Department of Biology, Baldwin Wallace University, Berea, OH How do Pork Loin and Free Range Lamb Muscle Fibers differ in mitochondrial activity? Amily Farraj, Brianna Colosi and Chynna Smith Department of Biology, Baldwin Wallace University, Berea, OH Introduction The overall purpose of this study was to learn about the different stages of cellular respiration individually. Since the three stages: glycolysis, the Krebs cycle and the electron transport chain are all catalyzed by different enzymes with different functions, we need to examine a tissue whose cells are specifically for one of the stages. One of the very few tissues in the human body with this kind of specialization is found in skeletal muscles, specifically the nicatinamide adenine dinucleotide dehydrogenase (NADH-D) enzyme activity in the electron transport chain (ETC). During the ETC, hydrogen protons are pumped from the matrix of the mitochondria into the cristae region. Eventually, the hydrogen protons are brought back into the matrix via a protein called ATP synthase which converts ADP into ATP. ATP is essential in muscles because of the contractions and constant movements they are making. Oxidative phosphorylation is the production of ATP using energy derived from the redox reactions of an electron transport chain (Reece, pg. 168). When cells use oxidative phosphorylation 28-32 ATPs are produced as a result and when cells use glycolysis around 2 ATP are produced. . To test which stage was more productive in producing ATP we looked at the differences between meat cut from pork loin and meat cut from free ranged lamb. During this experiment, NADH solution was added to a slide with the muscle fibers on it. The solution was removed but stained the fibers to make visible the enzymatic activity. Therefore, we were able to microscopically view the fibers which contained enzymatic activity. We hypothesized that the free range lamb muscles would have a consistent production of ATP because they are used to roaming freely verses the pork loin muscle which are limited in how often they are able to roam freely. This means that the pork loin would use more ATP in shorter intervals. Studying these enzymes gave us a clear understanding of their function in the electron transport chain and helped us understand how ATP was produced. Results   Figure 2 Figure 2 is a representation of the percent of pork loin muscle fibers found for each color after staining with the NADH solution. There is a greater percent of dark blue muscle fibers which carry more mitochondria. Conclusion In conclusion of our experiment on muscle fiber in pork loin and free range lamb mitochondrial activity we found variation. Pork loin muscle fibers were higher in mitochondrial activity while free range lamb had a steady average activity. This study was important because it compares to the maximum oxygen consumption in athletes and people who may not exercise regularly. Much like an athlete’s muscles free range lamb muscles do not contract as quickly and as frequently as those of the pork loin. Their environment allows the free range lamb to adapt to constant movement and exercise unlike the pork loin who may be confined to small areas. Pork loin muscles may only be active when they are let out of confinement for a certain amount of time. This leads us to believe that the pork loin muscles were found to have more mitochondrial activity in dark blue fibers because of their need to suddenly produce ATP. The ATP in pork loin is store in the mitochondria longer because it is not used regularly like the free range lamb. Our hypothesis was proven correct with the findings of this experiment. Figure 5 The average area of each of the colors of muscle fibers found in free range lamb is compared in Figure 5. These fibers were observed under a microscope at 40X with a field of view of 0.45mm. Although the white area averaged to approximately 3.7um for the free range lamb there was a larger percentage of white fibers present in comparison to the pork loin white muscle fibers. Materials and Methods To begin the experiment on muscle activity a previously prepared slide of muscle sections is obtained which was either pork loin or free range lamb. One drop of NADH solution is placed on the slide and set aside for 15-30 minutes. After the time has passed the slide is cleaned with distilled water four times. A drop of 50% glycerol was placed on top of the muscle fiber section and a cover slip was added to the slide. The slide was then sealed with nail polish and set aside to dry for 5 minutes. The slide was then viewed under a microscope at 40X magnification. A total of 60 fibers were counted based on their color and recorded. The three color categories were dark blue, light blue, and white. The field of view was then measured using a millimeters with a ruler. The field of view for pork loin muscle fibers was measured at 0.5mm, the field of view for free range lamb was measured at 0.45mm. At 40X a muscle fiber was selected and measured for its diameter. As shown in Figure 1. This was repeated ten times for each color of muscle fibers. The area was then calculated based on the diameters measure for each of the muscle fiber colors. The data collected was then analyzed and interpreted. Figure 1: View of muscle fiber at 40X used to measure diameter of pork loin muscles. Figure 3 Figure 3 is the average area of pork loin muscle fibers viewed under a microscope at 40X power with a field of view at 0.5mm. Each color was tested ten times and the average area was calculated based on those values. The dark blue muscle fibers had a smaller area average than those of the white muscle fibers, therefore meaning that the pork loin mitochondria are not consistent in using ATP. Figure 6 In Figure 6 there is a comparison between the dark verses light muscle fibers in pork loin and free range lamb. The ratio of area was greater for the pork loin than free range lamb. As mentioned in Figure 3 the free range lamb expends a constant rate of ATP which would explain why they don’t have as much dark muscle fibers which carry more mitochondrial activity. On the other hand pork loin had more dark muscle fibers which could conclude that they do not have a steady production of ATP needed because the animal is confined when active. When the animal’s muscles are used the ATP production would greatly increase because their muscles are not used to that movement. Figure 4 Figure 4 shows the present of free range lamb muscle fibers counted for each color after staining. More than half of the muscle fibers counted were light blue. This indicates that the free ranged lamb used their ATP at a constant rate, in comparison to Figure 3 where the pork loin muscle fibers were inconsistent in expending ATP. Acknowledgments We would like to thank the authors of Campbell Biology 9th edition for the useful information they provided. We would also like to thank Dr. Morris for the excellent instruction and for providing all the materials needed for this experiment and our T.A Nick for helping us with all our questions. We are most thankful for Baldwin Wallace University and their beautiful laboratory facility and equipment used to make this all a possibility. Literature cited For further information Please contact: afarraj12@mail.bw.edu, bcolosi09@mail.bw.edu, and csmith12@mail.bw.edu for further information on this experiment. Or you can visit our website at http://musclefiberproject.weebly.com . Reece, Jane B., et al. Campbell Biology Ninth Edition. Boston: Benjamin Cummings, 2011.Lab Handout: Chapter 1: Cellular Respiration