (n = 10), and bars are SD. Significant differences are marked with asterisks (P < 0.05).
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Because muscle weight was greater in the heavier chicks during the days immediately posthatch, additional determinations were carried out with 15-d embryos from eggs of different weights to determine whether differences in muscle cell activity were present prehatch. Embryos were removed on d 15 of incubation from eggs weighing 68 ± 1 and 52 ± 1 g, respectively, from the same maternal flock at 44 wk of lay. The embryos from the heavier eggs weighed more than those from the smaller eggs (Figure 7, left panel). Pectoral muscles were removed from the embryos, myoblasts were prepared and counted, and the rate of proliferation was determined. Total myoblast numbers were not different between large and small embryos (Figure 7, middle panel), whereas thymidine incorporation levels were greater in the larger embryos (Figure 7, right panel).
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and that each 1-g increment in BW at hatch leads to 8 to 13 g of increase at marketing (Wilson, 1991). In this study we attempted to isolate the factors involved in enhanced growth of chicks hatching from maternal flocks in different stages of lay or with greater BW. As in many previous studies (Shanawany, 1984; Sinclair et al., 1990; Tuft, 1991; Wilson, 1991; Vieira and Moran, 1998; 1999), this study demonstrates that heavier chicks yielded the highest BW at marketing. Growth of chicks hatching at different BW from the same maternal flock was correlated with hatching weight as previously reported (Shanawany, 1984). However, comparison of chicks hatching at the same BW from early or late stage maternal flocks indicated that the age of the maternal flock did not influence posthatch performance. We examined development of the GIT in chicks as a possible determining factor, because rapid growth in the immediate posthatch period may be decreased if sufficient nutrients are not available (Sklan, 2001). However, no correlations were observed between growth and the size and development of all the parameters of the GIT that were examined. In fact, some of the heavier chicks had relatively smaller small intestines and ceca. In previous studies in turkey poults, no consistent effects of egg weights or hen size were found on small intestinal weight at hatch or through 7 d (Applegate and Lilburn, 1999). Together, these data would suggest that the capacity of the small intestines to supply nutrients is not regulating growth in the growing broiler under the conditions used in this study. In addition to the weight of the intestines, we also examined the relative size of livers in chicks that hatched
with different BW. Hepatic size was relatively larger in chicks that exhibited greater growth, which may reflect some parameters of metabolic activity. Hepatic metabolism in the newly hatched chick is very active in taking up endocytosed yolk lipid remnants, remodeling lipids to lipoprotein particles, and exporting them for circulation. This process involves accumulation of some lipid classes, in particular cholesterol esters, close to and after hatch (Noble and Ogunyemi, 1989; Speake et al., 1998). Furthermore, due to the extensive β-oxidation occurring in the liver, mitochondrial density increases and in parallel tissue, size may also increase (Speake et al., 1998). It has been shown that skeletal muscle growth could be affected by nutrition and environment (Halevy et al., 2000, 2001, 2003; Mozdziak et al., 2002). These changes were due to alterations in satellite cell activity in early posthatch stages, which dictate mature muscle size. In this study we observed that heavier chicks had greater muscle mass with more satellite cells that exhibited greater proliferative activity. Moreover, satellite cells in these chicks underwent earlier differentiation because levels of myogenin and p21, both involved in muscle differentiation (Andres and Walsh, 1996), were substantially higher than in the smaller chicks. Myogenin levels peaked earlier in heavy chicks, on d 4 posthatch as compared to d 5 in lighter chicks. As reported previously, myogenin expression in the muscle reflects differentiation of satellite cells in vivo (Yablonka-Reuveni et al., 1990; Halevy et al., 1996; Adams et al., 1999; YablonkaReuveni and Paterson, 2001). IGF-I levels were higher in heavier chick muscles compared to in smaller chicks already at 1 d of age, reached over 2-fold the next day,
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FIGURE 7. Egg and embryo weights and number of myoblasts and thymidine incorporation per gram of pectoralis muscle in heavy (black bars) or light (gray bars) eggs at 15 d from a maternal flock of 48 wk of age. Results are means (n = 10), and bars are SD. Significant differences are marked with asterisks (P < 0.05).
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ACKNOWLEDGMENTS We are grateful to Bruce Paterson for providing the chicken myogenin antibody. O. H. is Incumbent of the Vigevani Senior Lectureship in Animal Sciences. This work was supported in part by the Israeli Poultry Marketing Board.
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Barton-Davis, E. R., D. L. Shoturma, and H. L. Sweeney. 1999. Contribution of satellite cells to IGF-I induced hypertrophy of skeletal muscle. Acta Physiol. Scand. 167:301–305. Campion, D. R. 1984. The muscle satellite cell: a review. Int. Rev. Cytol. 87:225–251. Coleman, M. E., F. DeMayo, K. C. Yin, H. M. Lee, R. Geske, and C. Montgomery. 1995. Myogenic vector expression of insulin-like growth factor I stimulates muscle cell differentiation and myofiber hypertrophy in transgenic mice. J. Biol. Chem. 270:12109–12116. Duclos, M. J., R. S. Wilkie, and C. Goddard. 1991. Stimulation of DNA synthesis in chicken muscle satellite cells by insulin and insulin-like growth factors: evidence for exclusive mediation by a type-I insulin-like growth factor. J. Endocrinol. 128:35–42. Florini, J. R., D. Z. Ewton, and S. A. Coolican. 1996. Growth hormone and insulin-like growth factor system in myogenesis. Endocrinol. Rev. 17:481–517. Gal-Levi, R., Y. Leshem, S. Aoki, T. Nakamura, and O. Halevi. 1998. Hepatocyte growth factor plays a dual role in regulating skeletal muscle satellite cell proliferation and differentiation. Biochim. Biophys. Acta 1402:39–51. Halevy, O., A. Geyra, M. Barak, Z. Uni, and D. Sklan. 2000. Early posthatch starvation decreases sattellite cell proliferation and skeletal muscle growth in chicks. J. Nutr. 130:858–864. Halevy, O., V. Hodik, and A. Mett. 1996. The effects of growth hormone on avian skeletal muscle satellite cell proliferation and differentiation. Gen. Comp. Endocrinol. 101:43–52. Halevy, O., A. Krispin, Y. Leshem, J. P. McMurtry, and S. Yahav. 2001. Early-age-heat exposure affects skeletal muscle satellite cell proliferation and differentiation in chicks. Am. J. Physiol. 281:R302–R317. Halevy, O., and O. Lerman. 1993. Retinoic acid induces adult muscle cell differentiation mediated by the retinoic acid receptor. J. Cell. Physiol. 154:566–572. Halevy, O., E. Monsonego, C. Marcelle, V. Hodik, A. Mett, and M. Pines. 1994. A new avian fibroblast growth factor receptor in myogenic and chondrogenic cell differentiation. Exp. Cell Res. 112:278–284. Halevy, O., Y. Nadel, M. Barak, I. Rozenboim, and D. Sklan. 2003. Early posthatch feeding stimulates satellite cell proliferation and skeletal muscle in turkey poults. J. Nutr. 133:1376–1382. Hodik, V., A. Mett, and O. Halevy. 1997. Mutual effects of growth hormones and growth factors on avian skeletal muscle satellite cells. Gen. Comp. Endocrinol. 108:161–170. Leshem, Y., D. B. Spicer, R. Gal-Levi, and O. Halevy. 2000. Hepatocyte growth factor (HGF) inhibits skeletal muscle cell differentiation: a rule for the bHLH protein twist and the cdk Inhibitor p27. J. Physiol. (Lond). 184:101–109. Mauro, A. 1961. Satellite cell of skeletal muscle fibers. J. Biophys. Biochem. Cytol. 9:493–495. Moss, F. P., R. A. Sommonds, and H. W. McNary. 1964. The growth and composition of skeletal muscle in the chicken. 2. The relationship between muscle weight and the number of nuclei. Poult. Sci. 43:283–290. Mozdziak, B. E., J. J. Ivans, and D. W. McCoy. 2002. Early posthatch starvation induces myonuclear apoptosis in chickens. J. Nutr. 132:901–903. Naya, F., and E. N. Olson. 1999. Mef2: A transcriptional target for signaling pathways controlling skeletal muscle growth and differentiation. Curr. Opin. Cell Biol. 11:683–688. Noble, R., and D. Ogunyemi. 1989. Lipid changes in the residual yolk and liver of the chick immediately after hatching. Biol. Neonate 56:228–236. Noy, Y., and D. Sklan. 1999. Energy utilization in newly hatched chicks. Poult. Sci. 78:1750–1756. Noy, Y., and D. Sklan. 2001. Yolk and exogenous feed utilization in the posthatch chick. Poult. Sci. 80:1490–1495.
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and then declined. Therefore, it is suggested that the higher proliferation and earlier differentiation of the satellite cells in the heavier chicks could be attributed to higher IGF-I levels in these chicks. IGF-I enhances satellite cell proliferation, differentiation, and muscle hypertrophy in mammals and chicks (Duclos et al., 1991; Coleman et al., 1995; Florini et al., 1996; Hodik et al., 1997; Barton-Davis et al., 1999). Moreover, in previous studies we have shown that IGF-I is the major growth factor involved in enhanced muscle growth following mild heat stress (Halevy et al., 2001) and immediate posthatch feeding (Halevy et al., 2003), which correlates with higher levels of myogenin in chicks and poults. However, we cannot exclude the role of other growth factors such as basic fibroblast growth factor in enhancing satellite cell proliferation in heavier chicks. Together, these findings are compatible with the pattern that heavier chicks have enhanced satellite cell proliferation and earlier differentiation, supporting greater muscle development during the critical period of satellite cell activity. In addition to the differences in satellite cell activity posthatch, it well may be that differences in muscle development are established before hatch. Myoblast proliferating activity was higher in 15 d embryos derived from larger eggs relative to activity from smaller eggs from the same maternal flock. The correlation between egg weight and BW at hatch is well documented (Shanawany, 1984; Wilson, 1991), and thus larger eggs produce heavier chicks at hatching, which grow to bigger chicks at marketing. This growth, at least in part, represents enhanced muscle growth due to more myoblasts being in the proliferative stage during embryonic development and more satellite cells at early posthatch development.
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SAS. 1986. SAS User’s Guide. Version 6 Edition. SAS Institute Inc., Cary, NC. Shanawany, M. M. 1984. Inter-relationship between egg weight, parental age, and embryonic development. Br. Poult. Sci. 25:449–455. Sinclair, R. W., F. E. Robinson, and R. T. Hardin. 1990. The effects of parent age and posthatch treatment on broiler performance. Poult. Sci. 69:526–534. Sklan, D. 2001. Development of the digestive tract of poultry. World’s Poult. Sci. J. 57: 415–428. Speake, B., A. Murray, and R. Noble. 1998. Transport and transformations of yolk lipids during development of the avian embryo. Progr. Lipid Res. 37:1–32. Tuft L. S., and L. S. Jensen. 1991. Effect of age of hen, egg weight, and sex on chick performance and lipid retention. Poult. Sci. 70:2411–2418. Uni, Z., Y. Noy, and D. Sklan. 1999. Posthatch development of small intestinal function in the poult. Poult. Sci. 78.215–222.
Vieira, S. L., and E. T. Moran, Jr. 1998. Broiler yields using chicks from egg weight extremes and diverse strains. J. Appl. Poult. Res. 7:339–346. Vieira, S. L., and E. T. Moran, Jr. 1999. Effects of egg of origin and chick post-hatch nutrition on broiler life performance and meat yields. World’s Poult. Sci. J. 55:127–142. Weintraub, H. 1993. The MyoD family and myogenesis: redundancy, networks, and thresholds. Cell 75:1241–1244. Wilson, H. R. 1991. Interrelationships of egg size, chick size, posthatching growth and hatchability. World’s Poult. Sci. J. 47:5–16. Yablonka-Reuveni, Z., T. M. Balestreri, and D. F. Bowen-Pope. 1990. Regulation of proliferation and differentiation of myoblasts derived from adult mouse skeletal muscle by specific isoforms of PDGF. J. Cell Biol. 111:1623–1629. Yablonka-Reuveni, Z., and B. Paterson. 2001. MyoD and myogenin expression patterns in cultures of fetal and adult chicken myoblasts. J. Histochem. Cytochem. 49:455–462.
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(Key words: chick, gastrointestinal tract, growth, satellite cells, skeletal muscle) 2003 Poultry Science 82:1778–1786
INTRODUCTION Broiler performance is influenced by the age of the maternal flock and the weight of the hatching chick (Shanawany, 1984; Sinclair et al., 1990; Wilson, 1991; Vieira and Moran, 1999). Broiler weights at marketing were reported in the 1980s to increase by 8 to 13 g for each gram of increase in the hatching weight (Wilson, 1991), and, although these values differ between strains, this trend seems be increasing in modern birds (Wilson, 1991). Although egg composition changes with egg weight, these changes do not appear to have significant effects on broiler growth or processing yields (Vieira and Moran, 1999), suggesting that other systems are involved in these process. The digestive system of the chick is anatomically complete at hatching but undergoes major morphological and functional changes in the immediate posthatch period (Sklan, 2001). In the hatchling, uptake of glucose and amino acids is limited
2003 Poultry Science Association, Inc. Received for publication April 28, 2003. Accepted for publication July 1, 2003. 1 To whom correspondence should be addressed: sklan@agri. huji.ac.il.
and increases rapidly within 4 d posthatch (Noy and Sklan, 1999). Morphological and functional development of the small intestine as well as growth are stimulated by early access to feed (Noy and Sklan, 2001). Hence we hypothesized that development of the gastrointestinal tract (GIT) may be a limiting factor in broiler performance in the posthatch period. An additional system that is observed to be at a critical stage at hatch and affecting subsequent performance is the skeletal muscle satellite cell (Halevy et al., 2000). Skeletal muscle growth in posthatch birds is determined by hypertrophy and accumulation of nuclei in muscle fibers. The latter process is attributed to myogenic precursor cells, located beneath the basal lamina of the fibers, termed satellite cells (Mauro, 1961). In response to external signals, these cells are capable of entering the cell cycle and proliferating, differentiating, and either fusing into existing fibers or fusing with each other to form new fibers (Campion, 1984). Myogenic differentiation involves the coordination of muscle-specific gene expression and withdrawal from the cell cycle. The
Abbreviation Key: GIT = gastrointestinal tract; IGF-I = insulin-like growth factor-I.
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ABSTRACT This study examined some of the factors connected with the greater marketing weight observed in chicks hatching with higher BW. Examination of chicks hatching from maternal flocks of different ages indicated that BW at hatch increased quadratically and yolk sac weight linearly with age, whereas components of the gastrointestinal tract showed no significant trend. Growth of pectoralis muscles and gastrointestinal tract were compared in chicks hatching at the same weight from maternal flocks of 28 and 64 wk of age and in chicks from the same maternal flock (44 wk old) hatching at different weights. The results indicated that no differences were found among chicks hatching with the same weight from maternal flocks of different ages. In contrast, in chicks from the same maternal flock hatching at different weights the gastrointestinal tract tended to compose a
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MATERIALS AND METHODS Experimental Procedures Experiment 1. Chicks (Ross 308) were obtained from a commercial hatchery within 1 h of clearing the shell (n = 10 males and 10 females); birds were from a mother flock aged 26 through 68 wk, every 2 wk. BW of chicks were determined, and the yolk sac and small intestines were weighed, the latter after gently extruding contents. Experiment 2. Male chicks (Ross 308) were taken within 1 h of clearing the shell either from a flock at 44 wk of age with hatching weights of 43.5 ± 0.5 g and 53.1 ± 0.5 g or from flocks of 28 and 62 wk of age with weights of 49.3 ± 0.4 and 49.3 ± 0.4 g, respectively. Chicks were divided into groups on the basis of BW equalizing the variance between treatments (n = 80) and transported within 30 min to the facility. Chicks were provided with commercial feed according to the manufacturer’s protocol,2 and water was available ad libitum in floor pens in temperature-controlled rooms. 2
Matmor Feed Mill, M.P. Evtach, Israel. New England Nuclear, Boston, MA. 4 Packard, Downers Grove, IL. 5 Pierce, Rockford, IL. 6 Schleicher and Schuell, Dassel, Germany. 7 Zymed, San Francisco, CA. 3
For the first 5 d of age, 5 chicks per day were taken, and intestines and pectoral muscles were removed for further analyses. Chicks were weighed at 7, 14, 21, 35, and 41 d when chicks were marketed. All experimental procedures were approved by the Animal Welfare Committee of the Faculty of Agriculture, The Hebrew University of Jerusalem. Experiment 3. Eggs from a breeder flock (Ross 308) 46 wk of age at 15 d of incubation and weighing 68.2 ± 0.9 and 51.8 ± 0.9 were taken for examination of myoblast proliferation in the pectoral muscle.
Cell Cultures Skeletal muscle satellite cells were cultured from the pectoral muscle of poults at various ages as described by Halevy and Lerman (1993). Tissues were removed from the chicks under sterile conditions, chopped, and incubated with pronase (1.4 g/L) followed by trypsin (2.5 g/L) and DNAse I (0.5 g/L) for an additional 10 min. Enrichment of satellite cell culture was achieved with several centrifugations and tituration of the resuspended pellet. Cells were then filtered through a mesh (200 µm) and preplated for 2 h on a plastic Petri dish. Cells were counted using a hemocytometer and plated at 5 × 104 cells/cm2 in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 100 mL/L horse serum (HS) on gelatin-coated (1 g/L) dishes and maintained at 37°C in a humidified atmosphere containing 95% air and 5% CO2. On all days cells were prepared under similar conditions by using 6 g of pectoralis muscle that had been pooled from the experimental birds. An enriched population of myogenic cells was recovered, and less than 5% of the cells were nonmyogenic (Halevy et al., 1994).
Thymidine Incorporation DNA synthesis was assessed by [3H]-thymidine incorporation (Halevy and Lerman, 1993). [3H]-thymidine3 was added (74 kBq/well) for 2 h of incubation. Radioactivity in dissolved precipitates was counted using a TriCarb 1600CA scintillation counter.4 Equal plating efficiency was verified by determining cell numbers in parallel wells.
Western Blot Analysis Western blot analysis was performed as described by (Leshem et al., 2000). Muscle extracts were sonicated and normalized for protein content (BCA kit5), and equal amounts of protein were separated by SDS-PAGE and transferred to nitrocellulose filters.6 Membranes were incubated overnight at 4°C with the appropriate primary antibodies and then washed and incubated for 1 h with horseradish peroxidase-conjugated goat antimouse or goat anti-rabbit IgG.7 Proteins were visualized by enhanced chemiluminescence. The following primary antibodies were used: anti-IGF-I monoclonal anti-
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MyoD family of basic helix-loop-helix (bHLH) transcription factors (MyoD, myf5, myogenin, MRF4), form heterodimers with ubiquitous bHLH nuclear proteins (E proteins) and act in cooperation with the MEF2 family members to activate skeletal muscle structural genes (Naya and Olson, 1999; Weintraub, 1993). Proliferation of satellite cells is regulated by several growth factors and hormones, including fibroblast growth factors (Florini et al., 1996), platelet-derived growth factor (Yablonka-Reuveni et al., 1990), growth hormone (Halevy et al., 1994), and hepatocyte growth factor (Allen et al., 1995; Gal-Levi et al., 1998). Whereas these mitogens inhibit satellite cell differentiation, insulin-like growth factor-I (IGF-I) has been found to induce this process, as well as to improve muscle hypertrophy (Coleman et al., 1995). In broiler chicks, the process of proliferation and differentiation of satellite cells is transient but crucial, occurring in the first days posthatch, after which the satellite cell population declines dramatically (Moss et al., 1964; Halevy et al., 2000). Recent studies with birds have shown that early access to feed posthatch enhanced satellite cell proliferation and stimulated skeletal muscle growth in broilers, supporting the key role of these cells in dictating muscle size (Halevy et al., 2000). Another recent study has shown that the smaller muscle size found in chicks without access to feed following hatch is due to enhanced apoptosis of myonuclei (Mozdziak et al., 2002). This study examined changes in the GIT and pectoral muscle in chicks hatching from maternal flocks of different ages and of different BW and their correlation with subsequent development.
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body (1:3,000),8 polyclonal antibodies against chicken myogenin (1:6,000; a kind gift from Bruce Paterson, NIH, Bethesda, MD), and polyclonal anti-p21 (1:1,500).9 Densitometric analysis was performed on bands using GelPro software. Protein levels in each lane were normalized to the levels of protein loaded on that lane or to the levels of α-tubulin as an internal standard.
Histology Segments of approximately 2 cm were taken from the midpoint of the duodenum (duodenum), from the midpoint between the point of bile duct entry and Meckel’s diverticulum (jejunum), and midway between Meckel’s diverticulum and the ileocecal junction (ileum). Segments were gently flushed twice with 0.9% NaCl to remove the intestinal contents, fixed in fresh 4% buffered formaldehyde, and routinely processed into 5mm hematoxylin-eosin-stained sections. Morphometric indices were examined using computer-aided light microscope image analysis as previously described (Uni et al., 1999).
Statistical Analyses Data were analyzed by analysis of variance using the general linear models procedures of SAS software (SAS, 1986). Differences between means were examined using
8
Upstate Biotechnology, Lake Placid, NY. BD Biosciences, Lexington, KY.
9
FIGURE 2. Body weight (top panels) and pectoralis weight in chicks for 5 d posthatch (bottom panels). (A, C) Performance of chicks of the same BW at hatching from maternal flocks at 28 (filled circles) and 64 wk (filled triangles) of age (early-late). (B, D,) Performance of chicks hatching at 43 ± 0.5 g (open circles) and 53.1 ± 0.5 g (open triangles) from a maternal flock aged 44 wk (small-big). Results are means (n = 10), and bars are SD. BW were significantly different between chicks from big or small eggs from d 1 through 5, and pectoralis weight was different from d 3 to 5 (P < 0.05).
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FIGURE 1. Body weight (open triangles), weight of yolk sac or gastrointestinal track (GIT; circles), and weight of small and large intestines (filled triangles) at hatch from a maternal flock at different stages of lay. Results are means (n = 20), and bars are SD.
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the Tukey test, and significance was P < 0.05 unless otherwise stated.
RESULTS Experiment 1 Changes in BW of the hatchling and of some internal organs were examined with increasing age of the maternal flock. BW of chicks at hatching increased quadratically (r = 0.92, P < 0.001) and reached a plateau in the older flocks (Figure 1). In contrast, the weight of the yolk sac increased linearly (r = 0.78, P < 0.001) with age of the maternal flock. No correlations were observed between the age of the maternal flock and either small intestinal weight or the weight of any of the small intestinal segments. BW at hatch was correlated with the weight of the yolk sac (r = 0.216, P < 0.01) but not with the weight of the small intestines (r = 0.075, P > 0.05).
Experiment 2 This experiment was carried out to elucidate the connection between hatching weight and chick develop-
ment. Four groups of chicks were compared: chicks hatching with equal weights from maternal flocks in early (1) or late (2) stages of lay or large (3) and small (4) chicks from the same maternal flock at 44 wk of age. Chicks were examined at different ages through marketing to determine relative development. The parameters determined in these chicks are summarized in Figures 2 through 4. BW was not different between chicks from maternal flocks in early or late stages of lay either close to hatch (Figure 2A) or through 41 d (Figure 3A). Sizes of the pectoral muscles until d 5 (Figure 2C) and pectoral muscles and abdominal fat (Figure 3C) were not different at 41 d between these groups of chicks. In contrast, chicks hatching with different weights from the same maternal flock in midlay had different BW and pectoral muscle until 5 d and through 41 d (Figures 2B and D and 3B), whereas abdominal fat was not different at 41 d (Figure 3D). In the period until d 5, development of the GIT (expressed as a proportion of BW) did not change with stage of lay (Figure 4C). Relative sizes of the gizzard and duodenum were not different between treatments at 5 d of age; however, the jejunum, ileum, and cecum composed a smaller proportion of the BW in large chicks compared to small chicks.
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FIGURE 3. Body weight of chicks through marketing and pectoralis (pect) and abdominal fat (fat) weight from maternal flocks at 28 wk (filled circles, crosshatched bars) and 64 wk (filled triangles, gray bars) of age (A, C) and from chicks hatching at 43 ± 0.5 (open circles, open bars) and 53.1 ± 0.5 g (filled circles and black bars) from a maternal flock aged 44 wk (B, D). Results are expressed as a proportion of weights of chicks from the 28-wk-old maternal flock (C) and of chicks weighing 43 ± 0.5 g at hatch (D). BW differed significantly among chicks from big and small eggs through marketing, and at marketing pectoralis size was different among these chicks (P < 0.05).
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In contrast, liver development was affected by the difference in BW, and liver composed a greater proportion of BW in large chicks as compared to small chicks (Figure 4B). Morphometric measurements of villus height and width and crypt depth did not indicate significant differences between groups of chicks and the total number of enterocytes and absorptive areas reflected intestinal size (data not shown). Because development of the GIT was not correlated with BW, whereas pectoral muscle development was, the number and proliferation of satellite cells from pectoral muscles were determined in the early posthatch period. The temporal pattern of satellite cell proliferation per gram of muscle, as measured by thymidine
incorporation into DNA, and of changes in cell numbers were similar in large and small chicks; however, the magnitude was greater in the large chicks (Figure 5A, C). This difference in proliferation and number of satellite cells was even greater when calculated for whole muscle (Figure 5B, D). Protein levels of the muscle regulatory factor myogenin and the cyclin-dependent kinase inhibitor p21, both of which are involved in muscle differentiation, were examined in pectoral muscle tissues. Higher levels of these proteins were observed in the muscles of the large chicks compared to the small chicks from d 1 posthatch (Figure 6). In the large chicks, myogenin levels peaked on d 4, whereas in the small chicks these levels
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FIGURE 4. BW (A), liver weight as a percentage of BW (B) and gastrointestinal tract (GIT) as a percentage of BW (C) of chicks with equal weights from maternal flocks 28 wk of age (crosshatched bars) and 64 wk of age (diagonal lines) and in chicks from a maternal flock aged 44 wk hatching at 43 ± 0.5 g (open bars) and weighing 53.1 ± 0.5 g (filled bars) at 5 d of age. Results are means (n = 10), and bars are SD. BW and liver percentage were significantly greater, and jejunal, ileal, and cecal percentages were lower in chicks hatching at 53 g as compared with chicks hatching at 43 g (P < 0.05). GIZ = gizzard; Duo = duodenum; Jej = jejunum; Il = ileum; Cec = cecum.
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were reached only on d 5. IGF-I levels peaked in both groups on d 2 but were 2-fold higher in the large compared to the small chicks.
Experiment 3
FIGURE 5. Thymidine incorporation levels in satellite cells per gram of tissue (A) or per total pectoralis muscle (B) and number of satellite cells per gram of tissue (C) or per pectoralis muscle (D) during first 5 d of age in chicks, from a maternal flock aged 44 wk, weighing 43 ± 0.5 (circles) and 53.1 ± 0.5 g (triangles) at hatch. Results are means (n = 10), and bars are SD. Significant differences are marked with asterisks (P < 0.05).
DISCUSSION This study indicates that hatching weight of chicks is a major determinant of marketing weight and that this growth process is regulated, at least in part, by muscle growth and not by the development of the GIT. Previous studies have suggested that hatching weight is the major predictor of marketing weight in chickens
FIGURE 6. Protein levels of insulin-like growth factor-I (IGF-1), p21, and myogenin in pectoralis muscle of chicks weighing 43 ± 0.5 (circles) and 53.1 ± 0.5 g (triangles) at hatch from a maternal flock aged 44 wk from d 1 through 5. Densitometric analysis of protein levels relative to αtubulin protein levels in pectoralis muscles derived from experimental chicks at various days of age. Results are means (n = 10), and bars are SD. Significant differences are marked with asterisks (P < 0.05).
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Because muscle weight was greater in the heavier chicks during the days immediately posthatch, additional determinations were carried out with 15-d embryos from eggs of different weights to determine whether differences in muscle cell activity were present prehatch. Embryos were removed on d 15 of incubation from eggs weighing 68 ± 1 and 52 ± 1 g, respectively, from the same maternal flock at 44 wk of lay. The embryos from the heavier eggs weighed more than those from the smaller eggs (Figure 7, left panel). Pectoral muscles were removed from the embryos, myoblasts were prepared and counted, and the rate of proliferation was determined. Total myoblast numbers were not different between large and small embryos (Figure 7, middle panel), whereas thymidine incorporation levels were greater in the larger embryos (Figure 7, right panel).
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and that each 1-g increment in BW at hatch leads to 8 to 13 g of increase at marketing (Wilson, 1991). In this study we attempted to isolate the factors involved in enhanced growth of chicks hatching from maternal flocks in different stages of lay or with greater BW. As in many previous studies (Shanawany, 1984; Sinclair et al., 1990; Tuft, 1991; Wilson, 1991; Vieira and Moran, 1998; 1999), this study demonstrates that heavier chicks yielded the highest BW at marketing. Growth of chicks hatching at different BW from the same maternal flock was correlated with hatching weight as previously reported (Shanawany, 1984). However, comparison of chicks hatching at the same BW from early or late stage maternal flocks indicated that the age of the maternal flock did not influence posthatch performance. We examined development of the GIT in chicks as a possible determining factor, because rapid growth in the immediate posthatch period may be decreased if sufficient nutrients are not available (Sklan, 2001). However, no correlations were observed between growth and the size and development of all the parameters of the GIT that were examined. In fact, some of the heavier chicks had relatively smaller small intestines and ceca. In previous studies in turkey poults, no consistent effects of egg weights or hen size were found on small intestinal weight at hatch or through 7 d (Applegate and Lilburn, 1999). Together, these data would suggest that the capacity of the small intestines to supply nutrients is not regulating growth in the growing broiler under the conditions used in this study. In addition to the weight of the intestines, we also examined the relative size of livers in chicks that hatched
with different BW. Hepatic size was relatively larger in chicks that exhibited greater growth, which may reflect some parameters of metabolic activity. Hepatic metabolism in the newly hatched chick is very active in taking up endocytosed yolk lipid remnants, remodeling lipids to lipoprotein particles, and exporting them for circulation. This process involves accumulation of some lipid classes, in particular cholesterol esters, close to and after hatch (Noble and Ogunyemi, 1989; Speake et al., 1998). Furthermore, due to the extensive β-oxidation occurring in the liver, mitochondrial density increases and in parallel tissue, size may also increase (Speake et al., 1998). It has been shown that skeletal muscle growth could be affected by nutrition and environment (Halevy et al., 2000, 2001, 2003; Mozdziak et al., 2002). These changes were due to alterations in satellite cell activity in early posthatch stages, which dictate mature muscle size. In this study we observed that heavier chicks had greater muscle mass with more satellite cells that exhibited greater proliferative activity. Moreover, satellite cells in these chicks underwent earlier differentiation because levels of myogenin and p21, both involved in muscle differentiation (Andres and Walsh, 1996), were substantially higher than in the smaller chicks. Myogenin levels peaked earlier in heavy chicks, on d 4 posthatch as compared to d 5 in lighter chicks. As reported previously, myogenin expression in the muscle reflects differentiation of satellite cells in vivo (Yablonka-Reuveni et al., 1990; Halevy et al., 1996; Adams et al., 1999; YablonkaReuveni and Paterson, 2001). IGF-I levels were higher in heavier chick muscles compared to in smaller chicks already at 1 d of age, reached over 2-fold the next day,
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FIGURE 7. Egg and embryo weights and number of myoblasts and thymidine incorporation per gram of pectoralis muscle in heavy (black bars) or light (gray bars) eggs at 15 d from a maternal flock of 48 wk of age. Results are means (n = 10), and bars are SD. Significant differences are marked with asterisks (P < 0.05).
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ACKNOWLEDGMENTS We are grateful to Bruce Paterson for providing the chicken myogenin antibody. O. H. is Incumbent of the Vigevani Senior Lectureship in Animal Sciences. This work was supported in part by the Israeli Poultry Marketing Board.
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and then declined. Therefore, it is suggested that the higher proliferation and earlier differentiation of the satellite cells in the heavier chicks could be attributed to higher IGF-I levels in these chicks. IGF-I enhances satellite cell proliferation, differentiation, and muscle hypertrophy in mammals and chicks (Duclos et al., 1991; Coleman et al., 1995; Florini et al., 1996; Hodik et al., 1997; Barton-Davis et al., 1999). Moreover, in previous studies we have shown that IGF-I is the major growth factor involved in enhanced muscle growth following mild heat stress (Halevy et al., 2001) and immediate posthatch feeding (Halevy et al., 2003), which correlates with higher levels of myogenin in chicks and poults. However, we cannot exclude the role of other growth factors such as basic fibroblast growth factor in enhancing satellite cell proliferation in heavier chicks. Together, these findings are compatible with the pattern that heavier chicks have enhanced satellite cell proliferation and earlier differentiation, supporting greater muscle development during the critical period of satellite cell activity. In addition to the differences in satellite cell activity posthatch, it well may be that differences in muscle development are established before hatch. Myoblast proliferating activity was higher in 15 d embryos derived from larger eggs relative to activity from smaller eggs from the same maternal flock. The correlation between egg weight and BW at hatch is well documented (Shanawany, 1984; Wilson, 1991), and thus larger eggs produce heavier chicks at hatching, which grow to bigger chicks at marketing. This growth, at least in part, represents enhanced muscle growth due to more myoblasts being in the proliferative stage during embryonic development and more satellite cells at early posthatch development.
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