Muscle development- Could environmental manipulations during embryogenesis of broilers change it? O. HALEVY1*, I. ROZNEBOIM1, S. YAHAV2 and Y. PIESTUN1,2 1
Faculty of Agriculture, Dept. of Animal Sciences, The Hebrew University of Jerusalem, P.O. Box 12, Rehovot, Israel, Institute of Animal Sciences, 2ARO The Volcani Center, Bet-Dagan, P.O. Box 6, Israel, Corresponding author:
[email protected] __________________________________________________________________________________ Growth rate and meat yield of commercial broilers and turkeys have improved each year, with greater input efficiency and an earlier market age. As a result, the period of embryonic development and early posthatch now makes up a greater proportion of the bird’s life. Therefore, any technology that could enhance muscle growth during this period would help maintaining the progress of growth performance in the future. Skeletal muscle fibers are formed during embryogenesis and continue to enlarge postnatally until the mature size has been reached. This postnatal myofiber growth entails an increase in myofiber protein accretion and in the number of myofiber nuclei. The primary source of these additional myofiber nuclei are the satellite cells, myogenic stem cells situated on the surface of the myofiber between the myofiber plasmalemma and its covering basement membrane. In chicks, satellite cells (also termed adult myoblasts) are first detected in their sublaminar position between embryonic day E13 and E16, when the basement membrane surrounding the myofibers is developed. The number of satellite cells peaks on the second and third day of age and rapidly declines at around 8 days of age, after which they become quiescent. Previously, we have shown that nutritional treatments, i.e., providing feed immediately posthatch, or environmental treatments, such as heat conditioning or monochromatic green-light illumination during the first days posthatch, increase muscle growth and breast muscle weight at marketing day. In all cases, the increase in muscle growth was due to changes at the cellular and molecular levels leading to increased satellite cell proliferation and differentiation. The significant effects on muscle growth resulting from the treatments in the first days posthatch raised the hypothesis that muscle growth could be affected during the embryonic development. In experiments in which fertile eggs were illuminated under monochromatic green light from E5, there was a positive effect on embryo development and posthatch muscle growth. Further studies revealed that this enhanced muscle weight was due to increased satellite cell numbers and enhanced fiber synchronization during early days posthatch. In another study, a temperature manipulation (at 38.5oC or 39.5oC and 65% humidity) from E16 to E18 for 3 h/day augmented muscle cell proliferation even at later days such as day 13 of age. These data suggest that under the conditions of this study, the temperature manipulation may alter muscle development by delaying satellite cell differentiation and allowing these cells to remain in the cell cycle longer. This results in more cell proliferation and subsequently, in enhanced muscle growth and meat production. Collectively, these data highlight the notion that environmental manipulations in broilers during embryonic development or early on posthatch can serve as a powerful tool in affecting muscle development and enhancing muscle growth and meat production.
_________________________________________________________________________________ Key words: Skeletal muscle, myoblasts, satellite cells, broilers, embryogenesis
Introduction Development and histogenesis of skeletal muscle proceeds from early embryogenesis through adulthood. Embryonic muscle-precursor cells undergo myogenic determination and give rise to myoblasts (Ordhal et al., 2000). Myoblasts proliferate, withdraw from the cell cycle, differentiate, and eventually fuse into multinucleated fibers. Previous studies have indicated that the developing muscle consists of multiple myogenic populations (Cossu and Molinaro, 1987; Stockdale, 1992). In the chick, embryonic myoblasts are most abundant on E5, whereas fetal myoblasts are most abundant between E8 and E12 (Stockdale, 1992). Individual myofibers become encased by a basement membrane during late embryogenesis (E15 onwards), and it is at this stage that it becomes possible to distinguish satellite cells by their morphology and location (Hartley et al., 1992). Satellite cells (or adult myoblasts), first identified by Mauro (1961), are the primary source of myogenic precursors in the postnatal muscle (Campion, 1984; Schultz and McCormik, 1994). These mononucleated cells lie under the basal lamina of the myofiber and are uniformly distributed throughout the length of the muscle (Campion, 1984). Skeletal muscle nuclei consist of a high percentage of proliferating satellite cells at hatch. However, in adults, the number of satellite cells decreases to less than 5% of total myofiber nuclei and they become largely quiescent (Hawke and Garry, 2001). Satellite cells re-enter the cell cycle in response to various muscular stresses and undergo proliferation followed by withdrawal from the cell cycle and fusion into existing or newly formed fibers (Bischoff, 1994). Several growth factors, including members of the fibroblast growth factor (FGF) family, (Florini et al., 1996; Olwin et al., 1994), platelet-derived growth factor (PDGF; Yablonka-Reuveni and Seifert, 1993) and hepatocyte growth factor (HGF; Allen et al., 1995; Gal-Levi et al., 1998) are able to stimulate satellite cell proliferation and inhibit differentiation. Insulin-like growth factor I (IGF-I) has been shown to stimulate proliferation as well as differentiation of satellite cells and increase myofiber hypertrophy (Adams and McCue, 1998; Florini et al., 1996; Paul and Rosenthal, 2002). Recent studies have demonstrated that mild heat stress (thermal conditioning, TC) for 24 h at 3 days of age induces compensatory growth, leading to improved performance and muscle growth in broilers (Yahav and Plavnik, 1999). This is due to enhanced proliferation and differentiation of satellite cells immediately after the TC period, which is affected by elevated levels of locally secreted IGF-I (Halevy et al., 2001). This study analyzes the effects of temperature manipulations (TM) during broiler embryogenesis on body and muscle weight at the early growth phase in posthatch broilers, as well as on muscle cell proliferation. TM during embryogenesis is uniform and perhaps more efficient at inducing alterations in myoblast proliferation. The embryonic days, E16 to E18 were chosen because this is when fetal myoblasts undergo massive differentiation and adult myoblasts proliferate (Hartley et al., 1992; Stockdale, 1992).
Materials and Methods Experimental procedure: Fertile Cobb strain broiler eggs (n = 240 and n = 540 for experiment I and II, respectively) were purchased from a local hatchery (Braun, Israel). The eggs were arranged in homological locations in two incubators. The incubators (Masalles, Spain, Type 65Hs) were identical and automatic. Incubation conditions from day 0 to day 21 were: 37.8°C and 56% RH for the control group. Thermal treatment of the eggs from E16 to E18 involved an increase in temperature to either 38.5°C (experiment I) or 39.5oC (experiment II) and in RH to 65% for 3 h (09:00-12:00) on each of those days. Immediately after the thermal treatments were terminated, incubation conditions were restored to regular levels. Eggs in both incubators were turned through 270° every hour. Data loggers (Microlog from Fourier Systems, UIL Ltd.) were placed in each incubator to monitor the conditions every 30 min. On E10, infertile and undeveloped eggs were removed after candling. On E19, the eggs were transferred to hatching trays located in each incubator. Upon hatching, each chick was weighed, sexed and wing-banded. Males were transferred to temperature-controlled brooders with free access to commercial diet and water.
Cell cultures:. Muscle cells from the pectoral muscle of male chicks were cultured at various ages as previously described (Halevy et al., 2000), and incubated in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% (v/v) HS. Cells were counted using a haemocytometer, and plated at 5x104 cells/cm2 on gelatin-coated dishes, and maintained at 37oC in a humidified atmosphere containing 95% air and 5% CO2. On all days, cell cultures were prepared from 6 g of muscle sampled from a pool of chopped muscles from 10 chicks. Thymidine incorporation assay: Cells were cultured in 24-well plates for 17 hours and 3 [ H]thymidine was added for additional 2 hours. The assay was performed as previously described (Halevy et al., 2001). PCNA analysis: Breast-muscle samples were removed from the same longitudinal region and immediately fixed in fresh 4% paraformaldehyde in PBS (pH 7.6), dehydrated and embedded in paraffin. Sections (5 µm) were cut, placed on glass slides, deparaffinized and rehydrated as previously described (Halevy et al., 2004). Muscle sections were immunostained with an antibody against proliferating cell nuclear antigen (PCNA, a marker for dividing cells), using a commercial kit from Zymed (San Francisco, CA) followed by counterstaining with haematoxylin as previously described (Halevy et al., 2001). Control slides, where the primary antibody was omitted, were processed in parallel. Three chicks were analyzed per each group; five sections were studied per each chick, monitoring five random fields per each section. Analysis of positive cells was performed based on digitized images as previously described (Halevy et al., 2001; 2006). Statistical analysis: Data were subjected to analysis of variance (one-way ANOVA) and to Student's t-test, by means of the JMP® software (SAS Institute, 2000).
Results Body weight (BW) was significantly higher as of day 9 of age in chicks that were thermally manipulated either at 38.5oC (Figure 1A) or 39.5oC (Figure 1B) compared to controls. Similar results were obtained for breast-muscle percentage of BW (data not shown) suggesting a potential effect of TM on muscle cell proliferation.
Figure 1: BW of control and thermally manipulated (TM) male chicks at various days posthatch. TM was conducted at 38.5oC (A) or 39.5oC (B) on embryonic days E16 to E18 for 3 hours daily. Results are means ± SE (n = 20 or n = 50 for A and B, respectively). *P < 0.05 vs. control at the same age.
Muscle cells were prepared from breast muscle on various days before and after hatch and thymidine incorporation into DNA as a parameter for cell proliferation was determined. Thymidine incorporation did not vary between the groups on E18 (data not shown) or at hatch when TM was at 38.5oC (Figure 2A). However, these levels were significantly higher in the TM versus control group on
day 1 onwards. Similar trend was observed when TM was raised to 39.5oC (Figure 2B). Thymidine incorporation levels remained higher even at day 13. Indeed, analysis of the expression levels of PCNA, a marker for cell proliferation, in breast muscle sections derived from the experimental chicks on day 9, revealed that the fold number of PCNA-expressing cells was 1.35-fold higher in the TM at 38.5oC (Figure 3A) and 2.5-fold higher the TM at 39.5oC (Figure 3B) than in the control group (P < 0.05).
Figure 2: Labeled thymidine incorporation into DNA in muscle cells derived form breast muscle of TM at 38.5oC (2A) or 39.5oC (2B) and control chicks at various ages before and after hatch. Results are means ± SE (n = 6). *P < 0.05 vs. control at the same age.
Discussion The results of this study suggest that short periods of heat exposure at various temperatures o (38.5 C or 39.5oC) during late-term embryogenesis affect BW and muscle growth on the early phase of growth in posthatch broilers. In both groups, higher muscle cell proliferation was evident in the TM relative to control chicks as observed by thymidine incorporation into DNA of muscle cells that were
Figure 3: PCNA analysis of cross-sections derived from the pectoralis muscle of 9-day-old controls and chicks that were thermally manipulated (TM) at 38.5oC (A) or 39.5oC (B). Data are means + SE of 4-5 fields of 3 independent chicks. *P < 0.05 vs. control.
derived from breast muscle. Previously, we reported that this analysis reflects the in vivo proliferation of muscle cells in chicks (Halevy et al., 2000, 2001). Indeed, higher population of proliferating cells in muscle was observed when cross-sections of breast muscle derived from control and TM chicks (38.5oC) were stained for PCNA. In both TM groups, the higher muscle cell proliferation was still observed at day 6 and day 9 while normally in broilers, this activity is markedly reduced and cells undergo differentiation (Halevy et al., 2001; 2004). This may suggest a higher potential for future myogenic cells leading to higher muscle growth due to TM for short intervals in late-term embryos. The kinetics of muscle cell proliferation varied according to the temperature of manipulation. Whereas, TM at 38.5oC had a later effect on inducing cell proliferation, TM at 39.5oC had an immediate effect and significant induction in cell proliferation was observed as early as after the first day of TM (data not shown). It may well be that the increase in temperature had a direct effect on cell proliferation. Alternatively, the TM at the higher temperature could have caused a more prominent effect on levels of systemic or locally-induced factors, known to affect muscle cell proliferation and muscle hypertrophy, such as thyroid hormones or IGF-I (Duclos et al., 1996; Adams and McCue, 1998; Adams et al., 2000). Increased plasma triiodothyronine hormone (T3) concentrations have been observed in chicks that were thermally manipulated at 3 days posthatch and this coincided with compensatory muscle growth (Yahav and Hurwitz, 1996; Yahav and McMurtry, 2001). In addition, levels of thyroid hormones were found to be significantly lower at hatch in chicks that had undergone TM at 38.5oC compared to untreated chicks on E16 through E18 (Yahav et al., 2004). In view of these results, it seems that the higher the temperature, the better the proliferation response. However, TM at 41oC on similar days revealed a lower proliferative response (unpublished results) suggesting that a TM-inducing effect on muscle cell proliferation is limited to a narrow range of temperatures. Collectively, the findings of these experiments suggest that TM of late-term embryos augment muscle cell proliferation. The mechanism underlying this increase is dependent on temperature, duration and timing of the manipulation.
Acknowledgements We thank D. Shinder and M. Lavi for their assistance. O. Halevy is a holder of the Charles Charkowsky Chair in Poultry Science and Animal Hygiene. This work was supported in part by the Israeli Poultry Marketing Board.
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