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Comparative Analysis: Muscle Fiber Typing and Histochemistry of the Gastrocnemius of a Rat and Chicken Gordon Cheung, Drew Daly, Alisa Dea, Kristin Rodriguez 5 June 2015 Abstract The purpose of this study is to utilize enzyme histochemistry to detect the presence of succinate dehydrogenase (SDH) in the muscle fibers of the gastrocnemius in a female rat and chicken to compare differing cell type frequencies and area between the species. The three muscle fiber types consist of: fast-twitch glycolytic (FG), fast-twitch oxidative (FOG), and slow-twitch oxidative (SO). The gastrocnemius from each species was dissected and stained using a reaction solution to allow for identification of the differing muscle fiber types by the level of staining power proportional to the presence of succinic dehydrogenase (SDH) in the mitochondria of each muscle fiber type. Our study found that the rat had a significantly greater mean fiber area in FG and FOG than the chicken, but no significant difference in SO mean fiber area. The rat had noticeably larger cell frequency of FOG fiber types than the chicken, while the chicken had more SO fibers than the rat. This demonstrates that these domestic vertebrates have developed unique muscle fiber characteristics to fit their physical needs in motor movement to adapt to the their environment. I.

Introduction Skeletal muscle is composed of a heterogenous collection of muscle fiber types. The

heterogeneity of the muscle fibers is the base of the flexibility which allows the same muscle to be used for various tasks, from continuous low-intensity activity (e.g., posture), to repeated submaximal contractions (e.g., locomotion), and to fast and strong maximal contractions (jumping, kicking) (Schiaffino & Reggiani, 2011). Contractile characteristics correlate with differences in morphological and biochemical properties, such as oxidative vs. glycolytic metabolism. Two of the key features of the different muscle fiber types are the number of mitochondria present in the muscle and the cross-sectional diameter of the fiber. Small diameters maximize oxygen diffusion while large diameters maximize force output. The three muscle fiber types consist of: fast-twitch glycolytic (FG), fast-twitch oxidative (FOG), and

slow-twitch oxidative (SO). FG fibers contain few mitochondria and contract rapidly (fasttwitch fiber), therefore, they are large diameter glycolytic fibers that fatigue fast. SO fibers are small diameter aerobic fibers (slow twitch) that contain more amounts of mitochondria in order to sustain contraction for longer periods of time compared to FG fibers. FOG fiber is an intermediate fiber type that is a fast contracting fiber with both aerobic and glycolytic metabolic capacities, with an intermediate fiber diameter (Blank, 2015). Over the past several decades, the number of techniques available for classifying muscle fibers has increased, resulting in several classification systems. Currently, muscle fibers are typed using three different methods: histochemical staining for myosin ATPase, myosin heavy chain isoform identification, and biochemical identification of metabolic enzymes (Binder, Scott, & Stevens, 2001). This study utilizes enzyme histochemistry for the presence of a specific enzyme, succinic dehydrogenase (SDH), that reflects the energy metabolism of the fiber. SDH is an enzyme complex, bound to the inner mitochondrial membrane of mammalian mitochondria and many bacterial cells. It is the only enzyme that participates in both the citric acid cycle and the electron transport chain. This allows for a staining technique to mark for mitochondria in tissues when incubated in a solution made of succinate and a reaction detector molecule such as nitroblue tetrazolium. As the enzyme-substrate reaction proceeds, nitroblue is reduced and precipitates out of solution as a blue compound, thus marking the mitochondria. Although mitochondria are too small to be seen as individual organelles under the light microscope, the greater their concentration within a cell, the darker the stain will be with this reaction to identify the three different muscle types (Sweeney, 2011). The test muscle of this study was the medial head of the gastrocnemius (MHG) of a rat (Rattus norvegicus sprague-dawley) and a whole gastrocnemius of a white leghorn chicken (Gallus gallus domesticus). The gastrocnemius is considered an antigravity muscle that works to oppose the effects of gravity and stabilize the body. Since the gastrocnemius is responsible for plantar flexion of the foot, it is able to contribute a major fraction of the wide dynamic range of force output in high force activities, such as running, jumping, and other "fast" movements of the leg, and to a lesser degree in walking and standing. This specialization is connected to the predominance of white muscle fibers (type II fast twitch) present in the gastrocnemius, as opposed to the soleus, which has more red muscle fibers (type I slow twitch) and is the primary active muscle when standing still, as determined by EMG studies (Binder, Scott, & Stevens).

An experiment done with rats found that muscle groups with anti-gravity function had a significantly higher percentage of type IIA muscle fibers and significantly lower percentage of type IIB muscle fibers. However, with the MHG they found that it contained more Type IIB fibers compared to other types (Eng, 2008). This is important because anti-gravity muscles may need more force generation rather than to function over a large range of motion in order to stabilize the body. Moreover, researchers found that the physiological cross-sectional area (PCSA) of the gastrocnemius had larger PCSAs along with shorter fibers to generate large forces over short distances (Eng, 2008). Comparatively, Chang and colleagues aimed to study muscle fiber changes between emasculated chickens and non-emasculated chickens. The researchers found through enzyme histochemistry that the gastrocnemius of non-emasculated chickens contained 28.21% Type I, 38.46% Type IIA, and 33.33% Type IIB (Chang, 2004). Furthemore, they found that the nonemasculated chickens had larger Type IIB PCSA followed by Type IIA and Type I (Chang, 2004). In another experiment done by Yue and colleagues, the authors looked into the effects of early feed restriction on the muscle fiber types in the lateral gastrocnemius of crossbred broiler chickens. Their mature chicken control group displayed a higher density of white fast-twitch myofibres at 51%, followed by slow-twitch myofibres at 25%, and then red fast-twitch myofibres at 23%; In average cross sectional area, the largest to smallest was the white fasttwitch myofibres (2960 µm2), red fast-twitch myofibres (2118 µm2), and the slow-twitch myofibres (1550 µm2) (Yue, 2007). Moreover, they have found that the changes seen in their chickens muscle fiber types were accompanied with changes of mRNA expression for growthrelated genes (Yue, 2007). In this study, the purpose was to compare the muscle fiber types located in the gastrocnemius medial head between different species, specifically the rat and chicken. The hypothesis of the study is that fast-twitch glycolytic (IIB) muscle fibers would be significantly greater in the rat than in the chicken gastrocnemius medial head. The null hypothesis is that there will be no significant difference between muscle fiber types between the rat and chicken gastrocnemius. II. Materials and Methods Animals

An adult female rat (Rattus norvegicus sprague-dawley) and an adult female white leghorn chicken (Gallus gallus domesticus) were provided for the Spring 2015 BIO 432 class by the Cal Poly Biology Department and Cal Poly Poultry Unit. The chicken was euthanized at the poultry unit before being involved in the experiment; however, the rat was euthanized by asphyxiation and acidosis using carbon dioxide in the Cal Poly informatory by Dr. Jason Blank. Both animals were caged animals. Day 1: Dissection of appropriate muscles and slide preparation Dissection of the rat’s right medial head of the gastrocnemius and the chicken’s right gastrocnemius muscle was performed and immediately placed on dry ice at its resting length for 15 minutes. After freezing, the muscles were taken out and wrapped in foil and properly labeled. The wrapped muscles were then placed back into the dry ice storage box until needed. As seen in Figure 1, a petri dish containing small pieces of dry ice was obtained to prevent muscle thawing while being sliced at cross sections using a razor and forceps for stabilization. Each muscle cross section was cut to about 1/10 of a millimeter and then placed in serial cross sections on individual Vectobond-coated slides. After being placed on slides, the muscles were allowed to dehydrate and adhere to the slide for at least thirty minutes. After dehydration, a pap-pen was used to create a hydrophobic barrier around the sections of muscles to allow the reagents to stay on the muscle sections and prevent them from dripping off the slide. The reaction solution (200mM Tris, 7mM Na+ succinate, 1.2mM nitroblue tetrazolium) was then applied to both slides using a transfer pipette to cover the cross sections. These slides were allowed to sit for 15 minutes. After staining, the slides were then rinsed with deionized water to stop the reaction. A 1:1 mixture of glycerol and water was then applied to the slides and covered with a cover-slip. Day 2: Analysis of Muscle Fibers Pictures of the slides were taken from under the Leica DM2500 microscope at low(40x) and high(100x) magnification. Microscope images were used to quantify the number of each fiber type in a given area to determine the numerical proportion of each fiber in that muscle. Using Adobe Photoshop, the cross sectional area of each fiber type was analyzed. Using JMP Pro 11, a repeated measures one-way ANOVA was conducted to determine any statistical

differences in muscle fiber type frequency and muscle fiber type cross sectional area between the rat and chicken.

Figure 1. Diagram of slicing cross-sections of a gastrocnemius muscle, creating a hydrophobic barrier with a pap-pen, and adding the reaction solution to the Vectobond-coated slides. II.

Results In Figure 1, the histochemical stained cross-sections of the gastrocnemius of the chicken

(left) and the MHG of the gastrocnemius of the rat (right) are shown at 100x magnification. The stained cross-section of the chicken looks smeared, and also multiple tissue layers can be seen. Histochemical slides appear more darkly stained in the chicken than the rat; however, the rat appears to have larger cell diameter. As seen in Figure 2, a repeated measures one way ANOVA revealed that there is a significant interaction between the muscle fiber type and the species (DF=2; F=15.3277; pvalue<0.0001). A Post-Hoc tukey test revealed that the rat has a significantly higher mean muscle fiber area than the chicken in FG and FOG muscle fibers, but no significant differences in SO muscle fiber types. The Post-Hoc Tukey test also revealed that there is a significant difference between SO fiber area of the rat compared to its FOG and FG fiber areas; however, in the chicken, there is no significant difference between its SO, FOG, and FG fiber areas. The rat had a noticeably larger cell frequency of FOG fiber types and a smaller cell frequency of SO fiber types compared to the chicken, but a slightly smaller proportion of FG fiber types compared to the chicken (Fig. 3). Qualitatively, the chicken gastrocnemius was noticeably larger and less red in color than the rat gastrocnemius.

In figure 4, the total area occupied by each fiber type in a given area of both animals are shown. The total area occupied by each fiber in the chicken seems to be only slightly greater in FOG fibers. In the rat, there is a much greater total area occupied by FOG fibers compared to its FG and SO fibers.

Figure 1. Histochemical SDH staining of cross sections from the gastrocnemius of a white Leghorn hen, Gallus gallus domesticus (left), and MHG of a Rattus norvegicus sprague-dawley (right) at 100x magnification showing A) slow oxidative fibers, B) fast oxidative-glycolytic fibers, and C) fast glycolytic fibers. Cells stained darker are indicative of greater mitochondrial count by the presence of succinic dehydrogenase.

Figure 2. Mean area differences of muscle fiber types between the gastrocnemius of a rat and chicken. There is a significant interaction between the muscle fiber type and the species (Df=2; F=15.3277; P-value<0.0001). Post-hoc tukey test revealed that the rat has a significantly higher mean muscle fiber area than the chicken in FG and FOG muscles fibers, but no significant differences in SO muscle fiber types. Significant differences between muscle fiber types are represented by letters. Error bars represent the standard error of mean.

Figure 3. Percentage of muscle fiber type in a given area of the gastrocnemius of a rat and chicken. Percentage values are shown for each fiber type. The majority of muscle fibers found in the rat tissue are fast oxidative-glycolytic and slow oxidative for the chicken.

Figure 4. Comparison between total area occupied by each fiber type in a given area of both a chicken and a rat. Total area was calculated by multiplying mean area of the specific fiber type by the number of cells. The rat occupied more area than the chicken with FOG muscle, but less area with the FG and SO muscles.


Discussion The purpose of this study was to analytically compare the difference in muscle fiber

types between the gastrocnemius of a rat and a chicken. With the rat, our experiment was inconsistent with research in that there was a larger proportion of type IIA muscles found in the medial head of the gastrocnemius rather than type IIB. Compared to previous research from Eng (2008), the medial head of the gastrocnemius of a same species rat was found to have 5.71% Type I, 9.8% Type IIA, 19.01% Type IIX, and 63.53% Type IIB, whereas our data showed

21.8% Type I, 57.1% Type IIA, and 21.1% Type IIB. There is a 38.1% difference in Type IIA fibers and a 42.4% difference between Type IIB fibers between Eng’s data and ours. This is relatively a large difference between the fiber frequency within species, and a variety of factors could account for this difference. One possible factor could be that Eng used male rats, whereas we used a female rat. In addition, the chicken results were also inconsistent with the research. Our experiment observed higher proportions of SO muscle fiber types followed by FOG and FG muscles rather than the Type IIA seen in the research. However, Chang and colleagues also looked at male chickens, whereas our experiment utilized a female chicken. Sex differences in chickens could account for differing muscle fiber types such as: male chickens observed to be more aggressive, how females stand when laying their eggs, or due to the higher levels of testosterone in males. It’s also important to note that the chicken used in our experiment was a domesticated chicken kept in a cage. The chicken was raised in a cage along with six other chickens, which limited the amount of space for the chicken to move freely. Thus, the chicken was confined to only standing or minimal walking to move around. With restrained movement, it’s plausible that the chicken’s growth from its development in a cage refrained its need to develop more FOG or FG muscle fibers. The SO muscles would allow the chicken to stand and maintain its posture within the cage for a long period of time without fatiguing. Genetic influence can also account for differences seen in the muscle fiber types between the species. Rats may be more physically built for running while chickens lead a more passive lifestyle compared to the rat. The size of muscle fibers are influenced by a number of factors including activity and innervation, growth, hormones, and nutrition (Armstrong, 1984). In Yue’s research they found that changes in mRNA expression for growth was accompanied with morphological changes in the muscle. The different behaviors and diet of the chicken and rat could have also triggered differing growth-related genes. Thus the gene expressions in the rats and chickens can alter their developmental patterns to suit their physical needs to adapt to their environments. Staining technique and tissue slice thickness could have both also accounted for our data being inconsistent with research. Since our chicken tissue slices were too thick, multiple layers of tissue were able to be seen. This caused the stained cross-section to look smeared, and also made it harder to distinguish between fiber types. For example, in our stained chicken cross-

section, bundles of what seems to be Type IIB cells can be seen throughout the slide. However, muscle fiber types are suppose to have a mosaic pattern, and bundles of a single fiber type tend not to be commonly seen. Follow up experimentation should be conducted to analyze various other muscle groups between the species. One weakness of our experiment was that we compared the MHG of a rat to the whole gastrocnemius of the chicken. Thus it was unclear whether our field of view under the microscope was the medial or lateral head. Future experimentation can look into the differences between the lateral and medial heads of the gastrocnemius between the species rather than only the medial head, and also between males and females. Other muscle groups can also be analyzed such as with the non-antigravity muscle groups such as the flexors. Furthermore, the biggest difference between the vertebrates is that birds have wings, so it would be interesting to investigate the differences between the pectoral muscles of the rat and chicken especially since chicken are non-migratory birds and do not utilize their wings continuously. Our study found that the rat had a significantly greater mean fiber area in FG and FOG compared to the chicken, but showed no significant difference in SO mean fiber area. The rat had noticeably larger cell frequency of FOG fiber types than the chicken, while the chicken had more SO fibers than the rat. Our results demonstrate that these domestic vertebrates have developed unique muscle fiber characteristics to fit their physical needs in motor movement for adaptation to the their environment.



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Research Project - Histochemical Fiber Typing