This research was funded by FAPESP (grants 2023/05002-3; 2023/02662-2 and 2024/09871-9), CAPES (Finance code 001), CNPq (304158/2022-4), and by PROPE (IEPe-RC grant number 149) of School of Veterinary Medicine and Animal Science, S&#227;o Paulo State University.

All procedures with animals complied with the ethical research standards established by the Animal Use Ethics Committee (CEUA) of the &#34;Universidade Estadual Paulista J&#250;lio de Mesquita Filho&#34; - UNESP Botucatu Campus, under protocol 0171/2018.
1. Experimental animals
<ol>
	<li>Finish 30 Nellore bulls (Bos indicus) and 30 F1 Angus-Nellore bulls (Bos taurus &#215; Bos indicus), aged 20-24 months, in a feedlot. House both groups of animals in collective pens measuring 5 m x 6 m with a concrete floor and equipped with shell-type water troughs, accommodating up to five animals per pen. Ensure that all animals belong to the same management group (born and raised on the same farm) and are submitted to the same feedlot period. 
	NOTE: In this study, the Nellore bulls had an average initial body weight of 370.7 kg while the F1 Angus-Nellore bulls had an average initial body weight of 380.8 &#177; 17 kg.</li>
	<li>Feedlot diet
	<ol>
		<li>Ensure that the finishing diet is comprised of 11.3% roughage (Tifton hay and sugarcane bagasse) and 88.7% concentrates (ground dry corn grain, soybean meal, corn wet distillers&#39; grains, dry corn gluten feed, and mineral core). Feed the animals for 120 days and provide the diets ad libitum twice a day (at 10:00 a.m. and 04:00 p.m.).</li>
	</ol>
	</li>
	<li>Slaughter
	<ol>
		<li>Register the final body weight (BWf) at the end of the experimental period. Process all animals at a nearby slaughterhouse, adhering to standard inspection procedures. Prior to slaughter, ensure that the animals undergo a minimum 16 h fast, abstaining from both feed and water. 
		NOTE: F1 Angus-Nellore bulls exhibited a final body weight of 615.09 &#177; 57.53 kg, while Nellore bulls had a weight of 545.47 &#177; 11.45 kg.</li>
	</ol>
	</li>
	<li>Evaluation of carcass traits
	<ol>
		<li>Weigh the beef carcasses initially and then subject them to a cooling period at 2-4 &#176;C for 48 h. Measurements include hot carcass weight (HCW), rib eye area (REA), and backfat thickness (BFT) at the 12th/13th rib interface, as recommended13. Determine the REA using the grid method with a small grid (18 cm x 13 cm) and measure the BFT in millimeters using a caliper.
		<ol>
			<li>Measure the REA in each carcass by using a reticulated grid (the same as used in the USDA Yield Grade classification system), divided into 1 cm&#178; squares with one dot in the middle. Add all squares within the ribeye tracing perimeter and those along the contour of the tracing passing through the middle dot.</li>
			<li>Measure the BFT at a specific position on the assessment site anywhere between the 12th/13th ribs. To determine this position, measure the length of the rib eye; then, starting at the medial border &#34;A&#34;, determine a point three quarters of the way along the rib eye and halfway across &#34;B&#34;. Take a caliper through this point and at right angles to the specified rib to the interface between the subcutaneous fat and intermuscular fat. Measure the subcutaneous fat by placing the caliper at a right angle to the line of the subcutaneous fat, from the interface point (Supplemental Figure S1).</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Sampling
	<ol>
		<li>Sample Longissimus thoracis (LT) from the left half-carcass (portion of &#177; 12.0 cm of meat), between the 11th and 13th ribs in the cranial direction. In the laboratory, section the meat samples into steaks of 2.54 cm.</li>
	</ol>
	</li>
	<li>Aging
	<ol>
		<li>Assess the meat quality traits after a 14 day wet-aging period at a temperature of 0-2 &#176;C in a biological oxygen demand (BOD) incubator. Use steaks of 2.54 cm thickness for the analysis of meat color, pH, intramuscular fat, purge loss, water-holding capacity, objective tenderness, and cooking losses. Pack the steaks separately in plastic bags for high vacuum and low oxygen permeability and after the aging time is reached, keep them frozen at -20 &#176;C until the time of analysis. Thaw the beef samples at 4 &#176;C for 24 h and expose them to oxygen for 30 min at 4 &#176;C (blooming time).</li>
	</ol>
	</li>
</ol>
2. Meat pH
<ol>
	<li>Measure the pH of the meat using a digital pH gauge equipped with a penetration probe. Calibrate it with pH 4.0 and 7.0 buffers at a room temperature of 25 &#176;C. Measure the meat pH at three locations of the LT muscle sample. Manually record the data readings and subsequently export the datasheet; calculate the average of the three readings for meat pH.</li>
</ol>
3. Intramuscular fat
NOTE: Intramuscular fat (IMF) content was determined using near infrared (NIR) spectroscopy14 and by gravimetric method15.
<ol>
	<li>Remove the subcutaneous fat from the LT muscle using a scalpel. Then, grind and homogenize the steak for 5 min using a mixer, incorporating approximately 180 g of the sample. Place the sample in a cup, position it inside the sample chamber, and perform subscanning of various zones of the test sample by rotating the sample cup; merge the zones for the final result.</li>
	<li>Take three readings for each sample. After homogenization, place the samples in the plate for subsequent reading. Set the apparatus to NIR transmission, with a moving grating monochromator scanning the region from 850 nm to 1050 nm.</li>
	<li>Export the data sheet and then calculate the average three reading for the IMF. Express the results as a percentage, using the formula: [(IMF average &#247; sample weight) &#215; 100].</li>
	<li>Combine homogenize the LT muscle samples (3.0 g) with a chloroform/methanol methanol/chloroform (2:1) solution for 2 min and subject them to centrifugation (700 &#215; g; 10 min; 20 &#176;C) to segregate the hydrophilic (upper), solid (middle), and hydrophobic (lower) phases.</li>
	<li>Filter the hydrophobic phase obtained post centrifugation using a filter paper on a funnel with slight suction. Transfer the filtrate (bottom phase; lipids in chloroform) to a flask labeled as lipid phase and transfer at least 5 mm of filtrate to a preweighed beaker flask after letting it stand for a few minutes. Then, record the volume of the chloroform layer (at least 150 mL) and aspirate the alcoholic layer.
	<ol>
		<li>Homogenize 100 g aliquots of the sample of fresh or frozen tissue for 2 min with a mixture of 100 mL of chloroform and 200 mL of methanol. Add 100 mL of chloroform to the mixture, blend for 30 s, add 100 mL of distilled water, and blend for another 30 s.</li>
		<li>Filter the homogenate through filter paper on a funnel with slight suction. Apply pressure with the bottom of a beaker when the residue becomes dry to ensure maximum recovery of solvent.</li>
		<li>Transfer the filtrate to a 500 mL graduated cylinder and let it stand for a few minutes to allow separation and clarification. Record the volume of the chloroform layer (at least 150 mL) and aspirate the alcoholic layer.</li>
		<li>Be sure to completely remove the top layer; the chloroform layer contains the purified lipid. For quantitative lipid extraction, recover the lipid trapped in the tissue residue by blending the residue and filter paper with 100 mL of chloroform.</li>
		<li>Filter the mixture through the funnel and rinse the blender jar and residue with a total of 50 mL of chloroform. Mix this filtrate with the original filtrate before removing the alcoholic layer. 
		NOTE: Filtration is typically rapid; apply pressure with the bottom of a beaker on the dry residue to ensure maximum recovery of solvent.</li>
	</ol>
	</li>
	<li>Dry the samples in an oven, cool them in a desiccator for at least 24 h, place them in an oven at 110 &#176;C until complete solvent evaporation, further cool them in a desiccator overnight, and finally reweigh them.</li>
	<li>Determine the IMF content by calculating the difference between the initial and final weights of the beaker.</li>
</ol>
4. Meat color
<ol>
	<li>Calibrate the device using a black and a white standard plate. Place the white calibration plate near the middle of the plate. When doing a calibration, use the area near the middle of the plate. Calibration is complete after the lamp flashes three times.</li>
	<li>Take measurements after 30 min at 4 &#176;C (blooming time). Obtain color readings from three different locations on the LT muscle sample, carefully avoiding connective tissue and fat.</li>
	<li>At room temperature (20 &#176;C), compute an average from these measurements, as recommended16.</li>
</ol>
5. Water losses
<ol>
	<li>Assess purge loss (PL) for all samples. Determine the PL of beef loin sections by measuring the variance between the initial weight before freezing and the final weight after freezing/thawing. 
	NOTE: Do not evaluate the PL of never-frozen control beef loins.</li>
	<li>Gauge the water-holding capacity (WHC) by the weight difference of a meat sample (approximately 1.0 g) before and after being subjected to a pressure of 10 kg for 5 min17.</li>
</ol>
6. Objective meat tenderness
NOTE: The measurement of Warner-Bratzler shear force (WBSF) was conducted as described18,19.
<ol>
	<li>Position samples on a grid attached to a glass refractory and cook them in an industrial electric oven until reaching a final temperature of 71 &#176;C. After cooking, cool, weigh, and refrigerate the samples at 4 &#176;C for 24 h.</li>
	<li>Determine cooking losses (CL) using the formula <img alt="Equation 1" src="/files/ftp_upload/66764/66764eq01.jpg" style="margin: auto;" />.
	<ol>
		<li>Determine drip loss by weighing the refractory before and after cooking the sample. To this end, place the samples on a grid over a glass refractory to allow drainage of meat juices and fat during cooking.</li>
		<li>Determine evaporation loss by weighing only the sample before and after cooking.</li>
		<li>Record raw and cooked weights and calculate the percentage of DL as the weight of drip after cooking divided by the weight of the thawed meat sample.</li>
		<li>Calculate the evaporation loss percentage (EVP) using the formula [100 - (weight after cooking) &#247; raw weight &#215; 100].</li>
	</ol>
	</li>
	<li>For the determination of WBSF, section eight cores with a diameter of 1.27 cm using a texture analyzer, equipped with a 3.07 mm Warner-Bratzler Shear Force blade and a V-shaped (60&#176; angle) cutting edge.
	<ol>
		<li>Report the results as the average of six values per sample, in kilograms (kg), after excluding the low and high extremes19.</li>
	</ol>
	</li>
</ol>
7. Biochemical assay
NOTE: Postmortem proteolysis was assessed by estimating the myofibril fragmentation index (MFI), following the original procedure outlined by Culler et al.20 and adapted for Bos indicus cattle by Borges et al.21.
<ol>
	<li>Homogenize fragments of approximately 3 g of LT samples (fat-stripped muscle tissue and connective tissue) in a buffer solution containing 100 mM potassium chloride, 20 mM potassium phosphate at pH 7, 1 mM ethylenediaminetetraacetic acid (EDTA), 1 mM magnesium chloride, and 1 mM sodium azide at 2 &#176;C, followed by centrifugation (1,000 &#215; g for 15 min at 4 &#176;C).
	<ol>
		<li>Resuspend the sediment in 10 volumes (v/w) of isolating medium using a stir rod, then sediment it again at 1,000 &#215; g for 15 min and decant the supernatant.</li>
		<li>Resuspend the sediment in 2.5 volumes (v/w) of isolating medium and separate the connective tissue and debris by passing it through a polyethylene strainer (18 mesh). Use an additional 2.5 volumes (v/w) to allow the myofibrils to pass through the strainer.</li>
		<li>Determine the protein concentration of the suspension of myofibrils using the biuret method of Gornall et al.22. Dilute an aliquot of the myofibril suspension with isolating medium to a protein concentration of 0.5 &#177; 0.05 mg/mL.</li>
		<li>Immediately measure the absorbance of this suspension at 540 nm. Determine MFI using spectrophotometry at 540 nm. Multiply the absorbance by 200 to obtain the MFI for each sample (and report it as an index without a measurement unit).</li>
	</ol>
	</li>
</ol>
8. Molecular biology assay
NOTE: For the analysis of myosin heavy chain (MyHC), the most abundant protein in bovine skeletal muscle, LT samples from both groups were processed following the protocol described in the literature23,24.
<ol>
	<li>Achieve electrophoretic separation using a gradient SDS-PAGE gel (7-10%) and a 4% stacking gel. Apply 25 &#181;L of each sample to the gel and run it at 70 V, 28 mA, and 4 &#176;C for 1 h, followed by a run at 180 V, 12 mA, and 4 &#176;C for 29 h.</li>
	<li>Employ two different buffers in the runs: the upper gel buffer comprising glycine, Tris(hydroxymethyl)aminomethane base, sodium dodecyl sulfate (SDS), and distilled water, while the lower gel buffer is identical to the upper buffer, with the addition of mercaptoethanol.</li>
	<li>Stain the gels with Coomassie Blue and capture images using appropriate software.</li>
	<li>Identify MyHC isoforms (MyHC-I, MyHC-IIa, MyHC-IIx/d) based on their molecular weights (223.900, 224.243, and 223.875 kDa, respectively). Conduct semi-quantitative analysis by densitometry of the bands corresponding to each isoform, utilizing appropriate software.</li>
	<li>Employ Rat soleus and Extensor digitorum longus (EDL) muscle as positive controls to classify MyHC isoforms, reserving one well in each gel for loading 40 &#181;L of the processed sample.</li>
	<li>For all data, perform the analysis of variance (ANOVA) by the F test, using the following model: 
	Yij = &#181; + ti + &#400;ij 
	where Yij is the observed value of the experimental unit referring to treatment i in repetition j; &#181; is the general effect of the mean; t is the treatment effect (genetic group), and&#160;&#949; is the experimental error.</li>
	<li>Compare the means by using the Student&#39;s t-test and adopt P-value &#60; 0.05 as the critical probability.</li>
</ol>

Assessing Meat Quality Traits in Bos indicus and Crossbred Bulls

The authors have nothing to disclose. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

During carcass evaluation, it is crucial to accurately measure growth and quality traits following a 48 h cooling period to obtain consistent and comparable data. The two biological models exhibited divergent carcass traits, particularly HCW, REA, and BFT, which are consistent with findings reported in other studies. The average HCW of Nellore bulls aligns with Brazilian market preferences, which prioritize greater meat production per animal unit with less fat content25. Conversely, crossbred catt...

Brazil has the largest commercial cattle herd globally, numbering approximately 220 million animals and ranking as the second-largest meat producer, yielding over 9 million metric tons of carcass equivalent annually1. The beef cattle production sector significantly contributes to the national agricultural system, with total annual sales surpassing R$ 55 billion. Since 2004, Brazil has been a key player in the global meat trade, exporting to over 180 countries, which represents ~50% of the world meat trade2.
Meat tenderness stands out as the paramount quality attribute influencing consumer satisfaction and meat consumption3.&#160;By employing biochemical and objective methods to measure meat tenderness, researchers aim to provide valuable insights into factors such as animal genetics, processing techniques, and storage conditions, ultimately enhancing the quality and consistency of meat products for consumers. Such information is useful because meat tenderness has gained increased importance in consumer decision-making during purchases. Moreover, meat tenderness assessment provides valuable information for quality control in meat production and processing industries. By consistently monitoring tenderness, producers can ensure that meat products meet desired standards and specifications. In this context, Brazilian beef cattle producers are progressively embracing intensive feedlot systems with crossbred animals to enhance capital turnover. This system accounts for about 10% tons of carcass produced annually in Brazil4,5.
The escalating demand for improved meat quality by consumers has prompted beef cattle producers to crossbreed with European breeds, primarily Aberdeen Angus6. This strategy aims to produce F1 Angus-Nellore hybrids, known for superior performance, desirable carcass traits, and enhanced meat quality compared to pure zebu animals7,8. In the tropical regions of Brazil, it is common practice to utilize non-castrated animals (bulls) of advanced maturity in finishing farms, potentially compromising meat quality attributes such as color, marbling, and tenderness. Notably, a survey reveals that 95% of animals finished in Brazilian feedlots are males, with 73% being Nellore, followed by 22% crossbred animals and 5% other genotypes9,10.
Understanding the biochemical mechanisms underlying meat tenderness is crucial for improving meat quality. One key aspect is postmortem proteolysis, which affects the structural integrity of muscle fibers11. The myofibril fragmentation index (MFI) is a widely used biochemical assay that quantifies the extent of myofibril degradation, providing insights into the tenderness of meat12. The MFI method involves measuring the fragmentation of myofibrillar proteins, which directly correlates with meat tenderness. This assay complements traditional meat quality assessments and offers a deeper understanding of the biochemical processes that contribute to variations in meat tenderness.
In this context, the present study investigated the skeletal muscle of Bos indicus versus crossbred (Bos taurus &#215; Bos indicus) bulls finished in feedlot, aiming to explain differences in meat quality traits.

<table><tbody><tr><td>Acetone</td><td>Merk, Darmstadt, Germany</td><td>CAS 67-64-1 | 100014</td><td>solutions used for the electrophoretic separations</td></tr><tr><td>Anti-MYH-1 Antibody</td><td>Merk, Darmstadt, Germany</td><td>MABT846</td><td>Rat soleus</td></tr><tr><td>Anti-Myosin antibody</td><td>Abcam, Massachusetts, United States</td><td>ab37484</td><td>Myosin heavy chain</td></tr><tr><td>Anti-Myosin-2 (MYH2) Antibody</td><td>Merk, Darmstadt, Germany</td><td>MABT840</td><td>Extensor digitorum longus (EDL)</td></tr><tr><td>Biological oxygen demand (BOD) incubator</td><td>TECNAL, S&amp;atilde;o Paulo, Brazil</td><td>TE-371/240L</td><td>Meat aging</td></tr><tr><td>Chloroform; absolute analytical reagent</td><td>Sigma-Aldrich, Missouri, United States</td><td>67-66-3</td><td>Intramuscular fat</td></tr><tr><td>CIELab system</td><td>Konica Minolta Sensing, Tokyo, Japan</td><td>CR-400 colorimeter</td><td>Meat color</td></tr><tr><td>Coomassie Blue</td><td>Sigma-Aldrich, Missouri, United States</td><td>C.I. 42655)</td><td>Myosin heavy chain</td></tr><tr><td>Electric oven</td><td>Ven&amp;acirc;ncio Aires, Rio Grande do Sul, Brazil</td><td></td><td>Meat tenderness</td></tr><tr><td>Ethanol</td><td>Merk, Darmstadt, Germany</td><td>64-17-5</td><td>solutions used for the electrophoretic separations</td></tr><tr><td>Ethylenediaminetetraacetic acid</td><td>Sigma-Aldrich, Missouri, United States</td><td>60-00-4</td><td>Post-mortem proteolysis</td></tr><tr><td>Glass flasks</td><td>Sigma-Aldrich, Missouri, United States</td><td></td><td>solutions used for the electrophoretic separations</td></tr><tr><td>Glycine</td><td>Sigma-Aldrich, Missouri, United States</td><td>G6761</td><td>Myosin heavy chain</td></tr><tr><td>Infrared spectroscopy - FoodScan</td><td>Foss NIRSystems, Madson, United States</td><td>FoodScan&amp;trade; 2</td><td>Intramuscular fat</td></tr><tr><td>Magnesium chloride</td><td>Sigma-Aldrich, Missouri, United States</td><td>&amp;nbsp;7786-30-3</td><td>Post-mortem proteolysis</td></tr><tr><td>Mercaptoetanol</td><td>Sigma-Aldrich, Missouri, United States</td><td>M6250</td><td>Myosin heavy chain</td></tr><tr><td>Methanol, absolute analytical reagent</td><td>Sigma-Aldrich, Missouri, United States</td><td>67-56-1</td><td>Intramuscular fat</td></tr><tr><td>pH meter</td><td>LineLab, S&amp;atilde;o Paulo, Brazil</td><td>AKLA 71980</td><td>Meat pH</td></tr><tr><td>PlusOne 2-D Quant Kit</td><td>GE Healthcare Product</td><td>Code 80-6483-56</td><td>Post-mortem proteolysis</td></tr><tr><td>Polypropylene</td><td>Sigma-Aldrich, Missouri, United States</td><td></td><td>solutions used for the electrophoretic separations</td></tr><tr><td>Potassium chloride</td><td>Sigma-Aldrich, Missouri, United States</td><td>7447-40-7</td><td>Post-mortem proteolysis</td></tr><tr><td>Potassium phosphate</td><td>Sigma-Aldrich, Missouri, United States</td><td>P0662</td><td>Post-mortem proteolysis</td></tr><tr><td>R software</td><td>Vienna, Austria</td><td>version 3.6.2</td><td>Data analysis</td></tr><tr><td>Sodium azide</td><td>Sigma-Aldrich, Missouri, United States</td><td>26628-22-8</td><td>Post-mortem proteolysis</td></tr><tr><td>Sodium dodecyl sulfate&amp;nbsp;(SDS)</td><td>Sigma-Aldrich, Missouri, United States</td><td>822050</td><td>Myosin heavy chain</td></tr><tr><td>Spectrophotometer</td><td>Perkin Elmer, Shelton, United States</td><td>Perkin Elmer&lt;br/&gt; Lambda 25 UV/Vis</td><td>Post-mortem proteolysis</td></tr><tr><td>Statistical Analysis System</td><td>SAS, Cary, North Carolina, United States</td><td>version 9.1,</td><td>Data analysis</td></tr><tr><td>Texture Analyzer</td><td>AMETEK Brookfield, Massachusetts, United&lt;br/&gt; States</td><td>CTX</td><td>Meat tenderness</td></tr><tr><td>Tris(hydroxymethyl)aminomethane</td><td>Sigma-Aldrich, Missouri, United States</td><td>77-86-1</td><td>Myosin heavy chain</td></tr><tr><td>Ultrafreezer</td><td>Indrel Scientific, Londrina, Paran&amp;aacute;, Brazil.</td><td>INDREL IULT 335 D - LCD</td><td>Sample storage</td></tr><tr><td>Ultrapure water</td><td>Elga PURELAB Ultra Ionic system</td><td>solutions used for the electrophoretic separations</td><td></td></tr><tr><td>Ultra-Turrax high shear mixer</td><td>Marconi &amp;ndash; MA102/E, Piracicaba, S&amp;atilde;o Paulo, Brazil</td><td></td><td>Post-mortem proteolysis</td></tr></tbody></table>

exploring the longissimus muscle: unraveling its correlation with meat quality in bos indicus and crossbred bulls

This study investigated muscle tissue in Bos indicus and crossbred bulls to explain differences in meat quality traits. Carcass traits, meat quality parameters, and biochemical and molecular investigations of myofibrillar proteins are described. Methods for evaluating pH, intramuscular fat (IMF), meat color (L*, a*, b*), water losses, tenderness, and molecular biology assays have been outlined. Specific procedures detailing calibration, sample preparation, and data analysis for each method are described. These include techniques such as infrared spectroscopy for IMF content, objective tenderness assessment, and electrophoretic separation of MyHC isoforms.
Color parameters were highlighted as potential tools for predicting beef tenderness, a crucial quality trait influencing consumer decisions. The study employed the Warner-Bratzler shear force (WBSF) method, revealing values of 4.68 and 4.23 kg for Nellore and Angus-Nellore (P &#60; 0.01), respectively. Total cooking losses and biochemical analyses, including myofibril fragmentation index (MFI), provided insights into tenderness variations. Muscle fiber types, particularly myosin heavy chain (MyHC) isoforms, were investigated, with a notable absence of MyHC-IIb isoform in the studied Zebu animals. The relationship between MyHC-I and meat tenderness revealed divergent findings in the literature, highlighting the complexity of this association. Overall, the study provides comprehensive insights into the factors influencing meat quality in Bos indicus and crossbred (Bos taurus &#215; Bos indicus) bulls, offering valuable information for the beef industry.

Table 1 displays the carcass traits of the two genetic groups investigated in this study. Notably, differences were identified (P &#60; 0.01) in HCW, REA, and BFT, with crossbred animals exhibiting greater values, suggesting a heterosis effect.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Variable&#185;</td>
			<td>Nellore</td>
			<td>F1 Angus x Nellore</td>
			<td>SEM</td>
			<td>P-value</td>
		</tr>
		<tr>
			<td...

Author Spotlight: Improving Beef Cattle Nutrition and Production with a Focus on Feed Efficiency and Meat Quality Traits Through Advanced Biochemical and Molecular Assays

We investigated skeletal muscle tissue in Bos indicus and crossbred bulls to explain differences in meat quality traits. Warner-Bratzler shear force (WBSF) was found to range from 4.7 kg to 4.2 kg. Myosin heavy chain isoforms revealed differences between animals, and myofibril fragmentation index provided further insights into tenderness (WBSF) variations.

Exploring the Longissimus Muscle: Unraveling its Correlation with Meat Quality in Bos indicus and Crossbred Bulls

This study investigated muscle tissue in Bos indicus and crossbred bulls to explain differences in meat quality traits. Carcass traits, meat quality ...

exploring-longissimus-muscle-unraveling-its-correlation-with-meat

Research

JoVE Journal

Biology

447 Views.  São Paulo State University, Botucatu (São Paulo). Ons onderzoek richt zich op het verbeteren van de voeding en productie van vleesvee, met een belangrijke focus op voerefficiëntie en vleeskwaliteitskenmerken. Onlangs hebben we verschillende voederefficiëntie-indexen van runderen onderzocht. Bijvoorbeeld de opname van restvoer, om inzicht te krijgen in de impact ervan op het karkas en de vleeskwaliteitskenmerken.We hebben biochemische en moleculaire tests toegepast, waaronder proteomics, lipidomics e...

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Species Determination and Quantitation in Mixtures Using MRM Mass Spectrometry of Peptides Applied to Meat Authentication

We describe a simple protocol for identifying and quantifying the two components in binary mixtures of species possessing one or more similar proteins. Central to the method is the identification of 'corresponding proteins' in the species of interest, in other words proteins that are nominally the same but possess species-specific sequence differences. When subject to proteolysis, corresponding proteins will give rise to some peptides which are likewise similar but with species-specific variants. These are 'corresponding peptides'. Species-specific peptides can be used as markers for species determination, while pairs of corresponding peptides permit relative quantitation of two species in a mixture. The peptides are detected using multiple reaction monitoring (MRM) mass spectrometry, a highly specific technique that enables peptide-based species determination even in complex systems. In addition, the ratio of MRM peak areas deriving from corresponding peptides supports relative quantitation. Since corresponding proteins and peptides will, in the main, behave similarly in both processing and in experimental extraction and sample preparation, the relative quantitation should remain comparatively robust. In addition, this approach does not need the standards and calibrations required by absolute quantitation methods. The protocol is described in the context of red meats, which have convenient corresponding proteins in the form of their respective myoglobins. This application is relevant to food fraud detection: the method can detect 1% weight for weight of horse meat in beef. The corresponding protein, corresponding peptide (CPCP) relative quantitation using MRM peak area ratios gives good estimates of the weight for weight composition of a horse plus beef mixture.

We present a protocol for identifying and quantifying the components in mixtures of species possessing similar proteins. Mass spectrometry detects peptides for identification, and gives relative quantitation by ratios of peak areas. As a tool food for fraud detection, the method can detect 1% horse in beef.

We describe a simple protocol for identifying and quantifying the two components in binary mixtures of species possessing one or more similar proteins. ...

Biochemistry

Behavioral and Locomotor Measurements Using an Open Field Activity Monitoring System for Skeletal Muscle Diseases

The open field activity monitoring system comprehensively assesses locomotor and behavioral activity levels of mice. It is a useful tool for assessing locomotive impairment in animal models of neuromuscular disease and efficacy of therapeutic drugs that may improve locomotion and/or muscle function. The open field activity measurement provides a different measure than muscle strength, which is commonly assessed by grip strength measurements. It can also show how drugs may affect other body systems as well when used with additional outcome measures. In addition, measures such as total distance traveled mirror the 6 min walk test, a clinical trial outcome measure. However, open field activity monitoring is also associated with significant challenges: Open field activity measurements vary according to animal strain, age, sex, and circadian rhythm. In addition, room temperature, humidity, lighting, noise, and even odor can affect assessment outcomes. Overall, this manuscript provides a well-tested and standardized open field activity SOP for preclinical trials in animal models of neuromuscular diseases. We provide a discussion of important considerations, typical results, data analysis, and detail the strengths and weaknesses of open field testing. In addition, we provide recommendations for optimal study design when using open field activity in a preclinical trial.

Open field activity levels are used to assess locomotive and behavioral activity levels. This protocol provides a well-designed, standardized protocol to use in preclinical trials for neuromuscular disorders.

The open field activity monitoring system comprehensively assesses locomotor and behavioral activity levels of mice. It is a useful tool for assessing ...

Behavior

Protocol for Assessing the Relative Effects of Environment and Genetics on Antler and Body Growth for a Long-lived Cervid

Cervid phenotype can be placed into one of two categories: efficiency, which promotes survival over extravagant morphometric growth, and luxury, which promotes growth of large weaponry and body size. Populations of the same species display each phenotype depending on environmental conditions. Although antler and body size of male white-tailed deer (Odocoileus virginianus) varies by physiographic region in Mississippi, USA and is strongly correlated with regional variation in nutritional quality, the effects of population-level genetics from native stocks and previous re-stocking efforts cannot be disregarded. This protocol describes how we designed a controlled study, where other factors that influence phenotype, such as age and nutrition, are controlled. We brought wild-caught pregnant females and six-month-old fawns from three distinct physiographic regions in Mississippi, USA to the Mississippi State University Rusty Dawkins Memorial Deer Unit. Deer from the same region were bred to produce a second generation of offspring, allowing us to assess generational responses and maternal effects. All deer ate the same high-quality (20% crude protein deer pellet) diet ad libitum. We uniquely marked each neonate and recorded body mass, hind foot, and total body length. Each subsequent fall, we sedated individuals via remote injection and sampled the same morphometrics plus antlers of adults. We found that all morphometrics increased in size from first to second generation, with full compensation of antler size (regional variation no longer present) and partial compensation of body mass (some evidence of regional variation) evident in the second generation. Second generation males that originated from our poorest quality soil region displayed about a 40% increase in antler size and about a 25% increase in body mass when compared to their wild harvested counterparts. Our results suggest phenotypic variation of wild male white-tailed deer in Mississippi are more related to differences in nutritional quality than population-level genetics.

Phenotypic differences among cervid populations may be related to population-level genetics or nutrition; discerning which is difficult in the wild. This protocol describes how we designed a controlled study where nutritional variation was eliminated. We found that phenotypic variation of male white-tailed deer was more limited by nutrition than genetics.

Cervid phenotype can be placed into one of two categories: efficiency, which promotes survival over extravagant morphometric growth, and luxury, which ...

Environment

Skeletal Muscle Gender Dimorphism from Proteomics

Gross contraction in skeletal muscle is primarily determined by a relatively small number of contractile proteins, however this tissue is also remarkably adaptable to environmental factors1 such as hypertrophy by resistance exercise and atrophy by disuse. It thereby exhibits remodeling and adaptations to stressors (heat, ischemia, heavy metals, etc.)2,3. Damage can occur to muscle by a muscle exerting force while lengthening, the so-called eccentric contraction4. The contractile proteins can be damaged in such exertions and need to be repaired, degraded and/or resynthesized; these functions are not part of the contractile proteins, but of other much less abundant proteins in the cell. To determine what subset of proteins is involved in the amelioration of this type of damage, a global proteome must be established prior to exercise5 and then followed subsequent to the exercise to determine the differential protein expression and thereby highlight candidate proteins in the adaptations to damage and its repair. Furthermore, most studies of skeletal muscle have been conducted on the male of the species and hence may not be representative of female muscle. 
In this article we present a method for extracting proteins reproducibly from male and female muscles, and separating them by two-dimensional gel electrophoresis followed by high resolution digital imaging6. This provides a protocol for spots (and subsequently identified proteins) that show a statistically significant (p &lt; 0.05) two-fold increase or decrease, appear or disappear from the control state. These are then excised, digested with trypsin and separated by high-pressure liquid chromatography coupled to a mass spectrometer (LC/MS) for protein identification (LC/MS/MS)5. This methodology (Figure 1) can be used on many tissues with little to no modification (liver, brain, heart etc.).

A straight-forward set of methods to isolate and determine the identity of the most abundant proteins expressed in skeletal muscle. About 800 spots are discerned on a two-dimensional gel from 10 mg muscle; this allows for the determination of gender-specific protein expression. These methods will give equivalent results in most tissues.

Gross contraction in skeletal muscle is primarily determined by a relatively small number of contractile proteins, however this tissue is also remarkably ...

Medicine

Measuring Liver Mitochondrial Oxygen Consumption and Proton Leak Kinetics to Estimate Mitochondrial Respiration in Holstein Dairy Cattle

Oxygen consumption, proton motive force (PMF) and proton leak are measurements of mitochondrial respiration, or how well mitochondria are able to convert NADH and FADH into ATP. Since mitochondria are also the primary site for oxygen use and nutrient oxidation to carbon dioxide and water, how efficiently they use oxygen and produce ATP directly relates to the efficiency of nutrient metabolism, nutrient requirements of the animal, and health of the animal. The purpose of this method is to examine mitochondrial respiration, which can be used to examine the effects of different drugs, diets and environmental effects on mitochondrial metabolism. Results include oxygen consumption measured as proton dependent respiration (State 3) and proton leak dependent respiration (State 4). The ratio of State 3 / State 4 respiration is defined as respiratory control ratio (RCR) and can represent mitochondrial energetic efficiency. Mitochondrial proton leak is a process that allows dissipation of mitochondrial membrane potential (MMP) by uncoupling oxidative phosphorylation from ADP decreasing the efficiency of ATP synthesis. Oxygen and TRMP+ sensitive electrodes with mitochondrial substrates and electron transport chain inhibitors are used to measure State 3 and State 4 respiration, mitochondrial membrane PMF (or the potential to produce ATP) and proton leak. Limitations to this method are that liver tissue must be as fresh as possible and all biopsies and assays must be performed in less than 10 h. This limits the number of samples that can be collected and processed by a single person in a day to approximately 5. However, only 1 g of liver tissue is needed, so in large animals, such as dairy cattle, the amount of sample needed is small relative to liver size and there is little recovery time needed.

Here, we share methods for measuring mitochondrial oxygen consumption, a defining concept of nutritional energetics, and proton leak, the primary cause of inefficiency in mitochondrial generation of ATP. These results can account for 30% of the energy lost in nutrient utilization to help evaluate mitochondrial function.

Oxygen consumption, proton motive force (PMF) and proton leak are measurements of mitochondrial respiration, or how well mitochondria are able to convert ...

Human Vastus Lateralis Skeletal Muscle Biopsy Using the Weil-Blakesley Conchotome

Percutaneous muscle biopsy using the Weil-Blakesley conchotome is well established in both clinical and research practice. It is a safe, effective and well tolerated technique. The Weil-Blakesley conchotome has a sharp biting tip with a 4 - 6 mm wide hollow. It is inserted through a 5 - 10 mm skin incision and can be maneuvered for controlled tissue penetration. The tip is opened and closed within the tissue and then rotated through 90 -180&#176; to cut the muscle. The amount of muscle obtained following repeated sampling can vary from 20 mg to 290 mg which can be processed for both histology and molecular studies. The wound needs to be kept dry and vigorous physical activity kept to a minimum for approximately 72 hr although normal levels of activity can restart immediately following the procedure. This procedure is safe and effective when close attention is paid to the selection of subjects, full asepsis and post procedure care. &#160;Both right and left vastus lateralis are suitable for biopsy dependent on participant preference.

Human Vastus Lateralis Skeletal Muscle Biopsy Using the Weil-Blakesley Conchotome

This video demonstrates the technique of percutaneous muscle biopsy of the human vastus lateralis using the Weil-Blakesley conchotome.

Percutaneous muscle biopsy using the Weil-Blakesley conchotome is well established in both clinical and research practice. It is a safe, effective and ...

Collection and Processing of Lymph Nodes from Large Animals for RNA Analysis: Preparing for Lymph Node Transcriptomic Studies of Large Animal Species

Large animals (both livestock and wildlife) serve as important reservoirs of zoonotic pathogens, including Brucella, Mycobacterium bovis, Salmonella, and E. coli, and are useful for the study of pathogenesis and/or spread of the bacteria in natural hosts. With the key function of lymph nodes in the host immune response, lymph node tissues serve as a potential source of RNA for downstream transcriptomic analyses, in order to assess the temporal changes in gene expression in cells over the course of an infection. This article presents an overview of the process of lymph node collection, tissue sampling, and downstream RNA processing in livestock, using cattle (Bos taurus) as a model, with additional examples provided from the American bison (Bison bison). The protocol includes information about the location, identification, and removal of lymph nodes from multiple key sites in the body. Additionally, a biopsy sampling methodology is presented that allows for a consistency of sampling across multiple animals. Several considerations for sample preservation are discussed, including the generation of RNA suitable for downstream methodologies like RNA-sequencing and RT-PCR. Due to the long delays inherent in large animal vs. mouse time course studies, representative results from bison and bovine lymph node tissues are presented to describe the time course of the degradation in this tissue type, in the context of a review of previous methodological work on RNA degradation in other tissues. Overall, this protocol will be useful to both veterinary researchers beginning transcriptome projects on large animal samples and to molecular biologists interested in learning techniques for in vivo tissue sampling and in vitro processing.

This protocol provides an overview of procedures for the isolation of RNA for the transcriptomic profiling of lymph node tissues from large animals, including steps in the identification and excision of lymph nodes from livestock and wildlife, sampling approaches to provide consistency across multiple animals, and considerations plus representative results for the post-collection preservation and processing for RNA analysis.

Large animals (both livestock and wildlife) serve as important reservoirs of zoonotic pathogens, including Brucella, Mycobacterium bovis, Salmonella, and ...

Immunology and Infection

Introducing an Angle Adjustable Cutting Box for Analyzing Slice Shear Force in Meat

Research indicates the fibre angle of the longissimus muscle can vary, depending upon location within a steak and throughout the muscle. Instead of using the original fixed 45 &deg; or 90 &deg; cutting angle for testing shear force, a variable angle cutting box can be adjusted so the angles of the knives correspond to the fibre angle of each sample. Within 2 min after cooking to an internal temperature of 71 &deg;C on an open-hearth grill set at 210 &deg;C, a 1 cm by 5 cm core is cut from the steak, parallel to muscle fibre direction, using 2 knife blades set 1 cm apart. This warm core is then subjected to the Slice Shear Force protocol (SSF) to evaluate meat texture. The use of the variable angle cutting box and the SSF protocol provides an accurate representation of the maximal shear force, as the slice and muscle fibres are consistently parallel. Therefore, the variable angle cutting box, in conjunction with the SSF protocol, can be used as a high-throughput technique to accurately evaluate meat tenderness in different locations of the longissimus muscle and, potentially, in other muscles.

Slice shear force is a reference method for beef texture analysis. Using an angle adjustable cutting box could increase its accuracy for research purposes. The results from different locations within the longissimus muscle show a high correlation with Warner-Bratzler shear force methodology and high potential adaptability for different muscles.

Research indicates the fibre angle of the longissimus muscle can vary, depending upon location within a steak and throughout the muscle. Instead of using ...

Obtaining Quality Extended Field-of-View Ultrasound Images of Skeletal Muscle to Measure Muscle Fascicle Length

Muscle fascicle length, which is commonly measured in vivo using traditional ultrasound, is an important parameter defining a muscle&#8217;s force generating capacity. However, over 90% of all upper limb muscles and 85% of all lower limb muscles have optimal fascicle lengths longer than the field-of-view of common traditional ultrasound (T-US) probes. A newer, less frequently adopted method called extended field-of-view ultrasound (EFOV-US) can enable direct measurement of fascicles longer than the field-of-view of a single T-US image. This method, which automatically fits together a sequence of T-US images from a dynamic scan, has been demonstrated to be valid and reliable for obtaining muscle fascicle lengths in vivo. Despite the numerous skeletal muscles with long fascicles and the validity of the EFOV-US method for making measurements of such fascicles, few published studies have utilized this method. In this study, we demonstrate both how to implement the EFOV-US method to obtain high quality musculoskeletal images and how to quantify fascicle lengths from those images. We expect that this demonstration will encourage the use of the EFOV-US method to increase the pool of muscles, both in healthy and impaired populations, for which we have in vivo muscle fascicle length data.

This study describes how to obtain high quality musculoskeletal images using the extended field-of-view ultrasound (EFOV-US) method for the purpose of making muscle fascicle length measures. We apply this method to muscles with fascicles that extend past the field-of-view of common traditional ultrasound (T-US) probes.

Muscle fascicle length, which is commonly measured in vivo using traditional ultrasound, is an important parameter defining a muscle&#8217;s force ...

Post-Myocardial Infarction Heart Failure in Closed-chest Coronary Occlusion/Reperfusion Model in G&#246;ttingen Minipigs and Landrace Pigs

The development of heart failure is the most powerful predictor of long-term mortality in patients surviving acute myocardial infarction (MI). There is an unmet clinical need for prevention and therapy of post-myocardial infarction heart failure (post-MI HF). Clinically relevant pig models of post-MI HF are prerequisites for final proof-of-concept studies before entering into clinical trials in drug and medical device development.
Here we aimed to characterize a closed-chest porcine model of post-MI HF in adult G&#246;ttingen minipigs with long-term follow-up including serial cardiac magnetic resonance imaging (CMRI) and to compare it with the commonly used Landrace pig model.
MI was induced by intraluminal balloon occlusion of the left anterior descending coronary artery for 120 min in G&#246;ttingen minipigs and for 90 min in Landrace pigs, followed by reperfusion. CMRI was performed to assess cardiac morphology and function at baseline in both breeds and at 3 and 6 months in G&#246;ttingen minipigs and at 2 months in Landrace pigs, respectively.
Scar sizes were comparable in the two breeds, but MI resulted in a significant decrease of left ventricular ejection fraction (LVEF) only in G&#246;ttingen minipigs, while Landrace pigs did not show a reduction of LVEF. Right ventricular (RV) ejection fraction increased in both breeds despite the negligible RV scar sizes. In contrast to the significant increase of left ventricular end-diastolic (LVED) mass in Landrace pigs at 2 months, G&#246;ttingen minipigs showed a slight increase in LVED mass only at 6 months.
In summary, this is the first characterization of post-MI HF in G&#246;ttingen minipigs in comparison to Landrace pigs, showing that the G&#246;ttingen minipig model reflects post-MI HF parameters comparable to the human pathology. We conclude that the G&#246;ttingen minipig model is superior to the Landrace pig model to study the development of post-MI HF.

The overall goal of the current study is to present the techniques of induction of myocardial infarction (MI) and post-myocardial infarction heart failure (post-MI HF) in closed-chest, adult G&#246;ttingen minipigs and the characterization of post-MI HF model in G&#246;ttingen minipigs as compared to Landrace pigs.

The development of heart failure is the most powerful predictor of long-term mortality in patients surviving acute myocardial infarction (MI). There is an ...

Exploring the Longissimus Muscle: Unraveling its Correlation with Meat Quality in Bos indicus and Crossbred Bulls

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