Escuela Agrícola Panamericana, Zamorano Food Science and Technology Department Food Science and Technology Major Graduation Research Project Influence of glycolic, oxidative, and mixed metabolism on growth in swine skeletal muscle Student María José Silva Hernández Advisors Adela Acosta, D.Sc. Morgan Zumbaugh, Ph.D. Honduras, November 2023 2 Authorities SERGIO ANDRÉS RODRÍGUEZ ROYO Rector ANA M. MAIER ACOSTA Vice-president and Academic Dean ADELA M. ACOSTA MARCHETTI Head of Food Science and Technology Department HUGO ZAVALA MEMBREÑO General Secretary 3 Contents List of Tables ........................................................................................................................................... 5 List of Figures .......................................................................................................................................... 6 List of Annex ............................................................................................................................................ 7 Abstract ................................................................................................................................................... 8 Resumen ................................................................................................................................................. 9 Introduction .......................................................................................................................................... 10 Materials and Methods ......................................................................................................................... 13 Experiment Description ........................................................................................................................ 13 Quick DNA Miniprep Plus Kit Protocol .................................................................................................. 15 Materials ............................................................................................................................................... 15 Reagents................................................................................................................................................ 15 Equipment ............................................................................................................................................. 15 Protocol ................................................................................................................................................. 16 TaqMan q-PCR for Mitochondrial DNA Quantification ......................................................................... 17 Materials ............................................................................................................................................... 17 Reagents................................................................................................................................................ 17 Equipment ............................................................................................................................................. 17 Protocol ................................................................................................................................................. 18 Hematoxylin & Eosin Staining ............................................................................................................... 20 Materials ............................................................................................................................................... 20 Reagents................................................................................................................................................ 21 Equipment ............................................................................................................................................. 21 4 Protocol ................................................................................................................................................. 21 Statistics Section ................................................................................................................................... 23 Results and Discussion .......................................................................................................................... 24 Mitochondrial DNA Quantification ....................................................................................................... 24 Hypertrophy and Adipose Tissue Growth ............................................................................................. 27 Analysis of the Cross-Sectional Area of Muscles .................................................................................. 28 Conclusions ........................................................................................................................................... 31 Recommendations ................................................................................................................................ 32 References ............................................................................................................................................ 33 Annexes ................................................................................................................................................. 35 5 List of Tables Table 1 Group of pigs according to age ............................................................................................... 14 Table 2 Equipment used in the DNA extraction protocol. ................................................................... 15 Table 3 Equipment used in the Mitochondrial DNA quantification protocol. ..................................... 17 Table 4 Pig primers/ Mitochondrial DNA ............................................................................................. 18 Table 5 Eukaryote primers / Nuclear DNA ........................................................................................... 18 Table 6 Samples used for standards .................................................................................................... 19 Table 7 Standards ................................................................................................................................. 19 Table 8 Cycling Parameters .................................................................................................................. 20 Table 9 Equipment used in H&E staining protocol. .............................................................................. 21 Table 10 Treatments ............................................................................................................................ 23 Table 11 Statistical Analysis PCR ........................................................................................................... 25 Table 12 Least Square Means for effect AGE ....................................................................................... 26 Table 13 Least Square Means for effect MUSCLE ................................................................................ 27 Table 14 Statistical Analysis CSA .......................................................................................................... 30 6 List of Figures Figure 1 Diagram for obtaining the Histological sample ...................................................................... 14 Figure 2 Diagram process of obtaining tissue section sample .............................................................. 21 Figure 3 Abundance of Mitochondrial DNA throughout the growth cycle .......................................... 25 Figure 4 Abundance of Mitochondrial DNA in different muscles ........................................................ 27 Figure 5 Body weight ........................................................................................................................... 28 Figure 6 Assessment of adipose tissue growth .................................................................................... 28 Figure 7 Assessment of muscle growth with different types of metabolism ...................................... 29 7 List of Annex Annex A DNA Extraction ....................................................................................................................... 35 Annex B Standars curve ........................................................................................................................ 36 Annex C Layout of the Standard 96-well plate .................................................................................... 37 Annex D Layout of the Pigs 96-well plate ............................................................................................ 38 Annex E Assess Mitochondrial DNA as an indicator of metabolic changes .......................................... 39 Annex F Least Squares Means of the analysis of the CSA of muscles ................................................... 40 8 Abstract The present work focuses on analyzing skeletal muscle metabolism in pigs, throughout different stages of growth and investigate how muscle fiber growth varies in three types of muscle with different metabolic profiles: Longissimus dorsi (glycolytic muscle), Latissimus dorsi (mixed muscle) and Masseter (oxidative muscle). The interaction between age and muscle has influence on hypertrophy muscle is also explored.The study was conducted with 25 selected castrated pig from the Kansas State University research facility. The pigs were fed according to a staggered feeding plan to meet their nutritional requirements at different stages of growth, were sampled at five slaughter time points from 20 to 180 days of age. The results revealed that there is not significant difference in Mitochondrial DNA abundance between oxidative and glycolytic muscle fibers. This contrasts with earlier research suggesting that oxidative muscle fibers tend to have a higher amount of Mitochondrial DNA. It is suggested that other factors than metabolic activity may influence this difference. In terms of the amount of Mitochondrial DNA (mtDNA), a peak was observed at 87 days of age, which could be related to the increase’s energy demands of muscle at this stage and the transition from a nursery diet to finisher diet. In addition, it was found that the interaction between age and muscle does significantly affect muscle growth. While locomotive muscle, such as Longissimus dorsi and Latissimus dorsi, showed continuous growth after 53 days, the Masseter muscle, related to chewing and located in the head, experienced significant growth after 120 days. Keywords: Fibers, hypertrophy, mtDNA, pig, nutrition. 9 Resumen El presente estudio se enfocó en el analisis del metabolismo del músculo esquelético en cerdos, a través de diferentes etapas de crecimiento e investigar cómo el crecimiento de la fibra muscular varía en tres tipos de músculo con diferentes perfiles metabólicos: Longissimus dorsi (músculo glicolico), Latissimus dorsi (músculo mixto) y Masseter (músculo oxidativo). El estudio se llevó a cabo con 25 cerdos castrados que fueron alimentados de acuerdo con un plan de alimentación para satisfacer sus necesidades nutricionales en diferentes etapas de crecimiento, fueron muestreados en cinco puntos de tiempo de sacrificio de 20 a 180 días de edad. Los resultados revelaron que no hay diferencia significativa en la abundancia de ADN mitocondrial entre las fibras musculares oxidativas y glicolicas. Contrastando con investigaciones anteriores que sugieren que las fibras musculares oxidativas tienden a tener una mayor cantidad de ADN mitocondrial. Sugiriendo que otros factores además de la actividad metabólica pueden influir en esta diferencia. En términos de la cantidad de ADN mitocondrial (ADNmt), se observó un pico a los 87 días de edad, que podría estar relacionado con el aumento en la demanda de energía del músculo en esta etapa y la transición de una dieta de inicio a una final. Además, se encontró que la interacción entre la edad y el músculo afecta significativamente el crecimiento muscular. Los músculos locomotores, como Longissimus dorsi y Latissimus dorsi, mostraron crecimiento continuo después de 53 días, el músculo Masseter, relacionado con la masticación, experimentó un crecimiento significativo después de 120 días. Palabras clave: ADNmt, cerdo, fibras, hipertrofia, nutrición. 10 Introduction According to the Organization of Economic Co-operation and development (OECD) and the Food Agriculture Organization of the United Nations (FAO), over the next ten years, global consumption of pork will rise to 127 million tons, making up 33% of the overall growth in meat. Pork consumption has increased quickly per person in Latin America as result of competitive prices that have brought pork to the international market (Organization for Economic Co-operation and Development (OECD)/ 2021). Meat demand is increasing globally because of demographic and economic growth. The swine business is currently in a production phase in which improving productive yields is critical to ensuring the sustainability of farms and the food chain. Improved swine farm performance and efficiency are connected to good health and nutritional management (Biovet 2021). The optimization of feed efficiency appears as a critical topic in swine husbandry, as it the significant consequences not only for production yields but also for farm economic viability. Given that feed expenses account for a significant portion of total production costs, ranging from 80 to 85%, producers must devise meticulously devised strategies to expedite attainment of optimal body weight within a shortened timeframe while simultaneously fostering increased feed efficiency (Campabadal 2009). The composition of tissues, particularly muscle and fat, is critical to the quality of meat products of animal origin, particularly pig. This composition is influenced by various factors including as diet, age, gender, and genetic type. Among these, nutrition is a simple instrument for modulating tissue composition and adapting it to quality criteria (Benítez et al. 2012). Skeletal muscle makes up between 30 to 65% of a pig’s body weight and 45% of its total protein, depending on the breed. Muscle fibers account for 75 to 90% of muscle volume, with the remainder made up of the adipose tissue, connective tissue, arteries and nerves (Graziotti et al. 2000). Pig breeders are increasingly interested in improving meat quality features, as it is difficult to improve meat quality using conventional selection methods. The quality of meat depends on the 11 composition of different types of muscle fiber and intramuscular fat content. Research of muscle transcriptional profiles is particularly interesting to understand the mechanisms of muscle development and find molecular methods to improve meat quality traits in pig breeding. In addition, muscle fiber are adaptable beings that can chance their phenotypic properties in response to changing functional needs. The early prenatal environment is critical for the development of the skeletal muscle. Several studies have revealed that intralitter variation in utero influences postnatal skeletal muscle growth in the pig (Dwyer et al. 1994). Muscle growth is recognized to be influenced by the number, size, and type of muscle fibers. In most mammalian species, the total number of the skeletal muscle fibers (hyperplasia) is fixed before birth. Skeletal muscle fibers only expand postnatally through hypertrophy (growth in size) (Karunaratne et al. 2005). According to previous studies, the increase in the number (hyperplasia) and size (hypertrophy) of myofibers contributes significantly to the postnatal muscle phenotype of pigs; However, the number and composition of muscle fiber vary significantly during prenatal development (Xu X et al. 2019). Due to the fact that genetic background has a significant impact on animal performance, a constructive interaction between genetics and nutrition is required to improve our understanding of what constitutes optimal responses to fiber-rich diets. A high-fat diet for pigs can cause variations in muscle fiber ratios due to Mitochondrial expansion or decrease, resulting in alterations in cellular metabolism (Vincent et al. 2015). Different breeds of pigs perform differently because proteins synthesis and Mitochondrial energy consumption of muscular tissue are not comparable. Furthermore, there is a link between the gene networks that control Mitochondrial energy metabolism and muscle growth (Pérez de Nanclares et al. 2017). Diet has a significant impact on muscle physiology and one of the best-known instances is the enzymatic activity of muscle in pigs fed a high protein diet, which results in increased glycolytic metabolism, which has ramifications for the animal’s growth. To meet the energetic requirement of 12 muscle, resources are used via biochemical reactions that allocate different nutrients to anabolic growth or catabolism. Substrates are oxidized during a condition of catabolism in which substrates are predominantly assigned to oxidative metabolic pathways, which creates carbon dioxide (CO2) and represents a loss of carbon that could be employed in anabolic reactions and muscle building (Kong and Adeola 2014). Whether nutrients are digested in muscle via glycolytic or oxidative mechanisms influences feed conversion. Understanding the cellular processes that govern food allocation to glycolytic or oxidative metabolic pathways will thus help us create novel strategies to increase swine feed efficiency (Sarri Espinosa 2018). Therefore, the objectives of this study are: Analyze the changes in skeletal muscle metabolism at various stages of the growth cycle of the pig depending on the type of muscle. Understand how muscle fibers growth differs in three skeletal muscles with different metabolisms: Longissimus dorsi (glycolytic muscle), Latissimus dorsi (mixed muscle) and Masseter (oxidative muscle). Investigate the metabolic changes that accompany skeletal muscle hypertrophy. 13 Materials and Methods Experiment Description The study used 25 castrated pigs chosen among the 1,500 pigs (300 pigs per litter of the same age) housed at Kansas State University (KSU) Swine Teaching and Research Center in Manhattan, Kansas. The pigs had Ad libitum access to feed and water, with a single automatic drinker and a trough in each pen. Staged feeding was implemented (Nursery diet 12-80 days and Finisher diet 80-280 days), to satisfy the nutrient requirements for these growing pigs to promote optimum growth. The pigs were brought to the KSU meat lab and slaughtered at 20 days (5.7 kg), 53 days (20.8kg), 87 days (42.2kg), 120 days (83.4kg) and 180 days of age (130.5kg) (Table 1). Within 15 minutes of exsanguination, three muscles samples were taken: Longissimus dorsi (LD), Latissimus dorsi (LAT) and Masseter (MS). The samples were split and stored in liquid nitrogen for further use in histology (CSA) or Mitochondrial isolation (PCR). The liquid nitrogen-frozen samples were kept at -80°C until further analysis. Histological samples were frozen in liquid nitrogen-cooled isopentane and stored at -80 °C after being immersed in plastic molds containing appropriate cutting material (Fisher Scientific, Hampton, NH) (Figure 1). Mitochondrial were isolated immediately for metabolite tracing (The protocol for this study was authorized by Kansas State University Institutional Animal Care and Use Committee). 14 Figure 1 Diagram for obtaining the Histological sample Note. Acquired from the manual Xenium In Situ for Fresh Frozen- Tissue Preparation Guide (10X Genomics, Inc. [date unknown]) Table 1 Group of pigs according to age Age (days) Age group Pig # Harvest group 20 1 P 16 4 P 17 P 18 P 19 P 20 53 2 P 11 3 P 12 P 13 P 14 P 15 87 3 P 6 2 P 7 P 8 P 9 P 10 120 4 P 1 1 P 2 P 3 P 4 P 5 180 5 P 21 5 P 22 P 23 P 24 P 25 Note. Pig #: identification given to each pig for study, P: pig and #: pig number. 15 Quick DNA Miniprep Plus Kit Protocol Materials Powder skeletal muscle samples Microcentrifuge tubes Zymo-Spin IIC-XLR column Collection tube Eon BioTek microplate Reagents Proteinase K storage buffer Proteinase K MQ water Solid tissue buffer Genomic Binding Buffer DNA Pre-wash buffer g-DNA wash buffer DNA elution buffer Equipment Table 2 presents the equipment used in the Quick DNA Miniprep Plus Kit protocol, along with their respective brads. Table 2 Equipment used in the DNA extraction protocol. Name of the equipment Brad Centrifuge 5810R Eppendorf Barnstead MaxQ 4000 Digital Orbital Incubator Shaker Thermo Fisher Scientific Mini vortex Thermo Fisher Scientific Eon plate reader BioTek 16 Protocol Incubator was preheated to 55°C and 80 rpm and 0.1g of skeletal muscle sample was weighed and placed in a microcentrifuge tube. In a bottle 1.040 µl of proteinase K storage buffer and 20 mg of proteinase k were mixed. The final concentration was 20 mg/ml, which was stored at -20°C after mixing. A solution of 190 µl of MQ water, 190 µl of solid tissue buffer (blue liquid) and 20 µl of proteinase K was added to the microcentrifuge tubes with the muscle sample, then taken individually to the vortex for 10-15 seconds and were left for an hour in conditions of 55°C to 80rpm in the incubator room. At the end of this time, each microcentrifuge tube was mixed with 800 µl of genomic binding buffer and vortex for 15 seconds. The mixture was transferred to a Zymo Spin IIC-XLR column in a collection tube, this step was repeated twice for the liquid amount, then all the mix was centrifuged at 12,000 rpm for one minute (x2), when these minutes passed the collection tube was discarded. 400 µl of DNA pre-wash buffer was added to the spin column in a new collection tube and centrifuged at 12,000 rpm for one minute and the collection tube was emptied. A 700 µl of g-DNA wash buffer was added to the spin column and centrifuged at 12,000 rpm for one minute and emptying the collection tube was emptied. 200 µl of g-DNA wash buffer was added to the spin column, centrifuged at 12,000 rpm for one minute and the collection tube with the supernatant was removed. The spin column was transferred to a clean microcentrifuge tube (1.5-1.7 mL), and a 50 µL DNA elution buffer was added directly into the matrix, it was left to rest for five minutes at room temperature and centrifuged at 14,000 rpm for one minute to elute the DNA, this step was repeated once again (Note: The eluted DNA was immediately used for molecular based applications or stored at <20 °C for future use). 17 When DNA abundance was analyzed 2µL of DNA and one blank of 2µL at ultrapure water were loaded into the Eon BioTek microplate and was read using the Eon BioTek reader. Gen5 application was opened and used the nucleic acid setting. TaqMan q-PCR for Mitochondrial DNA Quantification Materials MicroAmp™ Fast Optical 96-Well Reaction 4346906 MicroAmp™ Optical Adhesive Film 4311971 Pipettes and pipette tips with filter 1.6 mL autoclaved tubes Ice bucket with ice 10 M Primer and probe stocks DNA samples ON ICE Reagents DNase Away DEPC-treated RNase Equipment Table 3 presents the equipment used in the TaqMan q-PCR for Mitochondrial DNA Quantification protocol, along with their respective brads. Table 3 Equipment used in the Mitochondrial DNA quantification protocol. Name of the equipment Brand Centrifuge with adapter for 96-well plates No specific brand Fast Advanced Master Mix Applied Biosystems stored at -20oC freezer until 1st thaw, then at 4 °C TaqManTM 7500 Fast Real Time PCR System in rooms Thermo Fisher Scientific 18 Protocol Prepared 96-well plate layout that ran on the instrument and cleaned up the work area and pipets using DNase Away. To prepared PCR reaction mix: thawed TaqManTM Fast Advanced Master Mix on ice and blended gently while dipping the tube several times. Table 4 and Table 5 shows the total volumes of Reaction Mix’s both Mitochondrial DNA and Nuclear DNA. Table 4 Pig primers/ Mitochondrial DNA Reagent Initial (µMole/L) Final (µMole/L) Vol/Reaction (µL) # of reactions (µL) Total volume (µL) TaqMan 2X 1X 10 54 540 H2O 6.75 54 364.5 Primer F 100 1 0.5 54 27 Primer R 100 1 0.5 54 27 Probe 10 0.5 0.25 54 13.5 DNA up to 20ng 2 Table 5 Eukaryote primers / Nuclear DNA Reagent Initial (µMole/L) Final (µMole/L) Vol/Reaction (µL) # of reactions (µL) Total volume (µL) TaqMan 2X 1X 10 54 540 H2O 6 54 324 Primer F 100 1.5 0.75 54 40.5 Primer R 100 1.5 0.75 54 40.5 Probe 10 1 0.5 54 27 DNA up to 20ng 2 Note. The total number of reactions was calculated by multiplying the number of individual samples x 3 and adding 5% Added each component to a 1.6 mL tube labeled "RM mt" (reaction mix) and "RM EUK" in the order indicated above (largest volumes first, smallest volumes last). Changed the pipet tips between each component and after the addition of each component, pipet up and down a few times to rinse the tip and mix the solution. 19 The reaction solution was mixed gently inverted a few times and centrifuged briefly to bring all the liquid to the bottom of the tube. Finally, a serial dilution of 1:10 was made for 8 standard curve points. Standard 1 is pooled of all the sampler (Table 6) and the other standards are dilutions of the above (Table 7). Table 6 Samples used for standards Number of pigs Muscle 2 LD 29 LAT 5 MS 14 LAT 23 MS Table 7 Standards Standards DNA Quantity (µL) Water Quantity (µL) STD 1 10 from each sample = 100 0 STD 2 5 of STD 1 45 STD 3 5 of STD 2 45 STD 4 5 of STD 3 45 STD 5 5 of STD 4 45 STD 6 5 of STD 5 45 STD 7 5 of STD 6 45 STD 8 5 of STD 7 45 The following steps were taken for the standards: 20 μl of each standard mixture (Standard and reaction mix) and the plate was covered with MicroAmpTM Optical Adhesive Film, ensuring the edges were well covered. The plate was then briefly centrifuged before being placed in the thermocycler and ran the PCR reaction plate. The plate was placed in the thermocycler and the instrument was closed, verifying that the cycling parameters coincided with the above. Clicked "Start Run" and configured the plate under 20 "plate set-up" with the standards and samples in the assigned wells, when finished cleaned the work area using 10% ethanol, turned on the UV light and turned off the white light. For the duplicate sample: added 18 μl of the master mixture to each well (as marked on the plate layout), the pipet tips between tubes/wells were changed. All DNA samples were then briefly agitated and centrifuged before use. 2 μl of DNA was added to each well according to the tube/ plate layout and added the sample directly to the master mix at the bottom of each tube/well and pipetted up and down 2-3 times to rinse and mix, changed the pipet tips between each sample. Then the plate was centrifuged down to 1000rpm and placed on the machine running used 7500 Fast ABI; Taqman Assay. Finally, Table 8 shows the parameters used to verify the process. Table 8 Cycling Parameters Instrument Step Temperature (°C) Duration (s) Cycles 7500 Fast ABI Ampli TaqTm Fast DNA Polymerase Up activation 95 20 Hold Denature 95 3 40 Anneal/ extend 60 30 Hematoxylin & Eosin Staining Materials Tissue sections Microscope slides Slides cove Coplen jar Kimwipes Shandon Mounting media. 21 Reagents Hematoxylin 1% Deionized water Eosin 50% Ethanol (50%, 70%, 95%, 100%) MQ water Equipment Table 9 presents the equipment used in the Hematoxylin & Eosin Staining protocol, along with their respective brads. Table 9 Equipment used in H&E staining protocol. Name of the equipment Brand Cryostat microtome Thermo scientific Microscope Nikon Eclipse Ti2 Protocol Tissue sections of 5 µM were cut and thick on saline coated slides using a cryostat microtome, then stored the slides at -80 °C (Figure 2). Figure 2 Diagram process of obtaining tissue section sample Note. Acquired from the manual Xenium In Situ for Fresh Frozen- Tissue Preparation Guide (10X Genomics, Inc. [date unknown]) 22 The cuts were removed from the freezer and allowed to thaw/dry for 15 minutes. After 15 minutes, the slide was placed into the 1% prepared hematoxylin for three minutes, then placed in a coplen jar with running deionized water for five minutes. After this time submerged in prepared eosin (diluted 1:50) for one second (one dip). Then rinsed with deionized water for one minute. The slides were immersed in 50% ethanol for 10 seconds, followed by 70% ethanol for 10 seconds, 95% ethanol for 30 seconds and finally 100% ethanol for 30 seconds. The slide was quickly dipped into xylene seven times, then dried with a Kimwipe, making sure not to dry the area containing the section, as this would have cleaned the fabric. A few drops of mounting media were added to cover the entire whole slide and the slide cover was gently placed over the tissue section. The slide cover was left to dry and was ready for imaging after approximately 30 minutes. To take pictures of the slides with the sample the following steps were followed: The microscope cover sleeve was removed, and the different engines of the equipment were started in ascending order. The NIS-Elements AR 5.30.02 application was opened and the Nikon Ti2 button was clicked. Then the slide was placed face down and the live view button was pressed, ensuring that Fi3-DIA functions were activated and setting the target to 10x. The image was focused, and the capture was taken, making sure to save it in ND2 format. When the imaging was finished, the different engines of the microscope were switched off in descending order. The following steps were followed for image analysis: The computer was turned on, the NIS-Elements AR 5.30.02 application was introduced, and the passive mode button was clicked. Opened the folder of the images made in the previous steps and accessed the toolbar, selecting the tool to draw perimeters. 23 Zoomed in, if necessary, to find a piece of the image that could be clearly seen, and the edge of the fibers was drawn using the tool mentioned above (20 fibers per image). At the end the data was exported to an Excel sheet and analyzed the averages of the perimeters per image taken. Statistics Section The experiment followed a Completely Randomized Design (CRD) with effects on the variables muscle, age, and their interaction, separated into five treatments with five repeats (Table 10), yielding a total of 25 experimental units. When Pr ≤ 0.05, data were judged significant, and peer comparisons were separated using Tukey’s HSD. The data is show as Least Squares Means (LSMeans) ± Standard Error (SE). R Studio (version 4.2.1) was used to analyze all data. Table 10 Treatments Treatments Description Age (days) Types of muscles TRT 1 20 MS LAT LD TRT 2 53 MS LAT LD TRT 3 87 MS LAT LD TRT 4 120 MS LAT LD TRT 5 180 MS LAT LD Note. TRT: Treatments, MS (Masseter), LAT (Latissimus dorsi) and LD (Longissimus dorsi). 24 Results and Discussion Mitochondrial DNA Quantification The content of Mitochondrial DNA (mtDNA) in Longissimus dorsi (LD), Latissimus dorsi (LAT) and Masseter (MS) muscles with glycolytic, mixed, and oxidative metabolism, respectively, studied during five stages over a period of 180 days, was determined by selecting differences sequences and then performing a qPCR analysis. Table 11 shows the probabilities of muscle, age, and their interaction. The variance analysis (ANOVA) revealed that there were no significant differences between type of muscle (Pr= 0.936), while there were significant differences between ages (Pr=9.04e-05) allowing us to indicate that there is a difference in the quantity of mtDNA. The interaction between muscle types and age factor was not statistically significant (Pr= 0.298), indicating that the relationship between age and mtDNA abundance does not vary according to muscle type. Figure 3 provides a visual demonstration of the significance of the age facto. In this figure, a peak of growth in mtDNA abundance is observed at 87th day for all age groups. Table 12 shows the separation of means from the age factor. This table shows the average, minimum and maximum value of mtDNA abundance for each age group. The separation of means is important because it indicates that age 3 (87 days) is statistically different from other ages. The higher mtDNA content observe on 87th day can be associated with two reasons: Greater energy needs required by the muscle at this age, this assumed by the analysis performed by Pesce et al. (2001) which indicates: greater muscle development will have greater energy demand. We can compare this with the contractile activity of the heart muscle and the high energy demand required for this process, which generates a higher demand of mtDNA (Xie YM. et al. 2015). On the other hand, the transition from nursery diet (12-80 days) to a finisher diet (80-280 days) can generate a higher energy demand, because when transitioning to a diet with the objective of creating greater muscle growth. According to Whittemore (1993), and Fang LHu et al. (2019) the pig presents changes in 25 development, blood profiles and other parameter, thus causing the pig to enter into stress and sped more energy as a survival method to gain more weight and develop muscle. Table 11 Statistical Analysis PCR Df Sum Sq Mean Sq F value Pr(>F) Muscle 2 0.10 0.052 0.066 0.936 Age 4 22.61 5.653 7.139 9.04e-05 Muscle: Age 8 7.79 0.974 1.230 0.298 Residuals 60 47.51 0.792 Note. Df: degree freedom, Sum Sq: Sum of Squares, Mean Sq: Mean of Square, Pr (>F): probability Figure 3 Abundance of Mitochondrial DNA throughout the growth cycle Note.20 days: max 1.59, min:0.67; 53 days: max 1.98, min 1.07; 87 days: max 3.23, min 2.31; 120 days: max 2.32, min 1.40; 180 days max 2.07 min 1.15. b 1.13 b 1.52 a 2.77 ab 1.86 b 1.61 0 0.5 1 1.5 2 2.5 3 3.5 20 53 87 120 180 m tD N A C o p y N u m b e r Age (days) 26 Table 12 Least Square Means for effect AGE Age emmean SE Df Lower.CL Upper.CL Group 1 1.13 0.23 60 0.67 1.59 b 2 1.52 0.23 60 1.06 1.98 b 5 1.61 0.23 60 1.15 2.07 b 4 1.86 0.23 60 1.40 2.32 ab 3 2.77 0.23 60 2.31 3.23 a Note. SE: Standard Error, Df: degree freedom, Lower.CL: lower value, Upper CL: higher value. Figure 4 shows that, since the probability of mtDNA abundance in muscle was not statistically significant (Pr=0.936) (Table 11), all the bards representing the three types of muscles studied are at the same level. This can be sustained with the media separation shown on Table 13. These results differ with date obtained by Bakala et al. (2013), who reported that oxidative muscle fibers tend to have higher mtDNA copy number than glycolytic muscle fiber. It is also possible that there are other factors than metabolic activity influence mtDNA abundance un muscle cells. The date are also inconsistent with the analysis produced by: Hoeks and Schrauwen (2012) the abundance of mtDNA is an indicator of the metabolic activity if Mitochondria, which are cellular organelles responsible for energy production. Therefore, oxidative muscle fibers, which are primarily responsible for aerobic energy production, were expected to have higher mtDNA copy number than glycolytic muscle fibers, which are primarily responsible for anaerobic energy production. However, the results of our study did not support this hypothesis. It is possible that the differences in mtDNA abundance between oxidative and glycolytic muscle fibers are less marked that previously thought. 27 Figure 4 Abundance of Mitochondrial DNA in different muscles Note: Longisssimus dorsi (LD), Latissimius dorsi (LAT) and Masseter (MS). Table 13 Least Square Means for effect MUSCLE Muscle emmean SE Df Lower.CL Upper.CL Group LD 1.74 0.178 60 1.38 2.09 1 LAT 1.77 0.178 60 1.41 2.13 1 MS 1.83 0.178 60 1.47 2.18 1 Note. SE: Standard Error, Df: degree freedom, Lower.CL: lower value, Upper CL: higher value. Hypertrophy and Adipose Tissue Growth Assessment of muscle hypertrophy and adipose tissue in pigs was determined by measuring muscle Cross- Sectional Area (CSA) and Adipose Tissue Size (Backfat Thickness), respectively. Body weight increased gradually between the ages of 20 to 87 days, subsequently a rapid increase in body weight was observed between the ages of 87 to 180 days (Figure 5). While intensive growth of adipose tissue occurred between the ages of 120 to 180 days (Figure 6). According to the study conducted by Garrido de la Osa (2022) can indicate that the increase in body weight is due to an increase in muscle and adipose tissue. Intensive growth of adipose tissue occurs during the finishing period (finisher diet), when pigs are fed high-energy and low- fiber diets. 0.0 0.5 1.0 1.5 2.0 2.5 LD LAT MS m tD N A C o p y N u m b e r Muscle 28 Figure 5 Body weight Figure 6 Assessment of adipose tissue growth Analysis of the Cross-Sectional Area of Muscles Table 14 shows the probabilities of different factors, such as muscle, age, and interaction of these, to know their relationship with the cross section are. In this case, muscle and age values are e 5.7 d 20.8 c 42.2 b 83.23 a 130.27 0 20 40 60 80 100 120 140 0 20 40 60 80 100 120 140 160 180 200 B o d y w e ig h t (K g ) Age (Days) 29 statistically significant, indicating that the perimeter of muscle fibers varies depending on the muscle and the age in which it is found. In addition, an interaction between muscle and age was show. Figure 7 demonstrates that after 53 days that Longissimus dorsi (LD) and Latissimus dorsi (LAT) muscles grew at each point. Nevertheless, after 120 days Masseter (MS) muscle grew. Notably, between 87 and 120 days, MS muscle size seemed to be stuck. According to Ramírez (2004) Two reasons can be given this observation: The oxidative muscles (MS) have smaller fibers than glycolytic muscle (LD) and because the LD and LAT are locomotor muscles, whereas the MS muscle is a masticatory muscle. The locomotor muscle is subjected to a greater mechanical loas than the masticatory muscle, which may explain their greater growth and the location of the MS muscle is in the head may also contribute to its slower growth, is surrounded by other muscle and tissues, limit its ability to expand. Figure 7 Assessment of muscle growth with different types of metabolism Note. Longisssimus dorsi (LD), Latissimius dorsi (LAT) and Masseter (MS). 30 Table 14 Statistical Analysis CSA Df Sum Sq Mean Sq F value Pr (>F) Muscle 2 9551888 4775944 13.308 2.32e-05 Age 4 149459462 37364865 104.116 < 2e-16 Muscle:Age 8 22628365 2828546 104.116 8.65e-07 Residuals 50 17943905 358878 Note. Df: degree freedom, Sum Sq: Sum of Squares, Mean Sq: Mean of Square, Pr (>F): probability 31 Conclusions The results of the study show that changes in skeletal muscle metabolism in the pig depend on the type of muscle and age. In general, glycolytic, and mixed muscles showed greater metabolic activity than oxidative ones. The three types of muscle studied (Longissimus dorsi, Latissimus dorsi and Masseter) have distinct growth patterns. Longissimus dorsi, a glycolytic muscle, showed steady growth over time. Latissimus dorsi, a mixed muscle, showed accelerated growth after 53 days. Masseter, an oxidative muscle, showed slower growth after 53 days. There is a relationship between muscle hypertrophy, age, and muscle type. Depending on whether the muscle is glycolic, oxidative, or mixed will be the change in growth at different stages. 32 Recommendations Consider other factors that may influence the abundance of mtDNA in muscle cells, such as diet, exercise, or stress, may also influence this abundance. Evaluate how each of the nutrients incorporated in different diets throughout the life cycle affects muscle growth. 33 References 10X Genomics, Inc. Xenium in Situ for Fresh Frozen Tissues Tissue Preparation Guide; [accessed 2023 Sep 15]. https://cdn.10xgenomics.com/image/upload/v1671645454/support- documents/CG000579_Demonstrated_Protocol_XeniumInSituProtocolsFF_ TissuePreparationGuide.pdf. Benítez R, Núñez Y, Fernández A, Alves E, Martín Palomino P, Rodrigáñez J, Fernández A.I, Rodríguez C, López-Bote C, Silió L, et al. 2012. Influencia de la composición de la dieta sobre el perfil de ácidos grasos y la expresión en tejidos adiposo, muscular y hepático de cerdos ibéricos. Revista Complutense de Ciencias Veterinarias. 6 (1). https://core.ac.uk/download/pdf/38810084.pdf. Biovet. 2021. Mejora de la eficiencia en la producción porcina con pronutrientes acondicionadores intestinales. Tarragona: Biovet S.A; [updated 2/15/2021; accessed 2023 Aug 8]. https://biovet-alquermes.com/2021/02/15/mejora-de-la-eficiencia-en-la-produccion-porcina/. Campabadal C. 2009. Guía Técnica para Alimentación de Cerdos. Costa Rica: [publisher unknown]. 44 p. https://images.engormix.com/externalFiles/6_campabadal-guia-nutricion- cerdos.PDF. Dwyer CM, Stickland NC, Fletcher JM. 1994. The influence of maternal nutrition on muscle fiber number development in the porcine fetus and on subsequent postnatal growth. Journal of animal science. 72(4):911–917. eng. https://pubmed.ncbi.nlm.nih.gov/8014156/. doi:10.2527/1994.724911x. Fang LH, Jin YH, Do SH, Hong JS, Kim BO, Han TH, Kim YY. 2019. Effects of dietary energy and crude protein levels on growth performance, blood profiles, and carcass traits in growing-finishing pigs. Journal of Animal Science and Technology. 61(4):204–215. eng. https://www.ncbi.nlm.nih.gov/ pmc/articles/PMC6686147/. doi:10.5187/jast.2019.61.4.204. Garrido de la Osa, Nicolás. 2022. Estudio del crecimiento del tejido graso y muscular y de la expresión de genes relacionados con el metabolismo lipídico en tres estirpes de cerdo Ibérico finalizados en dos sistemas de alimentación; [accessed 2022]. https://dehesa.unex.es/bitstream/ 10662/15539/1/TDUEX_2022_Garrido_de%20la%20Osa.pdf. Graziotti G, Ríos C, Basso L. 2000. Las fibras musculares esqueléticas y la producción de carne en el cerdo. Argentina: Universidad de Buenos Aires; [accessed 2023 Aug 8]. 10 p. https:// www.produccion-animal.com.ar/produccion_porcina/00-carne_porcina/35-fibras_musculares.pdf. Hoeks J, Schrauwen P. 2012. Muscle mitochondria and insulin resistance: a human perspective. Trends in endocrinology and metabolism: TEM. 23(9):444–450. eng. https:// pubmed.ncbi.nlm.nih.gov/22726362/. doi:10.1016/j.tem.2012.05.007. Karunaratne JF, Ashton CJ, Stickland NC. 2005. Fetal programming of fat and collagen in porcine skeletal muscles. J Anat. 207(6):763–768. eng. doi:10.1111/j.1469-7580.2005.00494.x. Kong C, Adeola O. 2014. Evaluation of amino Acid and energy utilization in feedstuff for Swine and poultry diets. Asian-Australas J Anim Sci. 27(7):917–925. eng. doi:10.5713/ajas.2014.r.02. Organization for Economic Co-operation and Development (OECD)/. 2021. OECD-FAO Agricultural Outlook 2021-2030. Paris: [publisher unknown]. 337 p. https://www.fao.org/3/ cb5332en/cb5332en.pdf. 34 Pérez de Nanclares M, Trudeau MP, Hansen J, Mydland LT, Urriola PE, Shurson GC, Piercey Åkesson C, Kjos NP, Arntzen M, Øverland M. 2017. High-fiber rapeseed co-product diet for Norwegian Landrace pigs: Effect on digestibility. Livestock Science. 203:1–9. https://www.sciencedirect.com/ science/article/pii/S1871141317301907. doi:10.1016/j.livsci.2017.06.008. Pesce V, Cormio A, Fracasso F, Vecchiet J, Felzani G, Lezza AM, Cantatore P, Gadaleta MN. 2001. Age-related mitochondrial genotypic and phenotypic alterations in human skeletal muscle. Free radical biology & medicine. 30(11):1223–1233. eng. https://pubmed.ncbi.nlm.nih.gov/11368920/. doi:10.1016/s0891-5849(01)00517-2. Ramírez J. 2004. Características bioquímicas del músculo, calidad de la carne y de la grasa de conejos seleccionados por velocidad de crecimiento. https://www.tdx.cat/bitstream/handle/10803/ 5655/jart1de1.pdf. Sarri Espinosa L. 2018. Metabolismo proteico y tasas de renovación tisular en cerdos en crecimiento. Lleida: Universitat de Lleida. 41 p. https://repositori.udl.cat/server/api/core/bitstreams/ c30b0c94-2e0d-46c5-b0f4-089746aa8569/content. Vincent A, Louveau I, Gondret F, Tréfeu C, Gilbert H, Lefaucheur L. 2015. Divergent selection for residual feed intake affects the transcriptomic and proteomic profiles of pig skeletal muscle. Journal of animal science. 93(6):2745–2758. eng. doi:10.2527/jas.2015-8928. Whittemore C. 1993. The science and practice of pig production. The science and practice of pig production. English. Xie YM, Jin L, Chen XJ, He MN, Wang Y, Liu R, Li MZ, Li XW. 2015. Quantitative changes in mitochondrial DNA copy number in various tissues of pigs during growth. Genet Mol Res; [accessed 2023 Sep 16]. 14(1):1662–1670. eng. https://www.funpecrp.com.br/gmr/year2015/vol14-1/pdf/ gmr4495.pdf. doi:10.4238/2015.March.6.12. Xu X, Mishra B, Qin N, Sun X, Zhang S, Yang J, Xu R. 2019. Differential Transcriptome Analysis of Early Postnatal Developing Longissimus Dorsi Muscle from Two Pig Breeds Characterized in Divergent Myofiber Traits and Fatness. Animal Biotechnology. 30(1):63–74. eng. doi:10.1080/10495398.2018.1437045. 35 Annexes Annex A DNA Extraction Using the Quick-DNA Miniprep Plus Kit (Zymo Research 11-397B) following the manufacturer’s protocol for tissue with an overnight digestion period of 25-50mg of ground tissue at 56 oC. We elute with 50 μl of DNA elution buffer and then pass through the eluate again. Nanodrop and Dilute samples 1:1 as needed to get concentrations in a relatively close range to one another. They don’t have to be exactly the same concentration but +/- 20 g/l is OK. 36 Annex B Standars curve 1. Eight points are used in the standard curve 2. Two replicates are used for each point. Replicates are identical reactions, containing identical reaction components and volumes. 3. The starting quantity is dependent on the total ng/ μl of the pooled samples to make Standard 1 and the serial factor is 1:10). Vortex between each standard This makes 4 sets of standards: Standard DNA quantity Water Quantity (Ultra pure RNase-free) STANDARD 1 Pool 10 μl from 10 samples = 100 μl total (briefly vortex then nanodrop for quantity in ng/μl) 0 μl STANDARD 2 5 μl from standard 1 45 μl STANDARD 3 5 μl from standard 2 45 μl STANDARD 4 5 μl from standard 3 45 μl STANDARD 5 5 μl from standard 4 45 μl STANDARD 6 5 μl from standard 5 45 μl STANDARD 7 5 μl from standard 6 45 μl STANDARD 8 5 μl from standard 7 45 μl 4. Be sure to have a standard curve for each assay (or gene) to extrapolate copies/ng for samples. 5. Combine the Reaction Mix with each individual standard (36 μl MM + 4 μl standard) in separate tubes (8-tube PCR strip) and then add 20 μl of this mixture to each assigned well of the 96- well plate. 6. Each gene (mitochondrial and eukaryote) needs its own reaction mix and its own set of standards. 37 Annex C Layout of the Standard 96-well plate 96-well Plate 1 layout Note. Positive control: Isolated Mitochondria DNA, Negative control: MQ water 1 2 3 4 5 6 7 8 9 10 11 12 A Std 1 Std 1 Std 1 P1 LD P1 LD P1 LD Std 1 Std 1 Std 1 P1 LD P1 LD P1 LD B Std 2 Std 2 Std 2 P1 MS P1 MS P1 MS Std 2 Std 2 Std 2 P1 MS P1 MS P1 MS C Std 3 Std 3 Std 3 P1 LAT P1 LAT P1 LAT Std 3 Std 3 Std 3 P1 LAT P1 LAT P1 LAT D Std 4 Std 4 Std 4 P2 LD P2 LD P2 LD Std 4 Std 4 Std 4 P2 LD P2 LD P2 LD E Std 5 Std 5 Std 5 P2 MS P2 MS P2 MS Std 5 Std 5 Std 5 P2 MS P2 MS P2 MS F P2 LAT P2 LAT P2 LAT P2 LAT P2 LAT P2 LAT G Pos Control Pos Control Pos Control Pos Control Pos Control Pos Control H Neg Control Neg Control Neg Control Neg Control Neg Control Neg Control Nuclear DNA/ EUK mtDNA/ PIG 38 Annex D Layout of the Pigs 96-well plate 96-well Plate 2 layout Note. Positive control: Isolated Mitochondria DNA, Negative control: MQ water 96-well Plate 3 layout Note. Positive control: Isolated Mitochondria DNA, Negative control: MQ water 96-well Plate 4 layout Note. Positive control: Isolated Mitochondria DNA, Negative control: MQ water 1 2 3 4 5 6 7 8 9 10 11 12 A P3 LD P3 LD P3 LD P5 LD P5 LD P5 LD P3 LD P3 LD P3 LD P5 LD P5 LD P5 LD B P3 MS P3 MS P3 MS P5 MS P5 MS P5 MS P3 MS P3 MS P3 MS P5 MS P5 MS P5 MS C P3 LAT P3 LAT P3 LAT P5 LAT P5 LAT P5 LAT P3 LAT P3 LAT P3 LAT P5 LAT P5 LAT P5 LAT D P4 LD P4 LD P4 LD P6 LD P6 LD P6 LD P4 LD P4 LD P4 LD P6 LD P6 LD P6 LD E P4 MS P4 MS P4 MS P6 MS P6 MS P6 MS P4 MS P4 MS P4 MS P6 MS P6 MS P6 MS F P4 LAT P4 LAT P4 LAT P6 LAT P6 LAT P6 LAT P4 LAT P4 LAT P4 LAT P6 LAT P6 LAT P6 LAT G Pos Control Pos Control Pos Control Pos Control Pos Control Pos Control H Neg Control Neg Control Neg Control Neg Control Neg Control Neg Control Nuclear DNA/ EUK mtDNA/ PIG 1 2 3 4 5 6 7 8 9 10 11 12 A P7 LD P7 LD P7 LD P9 LD P9 LD P9 LD P7 LD P7 LD P7 LD P9 LD P9 LD P9 LD B P7 MS P7 MS P7 MS P9 MS P9 MS P9 MS P7 MS P7 MS P7 MS P9 MS P9 MS P9 MS C P7 LAT P7 LAT P7 LAT P9 LAT P9 LAT P9 LAT P7 LAT P7 LAT P7 LAT P9 LAT P9 LAT P9 LAT D P8 LD P8 LD P8 LD P10 LD P10 LD P10 LD P8 LD P8 LD P8 LD P10 LD P10 LD P10 LD E P8 MS P8 MS P8 MS P10 MS P10 MS P10 MS P8 MS P8 MS P8 MS P10 MS P10 MS P10 MS F P8 LAT P8 LAT P8 LAT P10 LAT P10 LAT P10 LAT P8 LAT P8 LAT P8 LAT P10 LAT P10 LAT P10 LAT G Pos Control Pos Control Pos Control Pos Control Pos Control Pos Control H Neg Control Neg Control Neg Control Neg Control Neg Control Neg Control Nuclear DNA/ EUK mtDNA/ PIG 1 2 3 4 5 6 7 8 9 10 11 12 A P11 LD P11 LD P11 LD P13 LD P13 LD P13 LD P11 LD P11 LD P11 LD P13 LD P13 LD P13 LD B P11 MS P11 MS P11 MS P13 MS P13 MS P13 MS P11 MS P11 MS P11 MS P13 MS P13 MS P13 MS C P11 LAT P11 LAT P11 LAT P13 LAT P13 LAT P13 LAT P11 LAT P11 LAT P11 LAT P13 LAT P13 LAT P13 LAT D P12 LD P12 LD P12 LD P14 LD P14 LD P14 LD P12 LD P12 LD P12 LD P14 LD P14 LD P14 LD E P12 MS P12 MS P12 MS P14 MS P14 MS P14 MS P12 MS P12 MS P12 MS P14 MS P14 MS P14 MS F P12 LAT P12 LAT P12 LAT P14 LAT P14 LAT P14 LAT P12 LAT P12 LAT P12 LAT P14 LAT P14 LAT P14 LAT G Pos Control Pos Control Pos Control Pos Control Pos Control Pos Control H Neg Control Neg Control Neg Control Neg Control Neg Control Neg Control Nuclear DNA/ EUK mtDNA/ PIG 39 Annex E Assess Mitochondrial DNA as an indicator of metabolic changes 40 Annex F Least Squares Means of the analysis of the CSA of muscles Least Squares Means for effect MUSCLE: AGE Muscle Age emmean SE Df Lower.CL Upper.CL Group MS 1 401 300 50 -201 1003 1 LAT 1 640 268 50 102 1178 1 LD 1 649 268 50 111 1187 1 LAT 2 870 268 50 332 1408 1 LD 2 1237 268 50 699 1776 1 MS 2 1329 268 50 791 1867 1 MS 4 1815 346 50 1120 2510 123 LAT 3 1850 300 50 1248 2452 12 MS 3 1892 300 50 1290 2493 12 MS 5 3054 268 50 2516 3592 234 LD 3 3199 268 50 2661 3737 234 LAT 4 3336 268 50 2798 3874 34 LD 4 3901 346 50 3206 4595 4 LAT 5 5801 300 50 5200 6403 5 LD 5 6306 346 50 5611 7000 5 Note: Ages: 1 (20 days), 2 (53 days), 3 (87 days), 4 (120 days) and 5 (180 days). Muscles: Masseter (MS), Latissimius dorsi (LAT) ans Longisssimus dorsi (LD). Least Squares Means for effect AGE Age emmean SE Df Lowe.CL Upper. CL Group 1 563 161 50 240 887 1 2 1146 155 50 835 1456 1 3 2314 167 50 1978 2649 2 4 3017 186 50 2644 3391 2 5 5054 177 50 4699 5409 3 Note: Ages 1 (20 days), 2 (53 days), 3 (87 days), 4 (120 days) and 5 (180 days). 41 Least Squares Means for effect MUSCLE Muscle emmean SE Df Lowe.CL Upper. CL Group MS 1698 133 50 1431 1965 1 LAT 2500 126 50 2247 27523329 2 LD 3058 135 50 2788 2649 3 Note: Masseter (MS), Latissimius dorsi (LAT) ans Longisssimus dorsi (LD).