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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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&#227;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&#226;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&#8482; 2</td>
			<td>Intramuscular fat</td>
		</tr>
		<tr>
			<td>Magnesium chloride</td>
			<td>Sigma-Aldrich, Missouri, United States</td>
			<td>&#160;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&#227;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&#160;(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 
			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 
			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&#225;, Brazil.</td>
			<td>INDREL IULT 335 D - LCD</td>
			<td>Sample storage</td>
		</tr>
		<tr>
			<td>Ultrapure water</td>
			<td colspan="2">Elga PURELAB Ultra Ionic system</td>
			<td>solutions used for the electrophoretic separations</td>
		</tr>
		<tr>
			<td>Ultra-Turrax high shear mixer</td>
			<td>Marconi &#8211; MA102/E, Piracicaba, S&#227;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

390 Views.  São Paulo State University, Botucatu (São Paulo). 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. 

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

Lens Transplantation in Zebrafish and its Application in the Analysis of Eye Mutants

The lens plays an important role in the development of the optic cup[1,2]. Using the zebrafish as a model organism, questions regarding lens development can be addressed. The zebrafish is useful for genetic studies due to several advantageous characteristics, including small size, high fecundity, short lifecycle, and ease of care. Lens development occurs rapidly in zebrafish. By 72 hpf, the zebrafish lens is functionally mature [3]. Abundant genetic and molecular resources are available to support research in zebrafish. In addition, the similarity of the zebrafish eye to those of other vertebrates provides basis for its use as an excellent animal model of human defects[4-7]. Several zebrafish mutants exhibit lens abnormalities, including high levels of cell death, which in some cases leads to a complete degeneration of lens tissues [8]. To determine whether lens abnormalities are due to intrinsic causes or to defective interactions with the surrounding tissues, transplantation of a mutant lens into a wild-type eye is performed. Using fire-polished metal needles, mutant or wild-type lenses are carefully dissected from the donor animal, and transferred into the host. To distinguish wild-type and mutant tissues, a transgenic line is used as the donor. This line expresses membrane-bound GFP in all tissues, including the lens. This transplantation technique is an essential tool in the studies of zebrafish lens mutants.

Lens development involves interactions with other tissues. Several zebrafish eye mutants are characterized by an abnormally small lens size. Here we demonstrate a lens transplantation experiment to determine whether this phenotype is due to intrinsic causes or defective interactions with tissues that surround the lens.

The lens plays an important role in the development of the optic cup[1,2]. Using the zebrafish as a model organism, questions regarding lens development ...

Exploring Cognitive Functions in Babies, Children &amp; Adults with Near Infrared Spectroscopy

An explosion of functional Near Infrared Spectroscopy (fNIRS) studies investigating cortical activation in relation to higher cognitive processes, such as language1,2,3,4,5,6,7,8,9,10, memory11, and attention12 is underway worldwide involving adults, children and infants 3,4,13,14,15,16,17,18,19 with typical and atypical cognition20,21,22. The contemporary challenge of using fNIRS for cognitive neuroscience is to achieve systematic analyses of data such that they are universally interpretable23,24,25,26, and thus may advance important scientific questions about the functional organization and neural systems underlying human higher cognition.
Existing neuroimaging technologies have either less robust temporal or spatial resolution. Event Related Potentials and Magneto Encephalography (ERP and MEG) have excellent temporal resolution, whereas Positron Emission Tomography and functional Magnetic Resonance Imaging (PET and fMRI) have better spatial resolution. Using non-ionizing wavelengths of light in the near-infrared range (700-1000 nm), where oxy-hemoglobin is preferentially absorbed by 680 nm and deoxy-hemoglobin is preferentially absorbed by 830 nm (e.g., indeed, the very wavelengths hardwired into the fNIRS Hitachi ETG-400 system illustrated here), fNIRS is well suited for studies of higher cognition because it has both good temporal resolution (~5s) without the use of radiation and good spatial resolution (~4 cm depth), and does not require participants to be in an enclosed structure27,28. Participants cortical activity can be assessed while comfortably seated in an ordinary chair (adults, children) or even seated in mom s lap (infants). Notably, NIRS is uniquely portable (the size of a desktop computer), virtually silent, and can tolerate a participants subtle movement. This is particularly outstanding for the neural study of human language, which necessarily has as one of its key components the movement of the mouth in speech production or the hands in sign language. 
The way in which the hemodynamic response is localized is by an array of laser emitters and detectors. Emitters emit a known intensity of non-ionizing light while detectors detect the amount reflected back from the cortical surface. The closer together the optodes, the greater the spatial resolution, whereas the further apart the optodes, the greater depth of penetration. For the fNIRS Hitachi ETG-4000 system optimal penetration / resolution the optode array is set to 2cm. 
Our goal is to demonstrate our method of acquiring and analyzing fNIRS data to help standardize the field and enable different fNIRS labs worldwide to have a common background.

Exploring Cognitive Functions in Babies, Children & Adults with Near Infrared Spectroscopy

Here we describe a data collection and data analysis method for functional Near Infrared Spectroscopy (fNIRS), a novel non-invasive brain imaging system used in cognitive neuroscience, particularly in studying child brain development. This method provides a universal standard of data acquisition and analysis vital to data interpretation and scientific discovery.

An explosion of functional Near Infrared Spectroscopy (fNIRS) studies investigating cortical activation in relation to higher cognitive processes, such as ...

Long-term Lethal Toxicity Test with the Crustacean Artemia franciscana

Our research activities target the use of biological methods for the evaluation of environmental quality, with particular reference to saltwater/brackish water and sediment. The choice of biological indicators must be based on reliable scientific knowledge and, possibly, on the availability of standardized procedures. In this article, we present a standardized protocol that used the marine crustacean Artemia to evaluate the toxicity of chemicals and/or of marine environmental matrices. Scientists propose that the brine shrimp (Artemia) is a suitable candidate for the development of a standard bioassay for worldwide utilization. A number of papers have been published on the toxic effects of various chemicals and toxicants on brine shrimp (Artemia). The major advantage of this crustacean for toxicity studies is the overall availability of the dry cysts; these can be immediately used in testing and difficult cultivation is not demanded1,2. Cyst-based toxicity assays are cheap, continuously available, simple and reliable and are thus an important answer to routine needs of toxicity screening, for industrial monitoring requirements or for regulatory purposes3. The proposed method involves the mortality as an endpoint. The numbers of survivors were counted and percentage of deaths were calculated. Larvae were considered dead if they did not exhibit any internal or external movement during several seconds of observation4. This procedure was standardized testing a reference substance (Sodium Dodecyl Sulfate); some results are reported in this work. This article accompanies a video that describes the performance of procedural toxicity testing, showing all the steps related to the protocol.

Long-term Lethal Toxicity Test with the Crustacean Artemia franciscana

This study concerns the development and standardization of a valuable methodological protocol to determine long-term (14 days) lethal toxicity exerted by chemical substances, industrial wastewater or sewage and liquid environmental samples on the saltwater crustacean, Artemia franciscana.

Our research activities target the use of biological methods for the evaluation of environmental quality, with particular reference to saltwater/brackish ...

Exploring Arterial Smooth Muscle Kv7 Potassium Channel Function using Patch Clamp Electrophysiology and Pressure Myography

Contraction or relaxation of smooth muscle cells within the walls of resistance arteries determines the artery diameter and thereby controls flow of blood through the vessel and contributes to systemic blood pressure. The contraction process is regulated primarily by cytosolic calcium concentration ([Ca2+]cyt), which is in turn controlled by a variety of ion transporters and channels. Ion channels are common intermediates in signal transduction pathways activated by vasoactive hormones to effect vasoconstriction or vasodilation. And ion channels are often targeted by therapeutic agents either intentionally (e.g. calcium channel blockers used to induce vasodilation and lower blood pressure) or unintentionally (e.g. to induce unwanted cardiovascular side effects).
Kv7 (KCNQ) voltage-activated potassium channels have recently been implicated as important physiological and therapeutic targets for regulation of smooth muscle contraction. To elucidate the specific roles of Kv7 channels in both physiological signal transduction and in the actions of therapeutic agents, we need to study how their activity is modulated at the cellular level as well as evaluate their contribution in the context of the intact artery.
The rat mesenteric arteries provide a useful model system. The arteries can be easily dissected, cleaned of connective tissue, and used to prepare isolated arterial myocytes for patch clamp electrophysiology, or cannulated and pressurized for measurements of vasoconstrictor/vasodilator responses under relatively physiological conditions. Here we describe the methods used for both types of measurements and provide some examples of how the experimental design can be integrated to provide a clearer understanding of the roles of these ion channels in the regulation of vascular tone.

Measurements of Kv7 (KCNQ) potassium channel activity in isolated arterial myocytes (using patch clamp electrophysiological techniques) in parallel with measurements of constrictor/dilator responses (using pressure myography) can reveal important information about the roles of Kv7 channels in vascular smooth muscle physiology and pharmacology.

Contraction  or relaxation of smooth muscle cells within the walls of resistance arteries  determines the artery diameter and thereby controls flow of ...

Assessment of Calcium Sparks in Intact Skeletal Muscle Fibers

Maintaining homeostatic Ca2+ signaling is a fundamental physiological process in living cells. Ca2+ sparks are the elementary units of Ca2+ signaling in the striated muscle fibers that appear as highly localized Ca2+ release events mediated by ryanodine receptor (RyR) Ca2+ release channels on the sarcoplasmic reticulum (SR) membrane. Proper assessment of muscle Ca2+ sparks could provide information on the intracellular Ca2+&#160;handling properties of healthy and diseased striated muscles. Although Ca2+ sparks events are commonly seen in resting cardiomyocytes, they are rarely observed in resting skeletal muscle fibers; thus there is a need for methods to generate and analyze sparks in skeletal muscle fibers.
Detailed here is an experimental protocol for measuring Ca2+ sparks in isolated flexor digitorm brevis (FDB) muscle fibers using fluorescent Ca2+ indictors and laser scanning confocal microscopy. In this approach, isolated FDB fibers are exposed to transient hypoosmotic stress followed by a return to isotonic physiological solution. Under these conditions, a robust Ca2+ sparks response is detected adjacent to the sarcolemmal membrane in young healthy FDB muscle fibers. Altered Ca2+ sparks response is detected in dystrophic or aged skeletal muscle fibers. This approach has recently demonstrated that membrane-delimited signaling involving cross-talk between inositol (1,4,5)-triphosphate receptor (IP3R) and RyR contributes to Ca2+ spark activation in skeletal muscle. In summary, our studies using osmotic stress induced Ca2+ sparks showed that this intracellular response reflects a muscle signaling mechanism in physiology and aging/disease states, including mouse models of muscle dystrophy (mdx mice) or amyotrophic lateral sclerosis (ALS model).

Described here is a method to directly measure calcium sparks, the elementary units of Ca2+ release from sarcoplasmic reticulum&#160;in intact skeletal muscle fibers. This method utilizes osmotic-stress-mediated triggering of Ca2+ release from ryanodine receptor in isolated muscle fibers. The dynamics and homeostatic capacity of intracellular Ca2+ signaling can be employed to assess muscle function in health and disease.

Maintaining homeostatic Ca2+ signaling is a fundamental physiological process in living cells. Ca2+ sparks are the elementary units of Ca2+ signaling in ...

Design and Analysis of Temperature Preference Behavior and its Circadian Rhythm in Drosophila

The circadian clock regulates many aspects of life, including sleep, locomotor activity, and body temperature (BTR) rhythms1,2. We recently identified a novel Drosophila circadian output, called the temperature preference rhythm (TPR), in which the preferred temperature in flies rises during the day and falls during the night 3. Surprisingly, the TPR and locomotor activity are controlled through distinct circadian neurons3. Drosophila locomotor activity is a well&#160;known circadian behavioral output and has provided strong contributions to the discovery of many conserved mammalian circadian clock genes and mechanisms4. Therefore, understanding TPR will lead to the identification of hitherto unknown molecular and cellular circadian mechanisms. Here, we describe how to perform and analyze the TPR assay. This technique not only allows for dissecting the molecular and neural mechanisms of TPR, but also provides new insights into the fundamental mechanisms of the brain functions that integrate different environmental signals and regulate animal behaviors. Furthermore, our recently published data suggest that the fly TPR shares features with the mammalian BTR3. Drosophila are ectotherms, in which the body temperature is typically behaviorally regulated. Therefore, TPR is a strategy used to generate a rhythmic body temperature in these flies5-8. We believe that further exploration of Drosophila TPR will facilitate the characterization of the mechanisms underlying body temperature control in animals.

Design and Analysis of Temperature Preference Behavior and its Circadian Rhythm in Drosophila

We recently identified a novel Drosophila circadian output, temperature preference rhythm (TPR), in which the preferred temperature in flies rises during the day and falls during the night. TPR is regulated independently from another circadian output, locomotor activity. Here&#160;we describe the design and analysis of TPR in Drosophila.

The circadian clock regulates many aspects of life, including sleep, locomotor activity, and body temperature (BTR) rhythms1,2. We recently identified a ...

Exploring Adipose Tissue Structure by Methylsalicylate Clearing and 3D Imaging

Obesity is a major worldwide public health issue that increases the risk to develop cardiovascular diseases, type-2 diabetes, and liver diseases. Obesity is characterized by an increase in adipose tissue (AT) mass due to adipocyte hyperplasia and/or hypertrophia, leading to profound remodeling of its three-dimensional structure. Indeed, the maximal capacity of AT to expand during obesity is pivotal to the development of obesity-associated pathologies. This AT expansion is an important homeostatic mechanism to enable adaptation to an excess of energy intake and to avoid deleterious lipid spillover to other metabolic organs, such as muscle and liver. Therefore, understanding the structural remodeling that leads to the failure of AT expansion is a fundamental question with high clinical applicability. In this article, we describe a simple and fast clearing method that is routinely used in our laboratory to explore the morphology of mouse and human white adipose tissue by fluorescent imaging. This optimized AT clearing method is easily performed in any standard laboratory equipped with a chemical hood, a temperature-controlled orbital shaker and a fluorescent microscope. Moreover, the chemical compounds used are readily available. Importantly, this method allows one to resolve the 3D AT structure by staining various markers to specifically visualize the adipocytes, the neuronal and vascular networks, and the innate and adaptive immune cells distribution.

Here, we describe a simple, inexpensive and fast clearing method to resolve the 3D structure of both mouse and human white adipose tissue using a combination of markers to visualize vasculature, nuclei, immune cells, neurons, and lipid-droplet coat proteins by fluorescent imaging.

Obesity is a major worldwide public health issue that increases the risk to develop cardiovascular diseases, type-2 diabetes, and liver diseases. Obesity ...

Divergence of Root Microbiota in Different Habitats based on Weighted Correlation Networks

The root microbiome plays an important role in plant growth and environmental adaptation. Network analysis is an important tool for studying communities, which can effectively explore the interaction relationship or co-occurrence model of different microbial species in different environments. The purpose of this manuscript is to provide details on how to use the weighted correlation network algorithm to analyze different co-occurrence networks that may occur in microbial communities due to different ecological environments. All analysis of the experiment is performed in the WGCNA package. WGCNA is an R package for weighted correlation network analysis. The experimental data used to demonstrate these methods were microbial community data from the NCBI (National Center for Biotechnology Information) database for three niches of the rice (Oryza sativa) root system. We used the weighted correlation network algorithm to construct co-abundance networks of microbial community in each of the three niches. Then, differential co-abundance networks among endosphere, rhizoplane and rhizosphere soil were identified. In addition, the core genera in network were obtained by the "WGCNA" package, which plays an important regulated role in network functions. These methods enable researchers to analyze the response of microbial network to environmental disturbance and verify different microbial ecological response theories. The results of these methods show that the significant differential microbial networks identified in the endosphere, rhizoplane and rhizosphere soil of rice.

Network analysis was applied to evaluate the association of various ecological microbial communities, such as soil, water and rhizosphere. Presented here is a protocol on how to use the WGCNA algorithm to analyze different co-occurrence networks that may occur in the microbial communities due to different ecological environments.

The root microbiome plays an important role in plant growth and environmental adaptation. Network analysis is an important tool for studying communities, ...

Exploring m6A and m5C Epitranscriptomes upon Viral Infection: an Example with HIV

The role of RNA modifications in biological processes has been the focus of an increasing number of studies in the last few years and is known nowadays as epitranscriptomics. Among others, N6-methyladenosine (m6A) and 5-methylcytosine (m5C) RNA modifications have been described on mRNA molecules and may have a role in modulating cellular processes. Epitranscriptomics is thus a new layer of regulation that must be considered in addition to transcriptomic analyses, as it can also be altered or modulated by exposure to any chemical or biological agent, including viral infections.
Here, we present a workflow that allows analysis of the joint cellular and viral epitranscriptomic landscape of the m6A and m5C marks simultaneously, in cells infected or not with the human immunodeficiency virus (HIV). Upon mRNA isolation and fragmentation from HIV- infected and non-infected cells, we used two different procedures: MeRIP-Seq, an RNA immunoprecipitation-based technique, to enrich for RNA fragments containing the m6A mark and BS-Seq, a bisulfite conversion-based technique, to identify the m5C mark at a single nucleotide resolution. Upon methylation-specific capture, RNA libraries are prepared for high-throughput sequencing. We also developed a dedicated bioinformatics pipeline to identify differentially methylated (DM) transcripts independently from their basal expression profile.
Overall, the methodology allows exploration of multiple epitranscriptomic marks simultaneously and provides an atlas of DM transcripts upon viral infection or any other cell perturbation. This approach offers new opportunities to identify novel players and novel mechanisms of cell response, such as cellular factors promoting or restricting viral replication.

Exploring m6A and m5C Epitranscriptomes upon Viral Infection: an Example with HIV

The role of RNA modifications in viral infections is just starting to be explored and could highlight new viral-host interaction mechanisms. In this work, we provide a pipeline to investigate m6A and m5C RNA modifications in the context of viral infections.

The role of RNA modifications in biological processes has been the focus of an increasing number of studies in the last few years and is known nowadays as ...

Application of Passive Head Motion to Generate Defined Accelerations at the Heads of Rodents

Exercise is widely recognized as effective for various diseases and physical disorders, including those related to brain dysfunction. However, molecular mechanisms behind the beneficial effects of exercise are poorly understood. Many physical workouts, particularly those classified as aerobic exercises such as jogging and walking, produce impulsive forces at the time of foot contact with the ground. Therefore, it was speculated that mechanical impact might be implicated in how exercise contributes to organismal homeostasis. For testing this hypothesis on the brain, a custom-designed ''passive head motion'' (hereafter referred to as PHM) system was developed that can generate vertical accelerations with controlled and defined magnitudes and modes and reproduce mechanical stimulation that might be applied to the heads of rodents during treadmill running at moderate velocities, a typical intervention to test the effects of exercise in animals. By using this system, it was demonstrated that PHM recapitulates the serotonin (5-hydroxytryptamine, hereafter referred to as 5-HT) receptor subtype 2A (5-HT2A) signaling in the prefrontal cortex (PFC) neurons of mice. This work provides detailed protocols for applying PHM and measuring its resultant mechanical accelerations at rodents' heads.

The present protocol describes a custom-designed ''passive head motion'' system, which reproduces mechanical accelerations at rodents' heads generated during their treadmill running at moderate velocities. It allows dissecting mechanical factors/elements from the beneficial effects of physical exercise.

Exercise is widely recognized as effective for various diseases and physical disorders, including those related to brain dysfunction. However, molecular ...