The effects of thermal and light exposure on the development of broiler chicken leg musculature

Modified incubation factors affect skeletal musculature development of 
 broiler chickens during embryonic and postnatal periods of development. These 
 changes appear to have great influence on the proliferation of myoblasts and 
 muscle growth after hatching. In this study, the histomorphological and 
 -morphometric parameters of broiler chicken leg musculature, after thermal 
 and light treatments during embryogenesis, were examined. The applied 
 treatments included thermal manipulation (exposure of fertilized eggs to 
 increased temperature), light manipulation (exposure of fertilized eggs to 
 monochromatic green light) and simultaneous thermal and light manipulations. 
 Following the specific parameters that were observed, it was determined that 
 in the late postnatal period of development, treated groups have a larger 
 diameter and smaller nucleocytoplasmic ratio of muscle cells compared to 
 those of the control group, in which fertilized eggs were incubated at a 
 constant temperature of 37.8°C, without light. Diameter was increased by 
 4.20%, 3.77% and 4.55% on day 42 after hatching in thermal, light and 
 combined thermal and light treated groups, respectively, compared to the 
 control. The nucleocytoplasmic ratio was increased by 37.5% on day 42 after 
 hatching in the control group compared to all treated groups. The volume 
 density of the muscle connective tissue was approximately at the same level 
 between the groups. It can be concluded that thermal and light treatments 
 stimulate the proliferation of myoblasts and enhance development and growth 
 of leg muscles in broiler chickens. [Projekat Ministarstva nauke Republike 
 Srbije, br. TR 31033i br. OI173009]


INTRODUCTION
In the previous studies it was shown that some different feed treatments and additives affect the growth performance of chickens (Shashidhara and Devegowda, 2003;Li et al., 2007;Amad et al., 2011;Zikic et al., 2011).Halevy et al. (2006c) reported that environmental manipulations during embryogenesis appear to have important relevance for muscle development and also affect chicken growth.Thermal treatments of the fertilized eggs, from the 16 th to 18 th embryonic days, resulted in higher myoblast prolif-eration (Piestun et al., 2009), prolonged cell cycle of satellite cells (Halevy et al., 2006b) and higher body weight (Collin et al., 2005), all of which caused enhanced meat production.Illumination of chicken embryos with monochromatic green light resulted in higher breast and body weight at the later posthatch stages (Rozenboim et al., 2004), and a two-fold higher number of adult myoblasts in the muscles (Halevy et al., 2006a).
Duration and timing of thermal manipulations, as well as the exposure of fertilized eggs to some different part of light spectrum, have a key role in chicken development.Previous studies showed that the most appropriate duration and timing of thermal manipulations in order to reach higher growth is 3 h per day, during the period from the 16 th to 18 th embryonic day (Yahav et al., 2004).The exposure of fertilized eggs to white and green monochromatic light increases embryo weight, which positively reflects embryonic and postnatal development (Halevy et al., 2006c).The most appropriate temperatures that can be used for thermal manipulations are from 38.5°C to 39.5°C (Piestun et al., 2008), while temperatures above 40ºC, due to them causing a lower proliferative response of myoblasts (Halevy et al., 2006b), are not of choice.
The aim of this study was to examine the histomorphological and morphometric parameters of broiler chicken leg musculature after thermal and light manipulations during incubation.

Experimental design
Eight hundred fertilized broiler eggs (Ross 308) of approximately the same weight (65.0±2.0 g), from one parent flock (47 weeks of age), were used in the study.The eggs were divided into 4 groups (control, thermally treated, light treated and simultaneously thermally and light treated), and incubated in 200egg incubators (type ISC32, Fenil, Beska, Serbia).Control eggs were maintained at 37.8°C and 58% relative humidity during the whole incubation period.Thermal manipulation at 39.0°C was applied to the second group, for 3 h/day (09:00 -12:00) on days 16, 17 and 18 of the embryonic period.Eggs from the third group, which were light treated, received monochromatic green light provided by LED (light-emitting diode) lamps of 560 nm light, at an intensity of 0.1 W/m 2 at eggshell level.Lighting was intermittent (15 min light, 15 min dark) from embryonic days 6-15; thereafter, lighting was continuous.Eggs from the fourth group were simultaneously thermally and light treated according to the regime of the second group (for thermal ma-nipulations) and the third group (for light manipulations).
On the tenth day of incubation, unfertilized eggs and dead embryos were identified by candling and removed from the experiment.Upon hatching, chickens were transferred to brooders with free access to commercial diet and water, and were grown to 42 days of age.
From each of four groups, 10 embryos were randomly selected on the embryonic days 9, 12, 15, 19 and 20, as well as 10 chickens at days 1, 3, 7, 14, 21 and 42 of age.From these embryos and chickens, samples of leg skeletal muscle tissue (M.biceps femoris) were taken.

Histological analysis
Histological analysis was performed using classical light, scanning electron and transmission electron microscopy.To perform classical light microscopy analysis, the muscle tissues were fixed in a 10% buffered formalin solution, followed by a sequence of dehydration and clearing.After that, the samples were embedded in Paraplast and cut into 5-μm thick sections using a microtome Leica RM 2145 (Leica Microsystems, Germany).Histological preparations were stained with hematoxylin and eosin (H&E) and Mallory method.H&E-stained preparations were used for determining diameter and nucleocytoplasmic ratio of muscle cells, while the Mallory method-stained preparations were used for showing connective tissue (Bancroft andGamble, 2008a, 2008b;Grizzle, 2009).
Classical light microscopy was performed using a light microscope Leica DMLS with a Leica DC 300 digital camera, and the software package IM 1000 (Leica Imaging Systems Ltd, Germany).The diameter of muscle cells was measured as the average of the longest lines drawn across the length and width of their cross-sections.For the analyses of the volume density of connective tissue and the nucleocytoplasmic ratio of muscle cells, the M 42 testing system described elsewhere was used (Burity, 2004;Weibel, 2007;Uscebrka et al., 2010), which basically consists of 21 line segments and 42 points in a testing area.The volume density (%) of connective tissue of leg muscle was calculated using the following formula: where Vv(ct) is the volume density of connective tissue of leg muscle, P(ct) the number of test points lying over the connective tissue of leg muscle, and P(m) the number of test points lying over the muscle.The nucleocytoplasmic ratio of muscle cell was calculated using the following formula: where N / C is the nucleocytoplasmic ratio of muscle cell, P(n) the number of test points lying over the cell nucleus, and P(c) the number of test points lying over the cell cytoplasm.
For electron microscopy histological analysis, the muscle tissue samples were initially fixed in 2.5% glutaraldehyde, followed by washing in sodium cacodylate buffer.The samples were then post-fixed in 2% osmium tetroxide, followed by washing in distilled water and dehydration in a series of rising ethanol concentrations.After these processes, for scanning electron microscopy the samples were subjected to critical point drying, mounted on aluminum stubs and coated with gold-palladium, while for the transmission electron microscopy, samples were embedded in epoxy resin, cut into 1-μm thick sections, placed on copper grids and stained with uranyl acetate and lead citrate (Silva et al., 1992).
Scanning electron microscopy was performed using a scanning electron microscope Jeol JSM-6460LV (JEOL, USA).Tissue samples were critical point dried in a CPD 030 "Critical Point Dryer" (BAL-TEC, Liechtenstein).Transmission electron microscopy was performed using a transmission electron microscope Morgagni 268 (FEI, USA).

Statistical analysis
Statistical significance of differences obtained in measurements was determined using two-way ANO-VA and the post hoc Tukey test for each of the parameters measured.It was considered significant at p <0.05.Statistical processing of data was carried out using the software package Statistica for Windows ® , ver.10.0 (StatSoft, Tulsa, OK, USA).

Diameter of leg muscle cells
Comparisons of the diameter of leg muscle cells showed no significant differences (p >0.05) between chicken groups during the embryonic period of development as well as on the first day post hatching (Table 1).During the postnatal period of development, significant differences in the diameter of leg muscle cells were determined between the control group and all treated groups on days 7, 21 and 42 after hatching (p <0.05).Namely, a greater diameter was measured in the muscle cells of the control on day 7 after hatching compared to all treated groups (diameter in the control group was increased by 13.29% compared to the thermal and simultaneously thermally and light treated group; and by 13.63% compared to the light treated group), while in muscle cells of all treated groups greater diameter was measured on days 21 and 42 after hatching in comparison with the control group (Table 2).The diameter was increased by 8.76%, 8.56% and 9.19% on day 21 after hatching (by 4.20%, 3.77% and 4.55% on day 42 after hatching) in the thermally-, light-and the simultaneously thermally and light-treated groups, respectively, as compared to the control group.
Comparing the values of the diameter of leg muscle cells (Table 1), significant differences (p <0.05) were determinated in the control and thermally treated group between days 15 and 19 of embryonic development (on day 19 the diameter was increased by 7.55% and 7.06% in the control and thermally treated groups, respectively), and in the control group between day 20 of embryonic develop-(1) (2) ment and the first day post hatching (diameter was increased by 8.75% on the first day post hatching).During the postnatal period of development, in all chicken groups significant differences (p <0.05) in the diameter of leg muscle cells were determined (Table 2) between days 3 and 7, 7 and 14, 14 and 21, and 21 and 42 after hatching (in all groups, diameter was increased by 32.34-45.42%on day 7 compared to day 3 after hatching, by 40.12-59.22%on day 14 compared to day 7 after hatching, by 28.69-40.26%on day 21 compared to day 14 after hatching, and by 131.95-142.68% on day 42 compared to day 21 after hatching).
The differences in the diameter of muscle cells of the M. biceps femoris between control and treated groups on day 42 after hatching can be seen in Fig. 1.In the same area, in muscle of the treated group a smaller number of muscle cells of greater diameter can be observed, while in muscle of the control group, a greater number of muscle cells of smaller diameter can be seen.

Volume density of connective tissue in leg muscle
Comparison of the volume density of connective tissue in the leg muscle showed no significant differ-  ences (p >0.05) between chicken groups during the embryonic period of development, the first day post hatching (Table 1), and during the postnatal period of development (Table 2).
In all chicken groups, significant differences (p <0.05) in the volume density of connective tissue in the leg muscle were determined (Table 1) between days 9 and 12, 15 and 19, and 19 and 20 of embryonic development (in all groups, volume density of con-nective tissue was decreased by 7.97%-8.74%on day 12 compared to day 9 of embryonic development, by 10.16-12.62% on day 19 compared to day 15 of embryonic development, and by 10.34-13.52% on day 20 compared to day 19 of embryonic development).
An approximately equal amount of muscle and connective tissue in M. biceps femoris between control and treated groups on day 42 after hatching can be seen in Fig. 2. For detection of muscle (coloured     red-purple on photomicrographs) and connective tissue (coloured green on photomicrographs) the Mallory staining method was used.
The distribution of connective tissue within the M. biceps femoris is shown in Fig. 3. Bundles of muscle cells exhibit low levels of differentiation on day 15 of embryonic development, with small amounts of connective tissue between them.On the other hand, on day 42 after hatching, bundles of muscle cells are fully differentiated with a large amount of connective tissue between them, while inside them clear boundaries between individual muscle cells exist.

Nucleocytoplasmic ratio of leg muscle cells
Comparison of the nucleocytoplasmic ratio of leg muscle cells showed no significant differences (p >0.05) between chicken groups during the embryonic period of development as well as on the first day post hatching (Table 1).During the postnatal period of development, significant differences in the nucleocytoplasmic ratio of leg muscle cells were determined between the control group and all treated groups on days 7, 21 and 42 after hatching (p <0.05).Namely, a higher nucleocytoplasmic ratio was measured in the muscle cells of all treated groups than on day 7 after hatching (nucleocytoplasmic ratio in all treated groups was increased by 20% compared to the control group), while a lower nucleocytoplasmic ratio was measured on day 21 and 42 after hatching compared to the same parameter in the control group (Table 2).The nucleocytoplasmic ratio was increased by 27.27% on day 21 after hatching (by 37.5% on day 42 after hatching) in the control group compared to all treated groups.
Comparing the values of nucleocytoplasmic ratio of leg muscle cells (Table 1), significant differences (p <0.05) were determined in all chicken groups between days 19 and 20 of embryonic development and between day 20 of embryonic development and the first day post hatching (in all groups, the nucleocytoplasmic ratio was decreased by 18.18-19.35%on day 20 compared to day 19 of embryonic development, and by 23.08-26.92%on the first day post hatching compared to day 20 of embryonic development), as well as in the group that was simultaneously thermally and light treated during incubation between days 15 and 19 of embryonic development (nucleocytoplasmic ratio was decreased by 16.21% on day 19 of embryonic development).During the postnatal period of development, significant differences (p <0.05) in the nucleocytoplasmic ratio of leg muscle cells were determined (Table 2) in all chicken groups between days 21 and 42 after hatching (in all groups nucleocytoplasmic ratio was decreased by 21.43-27.27%on day 42 after hatching), in all groups except the control between days 7 and 14, and 14 and 21 after hatching (in all groups except the control, the nucleocytoplasmic ratio was decreased by 16.67-22.22%on day 14 compared to day 7 after hatching, and by 21.43-26.67%on day 21 compared to day 14 after hatching), as well as in the control group between days 3 and 7 after hatching (the nucleocytoplasmic ratio was decreased by 16.67% on day 7 after hatching).
The position of the nuclei inside the muscle cell of M. biceps femoris is shown in Fig. 4. The nucleus is peripherally positioned inside the muscle cell on day 15 of embryonic development.On day 42 after hatching, the nucleus retains a peripheral position, while the muscle cell diameter is significantly enlarged, which causes the nucleus to take up a much smaller part of the muscle cell area compared to the embryonic period.

Effects of incubation treatments on morphometric parameters
There were no significant differences (p >0.05) regarding the diameter and nucleocytoplasmic ratio of leg muscle cells between the treatments, and volume densities of connective tissue in leg muscle, during the embryonic period of development and the first day post hatching (Table 1).During the postnatal period of development, there were significant differences (p <0.05) between the treatments in terms of diameter and nucleocytoplasmic ratio of the leg muscle cells (as described before, significant differences (p <0.05) in the diameter and nucleocytoplasmic ratio of leg muscle cells were determined between the control group and all treated groups on days 7, 21 and 42 after hatching), but not for the volume density of connective tissue in leg muscle (Table 2).
Statistical analysis showed differences (p <0.05) among the ages when comparing the diameter and nucleocytoplasmic ratio of the leg muscle cells and volume density of connective tissue in the leg muscle during the embryonic period of development and the first day post hatching (Table 1), as well as during the postnatal period of development (Table 2).Generally, the diameter of leg muscle cells increased, while the volume density of connective tissue of the leg muscle as well as the nucleocytoplasmic ratio of leg muscle cells decreased from the early embryonic days to the late postnatal period of development.
Significant interactions were not detected between treatment and age regarding the diameter and nucleocytoplasmic ratio of leg muscle cells, as well as the volume density of connective tissue in leg muscle during the embryonic period of development and the first day post hatching (Table 1).During the postnatal period of development, significant interactions (p <0.05) were detected between treatment and age in terms of diameter and nucleocytoplasmic ratio of the leg muscle cells, but not the volume density of connective tissue in the leg muscle (Table 2).

DISCUSSION
The results of our study show that on day 7 after hatching the leg muscle cells of the control group have a greater diameter compared those of all treated groups.The smaller diameter of the muscle cells in the treated groups could be explained by thermal and light exposure during incubation, which caused prolonged proliferation of myoblasts (Rozenboim et al., 2004;Collin et al., 2007).We observed that during the time when the proliferation of myoblasts was still underway in the treated groups, an increase occurred in the diameter of muscle cells of the control group.On day 14 after hatching, no significant differences in the diameter of leg muscle cells between all chicken groups were determined.Obviously, the accelerated growth of skeletal muscle cells in the treated groups occurs upon myoblast proliferation.The thermal and light treatments applied in our study affected not only the prolonged proliferation of myoblasts, but also accelerate growth of the leg muscle cells in the late postnatal development.Precisely, we observed a larger diameter of leg muscle cells in the treated groups compared to those of the control group on days 21 and 42 after hatching.Enhanced growth of muscle cells of broiler chickens at market age caused by thermal manipulation during embryogenesis was also noticed by Halevy et al. (2006c) who pointed out that increased incubation temperature had a delayed effect on satellite cell proliferation and differentiation, which also contributes to muscle growth in the postnatal period.
We detected no significant differences in the volume density of connective tissue in the leg muscle between the control and all treated groups.This could be due to the equal quantity of muscle tissue per unit area within the leg muscle in all chicken groups at the same age.On day 7 after hatching, the thermal and light treatments induced prolonged proliferation of myoblasts, causing the formation of a higher number of leg muscle cells of smaller diameter per unit area in all treated groups, while within the leg muscles of the control group, a smaller number of muscle cells of greater diameter was formed per the same unit area.On days 21 and 42 after hatching, the applied treatments caused an increase in the diameter of leg muscle cells, which was reflected in the formation of a smaller number of leg muscle cells of greater diameter per unit area.Within the leg muscles of the control group, a higher number of muscle cells of smaller diameter was formed per the same unit area.On the other days measurements were performed, approximately the same number of leg muscle cells of approximately the same diameter were formed per unit area in all chicken groups.The described relation between diameter and number of leg muscle cells per unit area is in agreement with the observations of other authors (Halevy et al., 2006a;Halevy et al., 2006b), who detected an increased number of myoblasts and augmented muscle cell proliferation caused by environmental manipulations during embryogenesis of broilers.These processes did not cause significant differences in the volume density of connective tissue in leg muscle between the control and treated groups.
The results of our study show that on day 7 after hatching, the leg muscle cells of the treated groups had a higher nucleocytoplasmic ratio compared to those of the control group.This is supposedly the consequence of the greater diameter of leg muscle cells of the control group, as well as the greater amount of cytoplasm within these cells, where the nucleus takes up a smaller part of the cell.Namely, the leg muscle cells of all treated groups had a smaller diameter and smaller amount of cytoplasm, where the nucleus takes up a bigger part of the cell.The nuclei had the same diameter in all chicken groups on day 14 after hatching, which was reflected in the absence of significant differences in the nucleocytoplasmic ratio of leg muscle cells between all groups.On days 21 and 42 after hatching, the nucleocytoplasmic ratio of leg muscle cells was higher in the control group compared to all treated groups.This is related to the smaller diameter and smaller amount of cytoplasm of the leg muscle cells of the control group compared to all treated groups, which is similar to results previously reported by Piestun et al. (2009).In that study, in broiler chickens that were thermaly manipulated during late-term embryogenesis, a higher diameter of muscle cells in the late postnatal period was also detected, which is related to the smaller nucleocytoplasmic ratio of these cells.
As the result of thermal and light treatments during embryogenesis, beside prolonged proliferation of myoblasts Ghatpande et al. (1995) detected accelerated morphogenesis and growth of broiler chickens, which is in agreement with our results.Namely, we observed that during the time that myoblasts proliferated in the treated groups, an increase in the diameter of leg muscle cells of the control group occurred, which was supported by the following results of our study: a significant increase in the diameter of leg muscle cells of the control group between day 20 of embryonic development and the first day post hatching, as well as a significant decrease in the nu-cleocytoplasmic ratio of lthe eg muscle cells of the same group between day 3 and day 7 after hatching.The accelerated growth of the broiler chickens in the treated groups was related to a significant decrease in the nucleocytoplasmic ratio of leg muscle cells of treated groups between days 7 th and 4, as well as between days 14 and 21 after hatching.
In this study, a decrease in amount of connective tissue in the leg muscle from day 9 of embryonic development to day 42 after hatching was determined in all chicken groups.This could be explained by several processes that occur during muscle tissue development from the early embryonic to late postnatal period, i.e. growth and differentiation of leg muscle cells (Bellairs and Osmond, 2005), formation of bundles of these cells (Junqueira et Carneiro, 2003), as well as the distribution of connective tissue in and outside the formed bundles of muscle cells (Eurell and Frappier, 2006;Samuelson, 2007).These processes lead to a decrease in the amount of connective tissue, which is highly present in the early embryonic period, and an increase in the amount of muscle tissue toward the end of incubation and the period after hatching.
Finally, it can be concluded that thermal and light manipulations during incubation cause changes in the development of the skeletal musculature of broiler chickens, as shown by the described differences in the diameter and nucleocytoplasmic ratio of leg muscle cells, as well as the approximately equal volume density of connective tissue in the leg muscle between the control and all treated groups.Applied thermal and light treatments caused prolonged proliferation of myoblasts and accelerated growth of the leg muscle cells in the late postnatal development, which was reflected by the values of the observed parameters.These findings emphasize the possibility of enhancing skeletal muscle growth of broiler chickens using thermal and light manipulations during incubation.

Fig. 1 .
Fig. 1.M. biceps femoris of broiler chickens of control group (A) and simultaneously thermally and light treated group (B) on day 42 post hatching stained with H&E.Scale bar = 10 μm.

Fig. 2 .
Fig. 2. M. biceps femoris of broiler chickens of control group (A) and simultaneously thermally and light treated group (B) on day 42 post hatching stained with the Mallory method.Scale bar = 10 μm.

Fig. 3 .
Fig. 3. Scanning electron micrograph of M. biceps femoris of simultaneously thermally and light treated group of chickens.Figure shows initial phase (A, day 15 of embryonic development, scale bar = 1 μm) and mature stage (B, day 42 after hatching, scale bar = 100 μm) of the formation of skeletal muscle cells bundles.
Fig. 3. Scanning electron micrograph of M. biceps femoris of simultaneously thermally and light treated group of chickens.Figure shows initial phase (A, day 15 of embryonic development, scale bar = 1 μm) and mature stage (B, day 42 after hatching, scale bar = 100 μm) of the formation of skeletal muscle cells bundles.

Fig. 4 .
Fig. 4. Transmission electron micrograph of M. biceps femoris of chickens of simultaneously thermally and light treated group.In leg muscle cell, myofibrils show little differentiation on day 15 of embryonic development (A, scale bar = 2 μm), while on day 42 post hatching (B, scale bar = 1 μm) myofibrils are fully differentiated.

Table 1 .
Examination of samples of leg muscle tissue during the embryonic period of development and first day post hatching: diameter of muscle cells (μm), volume density of connective tissue of leg muscle (%) and the nucleocytoplasmic ratio of muscle cells.Values are means ± SE.
Means within a column, with no common superscript small letter (a, b, c, d, e, f) differ significantly (p < 0.05)

Table 2 .
Examination of samples of leg muscle tissue during the postnatal period of development: diameter of muscle cells (μm), volume density of connective tissue of leg muscle (%) and nucleocytoplasmic ratio of muscle cells.Values are means ± SE.