Myostatin Research Articles

The impact of myostatin variants on growth traits in South African Bonsmara beef cattle

Double muscling occurs when the myostatin (MSTN) gene is deactivated due to a series of mutations, leading to uncontrolled muscle growth and excessive muscle fiber accumulation, as the gene can no longer effectively regulate muscle development. This study aimed to assess the impact of MSTN variants and their combinations on growth traits, namely direct birth weight (BWDIR), direct weaning weight (WWDIR), average daily gain (ADG) and feed conversion ratio (FCR) in the South African (SA) Bonsmara. Genomically enhanced estimated breeding value (GEBVs) for traits of interest, and MTSN genotypes for SA Bonsmara animals were available for the study. Thirteen MSTN variants (Nt821, Q204X, F94L, E226X, E291X, C313Y, Nt419, S105C, D182N, Nt414, Nt324, Nt267, and Nt748) were routinely genotyped using the IDBv3 SNP array. Genotypic frequencies of MSTN variants ranged from 1.18% for Q204X to 35.02% for Nt748. No association was observed between the Nt267 variant and any growth traits, while both Nt748 and Nt414 variants affected WWDIR, ADG and FCR (p < 0.05). The results of the effect of multiple variants on growth traits indicated that there was an additive effect when more than one MSTN variant was present in an individual. This study is the first study to report the impact of MSTN variants on traits of economic importance in the SA Bonsmara breed.

The combined influences of local heat application and resistance exercise on the acute mRNA response of skeletal muscle

IntroductionThe development and maintenance of the skeletal muscle is crucial for the support of daily function. Heat, when applied locally, has shown substantial promise in the maintenance of the muscle. The purpose of this study was to determine the combined effects of local heat application and acute resistance exercise on gene expression associated with the human muscle growth program.Materials and methodsParticipants (n = 12, 26 ± 7 years, 1.77 ± 0.07 m, 79.6 ± 15.4 kg, and 16.1 ± 11.6 %BF) completed an acute bilateral bout of resistance exercise consisting of leg press (11 ± 2 reps; 170 ± 37 kg) and leg extension (11 ± 1 reps; 58 ± 18 kg). Participants wore a thermal wrap containing circulating fluid (40°C, exercise + heat; EX + HT) during the entire experimental period and 4 h post-exercise, while the other leg served as an exercise-only (EX) control. Biopsies of the vastus lateralis were collected (Pre, Post, and 4hPost) for gene expression analyses.ResultsIntramuscular temperatures increased (Post, +2.2°C ± 0.7°C, and p &amp;lt; 0.001; 4hPost, +2.5°C ± 0.6°C, and p &amp;lt; 0.001) and were greater in the EX + HT leg post-exercise (+0.35°C ± 0.3°C, and p = 0.005) and after 4hPost (+2.1°C ± 0.8°C and p &amp;lt; 0.001). MYO-D1 mRNA was greater in the EX + HT leg vs. the EX (fold change = 2.74 ± 0.42 vs. 1.70 ± 0.28, p = 0.037). No other genes demonstrated temperature sensitivity when comparing both legs (p &amp;gt; 0.05). mRNA associated with the negative regulator, myostatin (MSTN), decreased post-exercise (p = 0.001) and after 4 h (p = 0.001). mRNA associated with proteolysis decreased post-exercise (FBXO32, p = 0.001; FOXO3a, p = 0.001) and after 4 h (FBXO32, p = 0.001; FOXO3a, p = 0.027).ConclusionThe elevated transcription of the myogenic differentiation factor 1 (MYO-D1) after exercise in the heated condition may provide a mechanism by which muscle growth could be enhanced.

Expression of Genes Related to Meat Productivity, Metabolic and Morphological Significance of Broiler Chickens with the Use of Nutritional Phytochemicals.

The study aimed to analyze gene expression linked to skeletal muscle growth and lipid metabolism in broiler chickens fed with plant extracts. Five groups of chickens were formed: four experimental groups and one control group. The diets of the experimental groups were supplemented with different plant extracts: chicory, St. John's wort, maral root, and creeping thyme, whereas the control group received feed without phytobiotic compounds. Weekly weighings were conducted (n = 36). The chickens were slaughtered at day 26 for tissue sampling of four birds from each group. Gene expression (MYOG, MSTN, FASN) related to muscle growth and fatty acid synthesis was analyzed using the β-actin ACTB gene as a reference. Blood samples were taken at day 35 for biochemical analysis and anatomical dissection was performed. The study revealed that using plant extracts from chicory, thyme, and maral root increased MYOG gene activity by 4.21, 7.45, and 8.93 times, respectively. T. serpyllum extract boosted the MSTN gene by 10.93 times, impacting muscle growth regulation. FASN gene expression for fatty acid synthesis increased significantly by 18.22-184.12 times with plant extracts. The best results regarding meat productivity of chickens were obtained when using R. carthamoides extract. The results of the study will serve as a basis for further development of a phytocomposition designed to increase the meat productivity of broiler chickens in the production of environmentally safe poultry products.

A MSTNDel73C mutation with FGF5 knockout sheep by CRISPR/Cas9 promotes skeletal muscle myofiber hyperplasia.

Mutations in the well-known Myostatin (MSTN) produce a 'double-muscle' phenotype, which makes it commercially invaluable for improving livestock meat production and providing high-quality protein for humans. However, mutations at different loci of the MSTN often produce a variety of different phenotypes. In the current study, we increased the delivery ratio of Cas9 mRNA to sgRNA from the traditional 1:2 to 1:10, which improves the efficiency of the homozygous mutation of biallelic gene. Here, a MSTNDel73C mutation with FGF5 knockout sheep, in which the MSTN and FGF5 dual-gene biallelic homozygous mutations were produced via the deletion of 3-base pairs of AGC in the third exon of MSTN, resulting in cysteine-depleted at amino acid position 73, and the FGF5 double allele mutation led to inactivation of FGF5 gene. The MSTNDel73C mutation with FGF5 knockout sheep highlights a dominant 'double-muscle' phenotype, which can be stably inherited. Both F0 and F1 generation mutants highlight the excellent trait of high-yield meat with a smaller cross-sectional area and higher number of muscle fibers per unit area. Mechanistically, the MSTNDel73C mutation with FGF5 knockout mediated the activation of FOSL1 via the MEK-ERK-FOSL1 axis. The activated FOSL1 promotes skeletal muscle satellite cell proliferation and inhibits myogenic differentiation by inhibiting the expression of MyoD1, and resulting in smaller myotubes. In addition, activated ERK1/2 may inhibit the secondary fusion of myotubes by Ca2+-dependent CaMKII activation pathway, leading to myoblasts fusion to form smaller myotubes.

Vitamin D combined with whole-body vibration training for the treatment of osteo-sarcopenia: study protocol for a randomized controlled trial.

Osteo-sarcopenia (OS) has become a global public health problem and a frontier research problem, as a combination of sarcopenia (SP) and osteoporosis (OP) diseases. The clinical performances include muscle weakness, systemic bone pain, standing difficulty, even falls and fractures, etc., which seriously affect the patient's life and work. The pathological mechanism of the OS may be the abnormal metabolism which disrupts the equilibrium stability of the musculoskeletal system. Therefore, this study combined vitamin D (Vit. D) and whole-body vibration training (WBVT) to intervene in subjects of OS, aiming to evaluate the effectiveness and safety of the diagnosis and treatment protocol and to explore the efficacy mechanism. We propose a multicenter, parallel-group clinical trial to evaluate the efficacy and safety of Vit. D combined with WBVT intervention in OS. Subjects who met the inclusion or exclusion criteria and signed the informed consent form would be randomly assigned to the WBVT group, Vit. D group, or WBVT+ Vit. D group. All subjects will be treated for 1 month and followed up after 3 and 6 months. The primary outcomes are lumbar bone mineral density (BMD) and appendicular skeletal muscle mass (ASM) measured by dual-energy X-ray absorptiometry (DXA) and handgrip strength measured by grip strength meter. Secondary outcomes include serum markers of myostatin (MSTN), irisin and bone turnover markers (BTM), SARC-CalF questionnaire, 1-min test question of osteoporosis risk, patient health status (evaluated by the SF-36 health survey), physical performance measurement that includes 5-time chair stand test, 6-m walk, and the short physical performance battery (SPPB). If Vit. D combined with WBVT can well relieve OS symptoms without adverse effects, this protocol may be a new treatment strategy for OS. After therapeutic intervention, if the serum marker MSTN/irisin is significant, both have the potential to become sensitive indicators for screening OS effective drugs and treatments, which also indicates that WBVT combined with Vit. D plays a role in improving OS by regulating MSTN/irisin. Chinese Clinical Trial Registry ChiCTR2400082269 . Registered on March 26, 2024.

Synergistic Effects of Korean Mistletoe and Apple Peel Extracts on Muscle Strength and Endurance.

Muscular strength and endurance are vital for physical fitness. While mistletoe extract has shown efficacy in significantly increasing muscle strength and endurance, its accessibility is limited. This study explores combining mistletoe and apple peel extracts as an effective muscle health supplement. Analyses of histology, RNA, and protein in the combined extract-treated mouse group demonstrated significant enhancements in muscle strength and endurance, evidenced by larger muscle fibers, improved mitochondrial function, and a higher ratio of type I and IIa muscle fibers. Combining half doses of each extract resulted in greater improvements than using each extract separately, indicating a synergistic effect. Pathway analysis suggests that the observed synergy arises from complementary mechanisms, with a mistletoe extract-induced decrease in myostatin (MSTN) and an apple peel extract-induced increase in IGF1, leading to a sharp rise in AKT, S6K, and MuRF1, which promote myogenesis, along with a significant increase in PGC-1α, TFAM, and MEF2C, which are critical for mitochondrial biogenesis. This research provides practical insights into developing cost-effective, natural supplements to enhance muscle performance and endurance, with potential applications in athletic performance, improving muscle growth and endurance in children, and addressing age-related muscle decline.

PSVII-4 A novel deletion in exon 3 of the myostatin (MSTN) gene is identified in a Raramuri criollo cattle herd

Abstract Several polymorphisms in the coding region of the myostatin (MSTN) gene have been associated with the double-muscled (DM) phenotype in different species. An experimental herd of purebred Raramuri criollo cattle in Chihuahua, Mexico, has been selectively managed for beef production during the last 15 yr. Recently, two calves where visually identified as DM animals. The aim of this study was to identify changes in the MSTN gene sequence, associated with the two DM individuals and to screen for carriers of the potential polymorphisms within this herd. The DM paternity of the calves was verified by the SeekSire bovine parentage testing of NEOGEN (Lincoln, NE). A 6.6 Kb fragment of the MSTN gene were amplified by PCR and sequenced using Illumina DNA prep kit in a MiniSeq platform. The resulting reads were analyzed using different bioinformatic programs. SNPs were generated in VCF files and filtered by the following criteria: variant confidence normalized by depth of variant samples (QD) &amp;lt; 2.0, mapping quality (MQ) &amp;lt; 40.0, strand bias(FS) &amp;gt; 60.0. The deduced myostatin protein and the three-dimensional model were obtained with bioinformatic tools. Once a novel variant was identified, a real-time PCR protocol was developed to genotype 100 and 97 herdmates, the allelic and genotypic frequencies were estimated. A 9-bp deletion was identified at the same position as the 11-bp deletion (g.821to 831del11) of the Belgian Blue MSTN allele (Figure 1). Although the latter allele has been found in three Spanish cattle breeds ancestrally related to Raramuri criollo, this is the first report of the 9-bp deletion in the MSTN gene. Such deletion results in the loss of three highly conserved amino acids residues (274, 275 and 276) in MSTN, but does not lead to a premature stop codon as in the case of the 11-bp deletion characteristic to DM Belgian Blue. The in silico three-dimensional MSTN conformation obtained for this allele has some structural differences compared with the protein reference model. The allelic frequencies were 0.85 and 0.15 for the MSTN+ and MSTNdel alleles, respectively, whereas the genotypic frequencies were 0.71, 0.01 and 0.28 for MSTN+/MSTN+, MSTNdel/MSTNdel MSTN+/MSTNdel genotypes, respectively. Controlled mattings to generate different genotypes in the research herd would allow to continue studying the effects of this MSTN gene variant on productive performance, forage behavior, husbandry and carcass and meat quality traits known to be affected by the DM phenotype in beef cattle.

Dietary Supplementation with Nano-Curcumin Improves the Meat Quality and Nutrition Value of Largemouth Bass (Micropterus salmoides) Fed with a High-Carbohydrate Diet

This study investigated the effects of curcumin nanoparticles on the flesh quality of largemouth bass on a high-carbohydrate diet. A total of 180 fish (11.01 ± 0.02 g) were fed three semi-purified diets: the Control group (LC) were supplemented with standard carbohydrate (10%), the experimental group (HC) were supplemented with 15% carbohydrate, and the experimental group were supplemented with 0.2% nano-curcumin added to 15% carbohydrate (HCN) for 6 weeks. Results showed that a* value, taurine, valine, isoleucine, histidine, cystine, fatty acids (C17:0 and C20:2n6), MDA (malondialdehyde) content, and SOD (Superoxide dismutase) activity were significantly elevated (p &lt; 0.05) in HC. Muscle fibers showed significant increases in horizontal diameter, longitudinal diameter, and cross-sectional area (p &lt; 0.05), as well as up-regulated expression levels of the MRFS (myogenic regulatory factor) family gene and MSTN (myostatin) (p &lt; 0.05), and a significant decrease in C16:1n7 in HC (p &lt; 0.05). Importantly, the HCN group enhanced the muscle quality of largemouth bass by elevating the L* value, valine, isoleucine, arginine and cystine, C20:2n6, decreasing (p &lt; 0.05) refrigeration loss, chewability, firmness and hardness, then MDA content and SOD activity, and downregulating (p &lt; 0.05) MSTN and MRFS family gene expression levels to improve largemouth bass muscle quality.

Exploring the Impact of Astaxanthin Supplementation in Conjunction with a 12-Week CrossFit Training Regimen on Selected Adipo-Myokines Levels in Obese Males.

Obesity is associated with an exacerbated metabolic condition that is mediated through impairing balance in the secretion of some adipo-myokines. Therefore, the objective of the present study was to explore the impact of astaxanthin supplementation in conjunction with a 12-week CrossFit training regimen on some selected adipo-myokines, insulin insensitivity, and serum lipid levels in obese males. This study is a randomized control trial design; 60 obese males were randomly divided into four groups of 15, including the control group (CG), supplement group (SG), training group (TG), and combined training and supplement group (TSG). The participants were subjected to 12 weeks of astaxanthin (AST) supplementation [20 mg/d capsule, once/d] or CrossFit training or a combination of both interventions. The training regimen comprised 36 sessions of CrossFit, each lasting 60 min, conducted three times per week. The metabolic indices, body composition, anthropometrical, cardio-respiratory, and also some plasma adipo-myokine factors, including decorin (DCN), activin A, myostatin (MST), transforming growth factor (TGF)-β1, and follistatin (FST), were examined 12 and 72 h before the initiation of the main interventional protocols, and then 72 h after the final session of the training protocol. There was no significant difference in the baseline data between the groups (p > 0.05). There were significant interactions between group x time for DCN (η2 = 0.82), activin A (η2 = 0.50), FST (η2 = 0.92), MST (η2 = 0.75), and TGFB-1 (η2 = 0.67) (p < 0.001 for all the variables). Significantly changes showed for DCN in TSG compared to TG and SG and also TG compared to SG (p = 0.0001); for activin A in SG compared to TG (p = 0.01) and TSG (p = 0.002); for FST in SG compared to TG and TSG (p = 0.0001), also in TSG compared to TG (p = 0.0001); for MST in SG, TG, and TSG compared to CG (p = 0.0001) and also in TSG compared to SG (p = 0.0001) and TG (p = 0.001); for TGFB-1 in SG, TG, and TSG compared to CG (p = 0.0001) and also TSG compared to SG (p = 0.0001) and TG (p = 0.001). The 12-week CrossFit training concurrent with AST supplementation reduced anthropometric and metabolic factors and also serum lipid levels while producing positive changes in body composition and cardiovascular factors. Increased FST and DCN and reduced activin A, MST, and TGF-β1 were other affirmative responses to both interventions.

APPLICATION OF CRISPR TO INCREASE MEAT YIELD IN CHICKEN

Meat production is one of the major purposes of the poultry industry. Meat is full of protein and accomplishes the protein requirement of the diet. This study was carried out to attempt to use CRISPR-mediated genome editing of chicken to increase meat yield. For this purpose, CRISPR was used to edit the gene sequence and then it was allowed to infect the PGC’s (Primordial germ cells). The successfully edited PGC’s having blocked exon 3 of MSTN gene were then injected into the blood bloodstream of 2.6 days of fertilized egg cells. The eggs were incubated up to hatch. The wild type was taken as control and the Mutated ones as T1. The CRD was used with three replications to find the significance of variation in To and T1. The weight of chicks for T0 (wild type) and T1 (Mutated) was significantly different. The average weight of three replications of each treatment was compared through a bar graph. it was found that the CRISPR-mediated edited chicks were more weighted than that of their wild types. The CRISP-mediated inhibition of MSTN has a significant effect on muscle growth in chickens. The more such genes may be targeted to get more meat yield.

Electroporation Delivery of Cas9 sgRNA Ribonucleoprotein-Mediated Genome Editing in Sheep IVF Zygotes.

The utilization of electroporation for delivering CRISPR/Cas9 system components has enabled efficient gene editing in mammalian zygotes, facilitating the development of genome-edited animals. In this study, our research focused on targeting the ACTG1 and MSTN genes in sheep, revealing a threshold phenomenon in electroporation with a voltage tolerance in sheep in vitro fertilization (IVF) zygotes. Various poring voltages near 40 V and pulse durations were examined for electroporating sheep zygotes. The study concluded that stronger electric fields required shorter pulse durations to achieve the optimal conditions for high gene mutation rates and reasonable blastocyst development. This investigation also assessed the quality of Cas9/sgRNA ribonucleoprotein complexes (Cas9 RNPs) and their influence on genome editing efficiency in sheep early embryos. It was highlighted that pre-complexation of Cas9 proteins with single-guide RNA (sgRNA) before electroporation was essential for achieving a high mutation rate. The use of suitable electroporation parameters for sheep IVF zygotes led to significantly high mutation rates and heterozygote ratios. By delivering Cas9 RNPs and single-stranded oligodeoxynucleotides (ssODNs) to zygotes through electroporation, targeting the MSTN (Myostatin) gene, a knock-in efficiency of 26% was achieved. The successful generation of MSTN-modified lambs was demonstrated by delivering Cas9 RNPs into IVF zygotes via electroporation.

Establishment of CRISPR-Cas9 ribonucleoprotein mediated MSTN gene edited pregnancy in buffalo: Compare cells transfection and zygotes electroporation

Genome editing is recognized as a powerful tool in agriculture and research, enhancing our understanding of genetic function, diseases, and productivity. However, its progress in buffaloes has lagged behind other mammals due to several challenges, including long gestational periods, single pregnancies, and high raising costs. In this study, we aimed to generate MSTN-edited buffaloes, known for their distinctive double-muscling phenotype, as a proof of concept. To meet our goal, we used somatic cell nuclear transfer (SCNT) and zygotic electroporation (CRISPR-EP) technique. For this, we firstly identified the best transfection method for introduction of RNP complex into fibroblast which was further used for SCNT. For this, we compared the transfection, cleavage efficiency and cell viability of nucleofection and lipofection in adult fibroblasts. The cleavage, transfection efficiency and cell viability of nucleofection group was found to be significantly (P ≤ 0.05) higher than lipofection group. Four MSTN edited colony were generated using nucleofection, out of which three colonies was found to be biallelic and one was monoallelic. Further, we compared the efficacy, embryonic developmental potential and subsequent pregnancy outcome of SCNT and zygotic electroporation. The blastocyst rate of electroporated group was found to be significantly (P ≤ 0.05) higher than SCNT group. However, the zygotic electroporation group resulted into two pregnancies which were confirmed to be MSTN edited. Since, the zygotic electroporation does not require complex micromanipulation techniques associated with SCNT, it has potential for facilitating the genetic modification in large livestock such as buffaloes. The present study lays the basis for inducing genetic alternation with practical or biological significance.

Increased nanosphere size in the cuticle layer of Japanese quail egg by mutation in the myostatin gene

Cuticle quality can affect food safety by protecting poultry eggs from bacterial infection in the modern poultry industry. However, genetic factors related to cuticle nanostructure are not much reported due to limited bird models. In the current study, the genome-edited quail targeting myostatin (MSTN) gene was used to investigate the effect of MSTN mutation on the cuticle nanostructure and quality. To analyze nanostructure of the cuticle layer of the MSTN mutant and wild-type (WT) quail eggs, scanning electron microscope (SEM) images was taken. Thickness of the cuticle layer did not differ between the MSTN mutant and WT groups, but the size of the nanospheres in the surface of the cuticle layer was increased by MSTN mutation. In addition, increased size of the nanospheres in the MSTN mutant group was also shown in the upper region of the cross-sectional cuticle layer. Notably, both groups showed similar small-sized nanospheres in the lower region of the cuticle layer and the size was increased as they ascended to the upper region. The data suggested that MSTN mutation increased the size of the nanosphere in the upper region of the cuticle layer at a late phase rather than increasing the size of nanospheres in the lower region of the cuticle layer at an early phase of cuticle formation. However, the number of Escherichia coli attached to the surface did not differ between the two groups indicating no association between nanosphere size and bacterial attachment in quail eggs. The current study demonstrated a new function of the MSTN gene on regulation of cuticle nanostructure, for the first time. These results advanced our knowledge on the association between genetic factors and cuticle nanostructure and can be served as a reference to study the mechanism of cuticle formation in the future study.

Myocyte enhancer factor 2 upregulates expression of myostatin promoter in Yesso scallop, Patinopecten yessoensis

Myostatin (MSTN) plays an important role in muscle development in animals, especially for mammals and fishes. However, little information has been reported on the regulation of MSTN in marine invertebrates, such as bivalves. In the present study, we cloned the MSTN promoter sequence of Yesso scallop Patinopecten yessoensis, identifying 4 transcription start sites, eleven TATA boxes and one E-box. Additionally, transcription factor binding sites, including myocyte enhancer factor 2 (MEF2) and POU homeodomain protein, were identified. The interaction between the MSTN promoter and MEF2 was analyzed to reveal the transcriptional activity of different fragment sizes of promoters through the dual-luciferase reporter assays. The highest transcriptional activity was found in recombinant plasmids with the most MEF2 binding sites, indicating that this transcription factor upregulates MSTN in Yesso scallop. This study provides new insight into the regulation of muscle growth and development in this species.

Assessment of Metabolic, Inflammatory, and Immunological Disorders Using a New Panel of Plasma Parameters in People Living with HIV Undergoing Antiretroviral Therapy-A Retrospective Study.

Background/Objectives: People living with HIV (PLWH) treated with combined antiretroviral therapy (cART) show a greater predisposition to metabolic and inflammatory disturbances compared to the general population. This study aimed to assess the effect of five years of cART use on the level of selected parameters related to carbohydrate and lipid metabolism and inflammation in PLWH compared to the uninfected. Methods: The levels of sirtuins (-1, -3, -6); irisin (IRS); myostatin (MSTN); peptide YY (PYY); glucagon-like peptide-1 (GLP-1); dipeptidyl peptidase IV (DPP-4); fetuin-A (FETU-A); pentraxin 3 (PTX3); chemokine stromal cell-derived factor 1 (SDF-1); regulated on activation, normal T cell expressed and presumably secreted (RANTES); and interleukins (-4, -7, -15) in the plasma of PLWH and a control group were evaluated by immunoassay methods. The results obtained after five years of antiretroviral therapy were compared with the levels obtained before and one year after cART. Results: Analysis of the parameters after five years of cART showed significantly higher levels in PLWH compared to the control group for SIRT-6, IRS, and IL-4 and significantly lower levels for RANTES and IL-7. There were significantly higher levels of SIRT-6, PYY, GLP-1, and PTX3 obtained after five years of cART compared to the results before therapy and after one year of cART. Conclusions: The results indicated changes occur in the expression of selected parameters during cART use in PLWH. Further research on the clinical usefulness of selected parameters and obtaining new information on the development of HIV-related comorbidities needs to be conducted.

CRISPR-Cas guide RNA indel analysis using CRISPResso2 with Nanopore sequencing data

ObjectiveInsertion and deletion (indel) analysis of CRISPR-Cas guide RNAs (gRNAs) is crucial in gene editing to assess gRNA efficiency and indel frequency. This study evaluates the utility of CRISPResso2 with Oxford Nanopore sequencing data (nCRISPResso2) for gRNA indel screening, compared to two common Sanger sequencing-based methods, TIDE and ICE. To achieve this, sheep and horse fibroblasts were transfected with Cas9 and a gRNA targeting the myostatin (MSTN) gene. DNA was subsequently extracted, and PCR products exceeding 600 bp were sequenced using both Sanger and Nanopore sequencing. Indel profiling was then conducted using TIDE, ICE, and nCRISPResso2.ResultsComparison revealed close correspondence in indel formation among methods. For the sheep MSTN gRNA, indel percentages were 52%, 58%, and 64% for TIDE, ICE, and nCRISPResso2, respectively. Horse MSTN gRNA showed 81%, 87%, and 86% edited amplicons for TIDE, ICE, and nCRISPResso2. The frequency of each type of indel was also comparable among the three methods, with nCRISPResso2 and ICE aligning the closest. nCRISPResso2 offers a viable alternative for CRISPR-Cas gRNA indel screening, especially with large amplicons unsuitable for Illumina sequencing. CRISPResso2’s compatibility with Nanopore data enables cost-effective and efficient indel profiling, yielding results comparable to common Sanger sequencing-based methods.

Dietary vitamin E further enhances the effectiveness of Clostridium autoethanogenum protein as an alternative source of soybean meal in grass carp (Ctenopharygodon idellus) diets based on growth performance, flesh quality and antioxidant status

The effects of replacing soybean meal with Clostridium autoethanogenum protein and supplementing it with different gradients of vitamin E in grass carp diets on growth performance, flesh quality and antioxidant capacity were evaluated in a 69-day feeding trial. Five isonitrogen and isolipid diets were configured with the soybean meal-based diet (SM) served as the basal control diet, and the Clostridium autoethanogenum protein-based diet (CAP) as the treatment control diet. The CAP diet was supplemented with 80 mg/kg, 160 mg/kg and 240 mg/kg vitamin E (namely, 80VE, 160VE and 240VE), respectively. Final body weight (FBW) and specific growth rate (SGR) were significantly better in the CAP group than in the SM group (P < 0.05), and the 160 VE group was significantly better than the CAP group (P < 0.05). In terms of muscle quality, muscle fat content was significantly higher in the CAP group than in the SM group (P < 0.05), and muscle vitamin E content was significantly higher in each vitamin E-supplemented group than in the CAP group (P > 0.05). The total odor activity value in muscle in the CAP group was not significantly different from the SM group (P < 0.05), but the vitamin E-supplemented groups were significantly higher than the CAP group (P < 0.05). Muscle hardness and gumminess were significantly higher in the CAP group than the SM group and muscle shear force and gumminess were significantly higher in the 80VE group than the CAP group (P < 0.05). Muscle development-related genes in the CAP group were not significantly different from those in the SM group (P > 0.05), and Muscle Myf5 mRNA levels in the 160VE and 240VE groups were significantly higher than those in the CAP group, while the expression of MSTN genes was suppressed (P < 0.05). In terms of antioxidant properties, muscle MDA content was significantly higher in the CAP group than in the SM group (P < 0.05), and muscle MDA content was significantly lower in the 160VE and 240VE groups than in the CAP group (P < 0.05), at the same time, CAT and T-SOD activities were significantly higher (P < 0.05). In conclusion, complete replacement of soybean meal by the Clostridium autoethanogenum protein in grass carp diets promoted growth performance and muscle quality, but negatively affected antioxidant capacity, and supplementation of 160 mg/kg vitamin E in the CAP diet could further enhance growth performance and improve antioxidant capacity.

Genetic Variations of MSTN and Callipyge in Tibetan Sheep: Implications for Early Growth Traits.

Tibetan sheep are vital to the ecosystem and livelihood of the Tibetan Plateau; however, traditional breeding methods limit their production and growth. Modern molecular breeding techniques are required to improve these traits. This study identified a single nucleotide polymorphism (SNP) in myostatin (MSTN) and Callipyge in Tibetan sheep. The findings indicated notable associations between MSTN genotypes and growth traits including birth weight (BW), body length (BL), chest width (ChW), and chest circumference (ChC), as well as a particularly strong association with cannon circumference (CaC) at 2 months of age. Conversely, Callipyge polymorphisms did not have a significant impact on Tibetan sheep. Moreover, the analyses revealed a significant association between sex and BW or hip width (HW) at 2 months of age and ChW, ChC, and CaC at 4 months of age. Furthermore, the study's results suggested that the genotype of MSTN as a GA was associated with a notable sex effect on BW, while the genotype of Callipyge (CC) showed a significant impact of sex on CaC at 2 months of age. These results indicated that the SNP of MSTN could potentially serve as a molecular marker for early growth traits in Tibetan sheep.

In ovo betaine injection improves breast muscle growth in newly hatched goslings through FXR/IGF-2 pathway

Betaine has been shown to enhance growth performance and increase breast muscle yield in ducks and broilers through various mechanisms, including the modification of DNA methylation. However, the impact of in ovo betaine injection on muscle growth in newly hatched goslings remains unclear. In this study, fifty eggs were injected with saline or betaine at 7.5 mg/egg prior to incubation, and the subsequent effects on breast muscle growth in the newly hatched goslings were investigated. Betaine significantly increased (P < 0.05) the hatch weight, breast muscle weight, and breast muscle index, accompanied by an augmentation in muscle bundle cross-sectional area. Concurrently, betaine significantly upregulated (P < 0.05) the expression levels of myogenic regulatory factors, including myogenin (MyoG) and paired box 7 (Pax7) both mRNA and protein, while downregulating (P < 0.05) the mRNA and protein levels of myostatin (MSTN). Histological analysis revealed a higher abundance of proliferating cell nuclear antigen (PCNA) and Pax7 immune-positive cells in the breast muscle of the betaine group, consistent with elevated PCNA and Pax7 mRNA and protein levels. Additionally, significantly increased (P < 0.05) contents of insulin-like growth factor 1 (IGF-1) and insulin-like growth factor 2 (IGF-2) were observed in the breast muscle of the betaine group, so was mRNA expression of IGF-1, IGF-2, and insulin-like growth factor 1 receptor (IGF-1R). Betaine also significantly in8creased (P < 0.05) global DNA methylation of the breast muscle, accompanied by enhanced mRNA and protein levels of methionine cycle and DNA methylation-related enzymes, Interestingly, the promoter regions of IGF-1, IGF-2, and IGF-1R genes were significantly hypomethylated (P < 0.05). Moreover, in ovo betaine injection significantly upregulated (P < 0.05) the protein level of farnesoid X receptor (FXR) in breast muscle and FXR binding to the promoter of IGF-2 gene. These findings suggest that in ovo betaine injection promotes breast muscle growth during embryonic development in goslings through the FXR-mediated IGF-2 pathway, ultimately improving hatch weight and breast muscle weight.

Expression and secretion of SPARC, FGF-21 and DCN in bovine muscle cells: Effects of age and differentiation.

Skeletal muscle growth is an economically important trait in the cattle industry. Secreted muscle-derived proteins, referred to as myokines, have important roles in regulating the growth, metabolism, and health of skeletal muscle in human and biomedical research models. Accumulating evidence supports the importance of myokines in skeletal muscle and whole-body health, though little is known about the potential presence and functional significance of these proteins in cattle. This study evaluates and confirms that secreted proteins acidic and rich in cysteine (SPARC), fibroblast growth factor 21 (FGF-21), myostatin (MSTN), and decorin (DCN) are expressed and SPARC, FGF-21, and DCN are secreted by primary bovine satellite cells from 3- (BSC3; n = 3) and 11- (BSC11; n = 3) month -old commercial angus steers. Cells were cultured and collected at zero, 12, 24, and 48 hours to characterize temporal expression and secretion from undifferentiated and differentiated cells. The expression of SPARC was higher in the undifferentiated (p = 0.04) and differentiated (p = 0.07) BSC11 than BSC3. The same was observed with protein secretion from undifferentiated (p <0.0001) BSC11 compared to BSC3. Protein secretion of FGF-21 was higher in undifferentiated BSC11 (p < 0.0001) vs. BSC3. DCN expression was higher in differentiated BSC11 (p = 0.006) vs. BSC3. Comparing undifferentiated vs. differentiated BSC, MSTN expression was higher in differentiated BSC3 (p ≤ 0.001) for 0, 12, and 24 hours and in BSC11 (p ≤ 0.03) for 0, 12, 24, and 48 hours. There is also a change over time for SPARC expression (p ≤ 0.03) in undifferentiated and differentiated BSC and protein secretion (p < 0.0001) in undifferentiated BSC, as well as FGF-21 expression (p = 0.007) in differentiated BSC. This study confirms SPARC, FGF-21, and DCN are secreted, and SPARC, FGF-21, MSTN, and DCN are expressed in primary bovine muscle cells with age and temporal differences.