Novel Polymorphisms in Caprine Myostatin Gene and its Relationship with Growth Traits in Saburai Does (Capra hircus).

<b>Background and Objective:</b> Saburai goat (<i>Capra hircus</i>) is a crossbred goat from Boer buck (75%) and Ettawa doe (25%) for meat production purposes. This study was carried out to detect the mutation points or Single Nucleotide Polymorphisms (SNPs) in the myostatin (<i>MSTN</i>) gene of Saburai with the forward sequencing method. <b>Materials and Methods:</b> A total of twenty-one blood samples of Saburai does (75% Boer, 25% Ettawa) were collected from the Villager Breeder Center (VBC) at Tanggamus Regency of Lampung Province, Indonesia. The DNA analysis consists of DNA isolation, PCR analysis and sequencing analysis. The record data were used for association study with the mathematical model: Y_ij = µ+G_i+Є_ij. <b>Results:</b> Research showed that one common SNP of g.217_218.indel.TTTTA (5'UTR) and three novel SNPs of c.386G>C (exon 1), g.641_642.indel.T (intron 2) and c.4957G>C (exon 3) were detected in the present study. In this study, a novel SNP on exon 1 and intron 2 of Saburai <i>MSTN</i> gene has a moderate PIC value (>0.30). In addition, a novel SNP on exon 1 and exon 3 of the Saburai <i>MSTN</i> gene was detected as a missense mutation of A55P and A43P, respectively. Goats with the heterozygous genotype have higher growth traits compared to goats with the homozygous genotype. <b>Conclusion:</b> The goats with heterozygous genotypes can be further developed to increase the productivity of Saburai goats.

Dual single guide RNAs-mediating deletion of mature myostatin peptide results in concomitant muscle fibre hyperplasia and adipocyte hypotrophy in pigs.

Myostatin (MSTN) is a major gene target for skeletal muscle overgrowth in animals. We hypothesized that deletion of the entire mature peptide encoded by MSTN in pigs would knock out its bioactive form and accordingly stimulate skeletal muscle overgrowth. Thus, we engineered two pairs of single-guide RNAs (sgRNAs) to target exons 1 and 3 of MSTN in primary fetal fibroblasts of Taoyuan black pigs. We found that sgRNAs targeting exon 3, which encodes the mature peptide, had higher biallelic null mutation efficiency than those targeting exon 1. Somatic cell nuclear transfer was conducted using the exon 3 mutation cells as donor cells to generate five cloned MSTN null piglets (MSTN-/-). Growth testing revealed that both the growth rate and average daily weight gain of MST-/- pigs were greater than those of wild-type (MSTN+/+) pigs. Slaughter data demonstrated that the lean ratio of MSTN-/- pigs was 11.3% higher (P < 0.01) while the back-fat thickness was 17.33% lower (P < 0.01) than those of MSTN+/+ pigs. Haematoxylin-eosin staining indicated that the increased leanness of MSTN-/- pigs resulted from muscle fibre hyperplasia rather than hypertrophy.HE staining showed markedly decreased adipocyte size in MSTN-/- pigs. We also critically examined the off-target and random integration by resequencing, which showed that the founder MSTN-/- pigs contained no non-target mutations or exogenous plasmid elements. This study is the first to report the successful knock out of the mature MSTN peptide using dual sgRNA-mediated deletion, leading to the most prominent alteration of meat production traits in pigs published thus far. This new strategy is expected to have a wide impact on genetic improvements in food animals.

Influence of Garlic (Allium sativum) Clove-Based Selenium Nanoparticles on Status of Nutritional, Biochemical, Enzymological, and Gene Expressions in the Freshwater Prawn Macrobrachium rosenbergii (De Man, 1879).

Selenium (Se) is one of the essential micronutrients for performing vital body functions. This study aims at examining the influence of dietary supplementation of garlic clove-based green-synthesized selenium nanoparticles (GBGS-SeNPs, 48-87 nm) on carcass minerals and trace elements, and growth, biochemical, enzymological, and gene expression analyses in the freshwater prawn, Macrobrachium rosenbergii post larvae (PL). The 96 h LC50 of this GBGS-SeNPs to M. rosenbergii PL was 52.23 mg L-1. Five different artificial diets without supplementation of GBGS-SeNPs (control, 0.0 mg kg-1) and with supplementations of GBGS-SeNPs starting from 100 times lower than the LC50 value (0.5, 1.0, 1.5, and 2.0 mg kg-1) were prepared and fed to M. rosenbergii PL for 90 days. A dose-dependent accumulation of Se was observed in the carcass of experimental prawns. GBGS-SeNPs, up to 1.5 mg kg-1 significantly influenced the absorption of other trace elements (Ca, Cu, and Fe) and mineral salts (K, Mg, Na, and Zn). GBGS-SeNPs-supplemented diets showed efficient food conversion ratio (FCR) of 1.32 g against 2.71 g, and therefore enhanced the survival rate (85.6% against 78.8% in control) and weight gain (WG) of 1.41 g against 0.46 g of control prawn. GBGS-SeNPs significantly elevated the activities of protease, amylase, and lipase, and the contents of total protein, essential amino acids (EAA), total carbohydrate, total lipid, monounsaturated fatty acids (MUFA), polyunsaturated fatty acids (PUFA), and ash. These indicate the growth promoting potential of GBGS-SeNPs in prawn. The insignificantly altered activities of glutamic oxaloacetate transaminase (GOT), glutamic pyruvate transaminase (GPT), superoxide dismutase (SOD), and catalase, and the content of malondialdehyde (MDA) up to 1.5 mg kg-1 suggest its acceptability in prawn. Moreover, a respective down- and upregulated myostatin (MSTN) and crustacean hyperglycemic hormone (CHH) genes confirmed the influence of GBGS-SeNPs on the growth of prawn. In contrast, 2.0 mg kg-1 GBGS-SeNPs supplementation starts to produce negative effects on prawn (FCR, 1.76 g; survival rate, 82.2%; WG, 0.84 g against respective values of 1.32 g, 85.6%; and 1.41 g observed in 1.5 mg kg-1 of GBGS-SeNPs-supplemented diet fed prawn). This study recommends a maximum of 1.5 mg kg-1 GBGS-SeNPs as dietary supplement to attain sustainable growth of M. rosenbergii. This was confirmed through polynomial and linear regression analyses.

Effect of myostatin gene mutation on erythrocyte osmotic fragility, hematological parameters and fatty acid composition of serum and erythrocyte membranes in piglets.

Fatty acid composition of serum and erythrocyte membrane, erythrocyte osmotic fragility and hematological parameters were estimated with the objective of determining effects of the gene mutation in one-week-old MSTN homozygous mutant (KO, MSTN-/-), heterozygous mutant (MSTN-/+) and wild type (WT, MSTN+/+) piglets (n = 4 each). Erythrocyte osmotic fragility, complete blood count (CBC), and fatty acid composition of serum and erythrocyte membrane were determined by flow cytometric analysis, automated hematology analyzer system, and liquid chromatography, respectively. Mean of median corpuscular fragility (MCF) was lower (P < 0.05, 0.001) in KO than MSTN-/+ and WT piglets. KO piglets had decreased (P < 0.05) white blood cell (WBC) count, lymphocyte (LYM) count, platelet (PLT) count, mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), red cell distribution width-standard deviation (RDW-SD), red cell distribution width-coefficient volume (RDW-CV), plateletcrit (PCT), mean platelet volume (MPV), platelet distribution width (PDW), platelet-large cell ratio (P-LCR), and an increased red blood cell (RBC) count when compared with MSTN-/+ and WT piglets. The ratios of unsaturated fatty acid (UFA) to saturated fatty acid (SFA) concentrations in serum and erythrocyte membranes of MSTN KO piglets were 2-fold and 4-fold higher compared to WT piglets (P < 0.001), respectively. In conclusion, MSTN KO piglets had a decreased erythrocyte osmotic fragility, and altered hematological profile and fatty acid composition of serum and erythrocyte membranes, as characteristic phenotype.

Genetic polymorphism of myostatin gene in Sumba Ongole (Bos indicus) cattle and its association with growth traits.

Objective: As one of the most valuable genetic resources of Ongole beef cattle globally, the Sumba Ongole (SO) cattle population is being studied in this investigation of myostatin (MSTN) gene polymorphism and its association with growth traits. Materials and Methods: Blood samples from 161 SO cattle were collected and analyzed. Deoxyribonucleic acid (DNA) was isolated. The DNA was electrophoresed and extracted, and finally, the annealing temperature was optimized, followed by amplification and sequencing. Next, we used a Basic local alignment search tool to assess the sequencing data. Results: The analysis revealed 22 single nucleotide polymorphisms (SNPs) in the MSTN gene in this region that showed genetic variation. Two SNPs, c.424 G > A, and c.467 G > C, were found to be significantly associated with SO cattle phenotypes of wither height, heart girth, and hip height (p < 0.05) but not with body weight or body length (p > 0.05). Conclusion: As a result of our findings, the MSTN gene polymorphism and its correlation with growth traits in SO cattle may be employed as a candidate marker in SO cattle and other beef cattle breeds.

Genetic Variations and Haplotypic Diversity in the Myostatin Gene of Different Cattle Breeds in Russia.

The myostatin gene (MSTN) in cattle has a number of polymorphisms associated with increased muscle mass. The aim of the current study was to determine the haplotype frequencies of F94L and nt821(del11) MSTN polymorphisms among cattle bred for meat in Russia, using DNA analysis. Using the earlier created test systems based on the AS-PCR and PCR-RFLP methods, six populations of Aberdeen Angus (n = 684), two populations of Limousin (n = 54), one population of Simmental (n = 55), and one population of Belgian Blue (n = 137) belonging to Russian farms were genotyped on nt821(del11) and F94LMSTN polymorphisms. The animal carriers of the mutant allele of nt821(del11)MSTN associated with the double-muscling genetic defect were found in one Aberdeen Angus population at a frequency of 2.18%, but were not found in the Limousin and Simmental populations. However, 100% of the Belgian Blue population were heterozygous carriers of nt821(del11)MSTN. The frequencies of the A allele F94LMSTN desirable for productivity traits in the Limousin populations were the highest and accounted for 0.97 and 1 in populations one and two, while in the Aberdeen Angus, Simmental, and Belgian Blue populations, these figures were considerably lower at 0.04-0.08, depending on the population. The obtained data show the high genetic potential of Russian beef cattle, and facilitate an improvement in meat productivity by preserving the health of animals.

Genetic variation in the first intron and exon of the myostatin gene in several Indonesian cattle populations.

BACKGROUND AND AIM: Myostatin (MSTN), a member of the transforming growth factor-b family, is a negative regulator of muscle mass. This study aimed to detect the genetic variation of the 1160 bp fragment of exon 1 and part of intron 1 of the MSTN gene in several cattle populations raised in Indonesia. MATERIALS AND METHODS: Polymerase chain reaction products of the MSTN gene amplified from 92 animals representing 10 cattle populations (Peranakan Ongole [PO], Belgian Blue x PO cross, Rambon, PO x Bali cross, Jabres, Galekan, Sragen, Donggala, Madura, and Bali) were sequenced, compared, and aligned with bovine MSTN of Bos taurus (GenBank Acc. No. AF320998.1) and Bos indicus (GenBank Acc. No. AY794986.1). RESULTS: Four nucleotide substitutions (nt 1045 and 1066 in intron 1; nt 262 and 418 in exon 1) and two indels (nt 807 and 869 in intron 1) were synonymous mutations. Among these substitutions, only the nt 262G>C and nt 418A>G loci were polymorphic in all populations, except Bali cattle. The frequencies of the nt 262C (0.82) and nt 418A (0.65) alleles were highest. For the nt 262G>C locus, the CC genotype had the highest frequency (0.66) followed by GC (0.30) and CC (0.03). For the nt 418A>G locus, the AG genotype had the highest frequency (0.52) followed by AA (0.39) and GG (0.09). CONCLUSION: The results, showing genetic variations in exon 1 and intron 1 of the MSTN gene, might be helpful for future association studies.

Genetic variations in the Myostatin gene affecting growth traits in sheep.

BACKGROUND AND AIM: Sheep productivity in developing countries is crucial, as this animal is an essential source of meat and wool. Myostatin (MSTN) plays an important role in the regulation of muscle mass through the regulation of muscle growth, differentiation, and regeneration. The present study sought to investigate genetic variation in the first intron of the MSTN gene and the association of variants with growth traits in major sheep breeds in Egypt (Barki, Ossimi, and Rahmani) and Saudi Arabia (Najdi) using polymerase chain reaction (PCR) and sequencing. MATERIALS AND METHODS: Blood samples were collected, and DNA was extracted from 75 animals. A 386 bp fragment in the first intron of the MSTN gene was amplified using PCR. Polymorphic sites were detected using direct sequencing and then correlated with growth traits using a general linear model. RESULTS: Sequence analysis of the first intron of MSTN gene identified six single-nucleotide polymorphisms (SNPs) in the studied breeds. Four mutual SNPs were determined: c.18 G>T, c.241 T>C, c.243 G>A, and c.259 G>T. In addition, two SNPs c.159 A>T and c.173 T>G were monomorphic (AA and TT, respectively) in the Ossimi, Rahmani, and Najdi breeds and polymorphic in the Barki breed. The association analysis revealed that the c.18 G>T and c.241 C>T significantly associated (p<0.05) with birth weight and average daily weight gain, respectively. CONCLUSION: Our results strongly support MSTN as a candidate gene for marker-assisted selection in sheep breeding programs. Furthermore, the identified variants may be considered as putative markers to improve growth traits in sheep.

Associations between the Bovine Myostatin Gene and Milk Fatty Acid Composition in New Zealand Holstein-Friesian × Jersey-Cross Cows.

The myostatin gene (MSTN), which encodes the protein myostatin, is pleiotropic, and its expression has been associated with both increased and decreased adipogenesis and increased skeletal muscle mass in animals. In this study, the polymerase chain reaction, coupled with single strand conformation polymorphism analysis, was utilized to reveal nucleotide sequence variation in bovine MSTN in 410 New Zealand (NZ) Holstein-Friesian × Jersey (HF × J)-cross cows. These cows ranged from 3 to 9 years of age and over the time studied, produced an average 22.53 ± 2.18 L of milk per day, with an average milk fat content of 4.94 ± 0.17% and average milk protein content of 4.03 ± 0.10%. Analysis of a 406-bp amplicon from the intron 1 region, revealed five nucleotide sequence variants (A-E) that contained seven nucleotide substitutions. Using general linear mixed-effect model analyses the AD genotype was associated with reduced C10:0, C12:0, and C12:1 levels when compared to levels in cows with the AA genotype. These associations in NZ HF × J cross cows are novel, and they suggest that this variation in bovine MSTN could be explored for increasing the amount of milk unsaturated fatty acid and decreasing the amount of saturated fatty acid.

The evaluation of RS1805086 and RS1805065 polymorphisms in Mstn gene and anthropometric properties of national and amateur arm wrestlers / Evaluación de los polimorfismos RS1805086 y RS1805065 en el gen Mstn y las propiedades antropométricas de los luchadores de brazo nacionales y amateurs

The aim of this study is to investigate rs1805086 and rs1805065 polymorphisms of MSTN gene of national and amateur Turkish arm wrestlers and people leading a sedentary lifestyle, and the anthropometric properties such as hand, wrist, and forearm circumferences of national and amateur Turkish arm wrestlers are aimed to be explored. In this study, a total of 79 volunteers who were 24 national (7 females, 17 males) Turkish arm wrestlers, 21 amateur (7 females, 14 males) Turkish arm wrestlers and 34 sedentary people (12 females, 22 males) participated. To analyse the data, Statistical Package for the Social Sciences, SPSS 22 (SPSS Inc., Chicago, IL, USA) was used. As a result of the study, when data on rs1805086 and rs1805065 polymorphisms of MSTN gene were examined respectively, it was found out that MSTN 153KK genotype was 100.0% dominant in both national (n=24) and amateur (n=21) arm wrestlers, and it was 94.12 % dominant in sedentary people. KR genotype was observed in 5.88 % of the sedentary people. The data from the other rs1805065 polymorphism of MSTN gene showed that all participants (n = 45, 100.0 %) were carriers of normal homozygous genotype. Furthermore, for both female group and male group, there found to be statistically significant difference in terms of anthropometric properties. It can be concluded that though there was no significant difference between national and amateur Turkish arm wrestlers in terms of their MSTN gene characteristics; in terms of anthropometric properties, significant differences were discovered. It was found out that on these athletes, not MSTN gene polymorphisms but anthropometric properties were effective.

El objetivo de este estudio fue investigar los polimorfismos rs1805086 y rs1805065 del gen MSTN de luchadores de brazos turcos, nacionales y aficionados, y personas que llevan un estilo de vida sedentario, y las propiedades antropométricas además de las circunferencias de manos, muñecas y antebrazos de los luchadores de brazos turcos nacionales y aficionados. En este estudio, participaron un total de 79 voluntarios: 24 luchadores de brazos turcos nacionales (7 mujeres, 17 hombres), 21 luchadores de brazos turcos aficionados (7 mujeres, 14 hombres) y 34 personas sedentarias (12 mujeres, 22 hombres). Para analizar los datos, se utilizó el Paquete Estadístico para las Ciencias Sociales, SPSS 22 (SPSS Inc., Chicago, IL, EE. UU.). Como resultado del estudio, cuando se examinaron los datos sobre los polimorfismos rs1805086 y rs1805065 del gen MSTN respectivamente, se descubrió que el genotipo MSTN 153KK era 100,0 % dominante en luchadores de brazos nacionales (n = 24) y aficionados (n = 21) , y era 94,12 % dominante en personas sedentarias. El genotipo KR se observó en el 5,88 % de las personas sedentarias. Los datos del otro polimorfismo rs1805065 del gen MSTN mostraron que todos los participantes (n = 45; 100,0 %) eran portadores del genotipo homocigoto normal. Además, tanto para el grupo femenino como para el masculino, se encontró una diferencia estadísticamente significativa en términos de propiedades antropométricas. Se puede concluir que, aunque no hubo una diferencia significativa entre los luchadores de brazos turcos nacionales y aficionados en términos de sus características genéticas MSTN; en términos de propiedades antropométricas, se descubrieron diferencias significativas. Se descubrió que, en estos atletas, no fueron los polimorfismos del gen MSTN sino las propiedades antropométricas las efectivas.

Effects of Intronic SNPs in the Myostatin Gene on Growth and Carcass Traits in Colored Polish Merino Sheep.

Myostatin acts as a negative regulator of muscle growth; therefore, its role is important with regard to animal growth and meat production. This study was undertaken with the objective to detect polymorphisms in the first intron and c.*1232 position of the MSTN gene and to analyze effects of the detected alleles/genotypes on growth and carcass traits in Colored Polish Merino sheep. In total, 23 traits were analyzed, i.e., seven describing lamb growth and 16 carcass traits. Single nucleotide polymorphisms (SNPs) in the first intron and the c.*1232 position were identified using polymerase chain reaction single-strand conformation polymorphism (PCR-SSCP) and PCR-restriction fragment length polymorphism (PCR-RFLP) methods, respectively. The MIXED procedure of the SAS software package was used to analyze allelic and genotypic effects of the MSTN gene on growth and carcass traits. Polymorphisms were only detected in the first intron of the MSTN gene. All investigated sheep were monomorphic G in the c.*1232 position. The MSTN genotype was found to have significant effect on body weight at 2nd day of life (BW2) and loin and fore shank weights. Significant allelic effects were detected with respect to BW2, scrag, leg, fore, and hind shank weights. These results suggest that polymorphisms in the first intron of the MSTN gene are relevant with respect to several carcass traits and BW2 in Colored Polish Merino sheep.

Novel report on mutation in exon 3 of myostatin (MSTN) gene in Nilagiri sheep: an endangered breed of South India.

The variability in breeding program leads to rapid loss of genetic potential for which National Bureau of Animal Genetic Resources is emphasized to conserve the indigenous breeds. The variation in myostatin (MSTN) gene and its association with growth traits will throw light on its potential use as marker in selection. Hence, the study was conducted to detect polymorphism in exon 3 of MSTN, one of the most important growth regulatory gene and its association with growth in Nilagiri sheep breed. Blood samples were collected from Nilagiri sheep (n = 103) of South India and growth data up to 1 year of age was recorded. Genomic DNA was isolated and amplified for part of MSTN gene; PCR products were genotyped by restriction digestion (MspI) and confirmed by sequencing. Restriction digestion has revealed a single nucleotide polymorphism at locus G5622C in exon 3 which was confirmed by sequencing. The wild-type DNA molecule (MM) cleaved by MspI produced 301-bp and 314-bp fragments and those with mutation (mm) would remain undigested. The genotypic frequencies were MM (0.689) and Mm (0.311) with complete absence of mm genotype; and allelic frequencies were M (0.8445) and m (0.1555). The locus was in Hardy-Weinberg equilibrium. The association analysis revealed that there was no significant difference in mean birth, weaning, 6-, 9-, and 12-month weight between MM and Mm genotypes at g.5622G>C locus of exon 3 of MSTN gene. This is the first report of mutation in exon 3 of MSTN gene. The non-significant effect and absence of mm genotype at this locus needs further studies based on large population size and haplotype analysis.

Genotypic and allelic effects of the myostatin gene (MSTN) on carcass, meat quality, and biometric traits in Colored Polish Merino sheep.

The aim of this study was to identify polymorphisms in the first intron and c.*1232G>A position of the MSTN gene and analyze associations between the detected alleles/genotypes and carcass, meat quality, and biometric traits in Colored Polish Merino sheep. We analyzed 44 traits using the MIXED procedure of the SAS software. Five alleles (MSTN-A, MSTN-B, MSTN-C, MSTN-E and MSTN-E1) were detected. Significant genotypic effects were detected with regard to chest depth (live lamb) and fat depth over ribs, drip loss, subjective meat flavor and color, whereas significant allelic effects were found for chest depth (live lamb), pre-slaughter weight, hot carcass weight, cold carcass dressing out, leg depth (carcass), eye of loin width and area, intramuscular fat (IMF) content, water-holding capacity, and subjective meat tenderness, flavor and color. The results suggest MSTN gene polymorphisms may be considered a genetic marker of carcass quality, meat quality, and biometric traits in sheep.

CRISPR/Cas9-mediated sheep MSTN gene knockout and promote sSMSCs differentiation.

Myostatin (MSTN) is an important gene involved in the regulation of embryonic muscle cells and adult muscle development; it has a good application prospect in transgenic animal production by improving the yield of muscle. The purpose of this study is to construct MSTN gene knockout vector using clustered regularly interspaced short palindromic repeats ( CRISPR)/CRISPR-associated protein 9 ( Cas9). The knockout efficiency was evaluated in sheep ear fibroblasts (SEFs) by cleavage activity of transcription of guide RNA ( gRNA), luciferase-single-strand annealing assay, T7 endonuclease I assay (T7E1), and TA clone sequence (10/38); and above all, detection showed that the cleavage activity of CRISPR/Cas9-mediated MSTN reached 29%. MSTN-Cas9/gRNA4 was transfected into sheep skeletal muscle satellite cell (sSMSC) to confirm the function of MSTN in myotomes formation induced by starvation in low-serum medium. The results showed that myotubes formation efficiency were 11.2 ± 1.3% and 19.5 ± 2.1% in the control group and knockout group, respectively. The average length of myotomes was 22 ± 5.3 and 47 ± 3.6 µm, displaying that MSTN knockout can promote sSMSC differentiation in number and length. The unlabeled MSTN-Cas9/gRNA4 was transfected into SEFs and monoclonal positive cells was obtained after 48 hours transfection. The MSTN-positive cells were used as donor cells to perform somatic cell nuclear transplantation to produce transgenic sheep. A total of 20 embryos were transplanted into surrogate mothers, four of them normally produce offspring. The genomic DNA of surviving lambs were used as a template, three positive individuals were identified by T7E1 digestion. All the results demonstrated that the CRISPR/Cas9 system has the potential to become an important and applicable gene engineering tool in animal breeding.

Molecular characterization of exon 3 of caprine myostatin gene in Marwari goat.

AIM: To estimate genetic variability in exon 3 of caprine myostatin gene in Marwari goats. MATERIALS AND METHODS: A total of 120 blood samples from unrelated Marwari goats were randomly collected from different villages of Bikaner (Rajasthan), India. Genomic DNA was extracted from whole blood using blood DNA isolation kit (Himedia Ltd.) as per manufacturer's protocol. The quality of extracted genomic DNA was checked on 0.8% agarose gel. Specifically designed a primer set for caprine myostatin (MSTN) gene (Genebank accession no. DQ167575) was used to amplify the exon 3 region of MSTN gene in Marwari goat. The genetic variability in exon 3 of MSTN gene in Marwari goat was assessed on 8% polyacrylamide gel electrophoresis to detect single strand conformation polymorphism (SSCP) pattern. RESULTS: The exon 3 of MSTN gene in Marwari goat showed two types of conformation patterns on 8% polyacrylamide gel. One of the patterns showed only two bands and was considered as genotype AA, whereas another pattern having an extra band was designated as genotype AB. The frequencies of AA and AB genotype for exon 3 region of MSTN gene were calculated as 0.90 and 0.10, respectively. CONCLUSION: Low level of polymorphism was observed at exon 3 region of MSTN gene in Marwari goat through SSCP analysis. This information could be utilized in future breeding plan to exploit the unique characteristics of Marwari goat of Rajasthan.

The effect of eight weeks resistance and aerobic training on myostatin and follistatin expression in cardiac muscle of rats.

Introduction: The clinical studies have shown that the myostatin gene expression and its serum density occur more frequently in heart patients than in healthy individuals. The purpose of this study is to investigate the influence of 8-week resistance and aerobic exercise on the myostatin and follistatin gene expression of myocardium muscle of healthy male Wistar rats. Methods: In this experimental study, 20 five-week-old adult Wistar rats (250 ± 26.5 g) were divided into three groups: healthy control group (n = 6), resistance exercise group (n = 7), and aerobic exercise group (n = 7). The resistance and aerobic exercise plan consisted of 8 weeks and 3 sessions per week. The resistance exercise group performed climbing a one-meter 26-stair ladder with a slope of 85 degrees for 3 sets of 5 repetitions per session. The aerobic exercise group performed running at a speed of 12 meters per minute for 30 minutes during the first sessions gradually increasing up to a speed of 30 meters per minute for 60 minutes during the final sessions (equivalent to 70% to 80% of maximum oxygen consumption). The differences between the groups were evaluated using a one-way analysis of variance (ANOVA) test. When appropriate, LSD post-hoc test was used. The significance level for the study was less than 0.05. Results: The results of this study shows that after 8 weeks of exercise, there is no significant difference between myostatin mRNA gene expression levels of the heart muscle among the three groups of control, resistance exercise, and aerobic exercise (P = 0.172, F = 1.953). However, the mean differences between follistatin mRNA levels of the heart muscle among the three groups of control, resistance exercise, and aerobic exercise are statistically significant (F = 38.022, P = 0.001). Furthermore, the ratio of follistatin to myostatin mRNA gene expression of the heart muscle (P = 0.001, F = 10.288) shows significant difference among the three groups. Conclusion: Our results indicate that the resistance and aerobic exercise could cause a decrease in myostatin and an increase in follistatin levels, thus preventing many muscular physiological disorders such as arthritis and muscle weakness.

The effect of leader peptide mutations on the biological function of bovine myostatin gene.

The growth of muscle fibers can be negatively regulated by bovine myostatin. The first two exons of myostatin gene code for the N-propeptide and its third exon codes for the C-polypeptide. Myostatin is secreted as a latent configuration formed by dimerization of two matured C peptides non-covalently linked with the N terminal pro-peptide. Pro-peptide has two distinct functions in guiding protein folding and regulating biological activity of myostatin. When the structure of the leader peptide is altered via mutations resulting in more tight binding with the mature peptide, myostatin function is inhibited, resulting in the changes of P21 and CDK2 expression levels which are related to the regulation of cell cycle. In the present study, the coding region of bMSTN (bovine myostatin) gene was amplified and mutated (A224C and G938A) through fusion PCR, and the mutated bMSTN gene (bMSTN-mut) was inserted in frame into the pEF1a-IRES-DsRed-Express2 vector and transfected into bovine fibroblast cells. The expression levels of bMSTN-mut, P21 and CDK2 (cyclin dependent kinase 2) were examined with qPCR and Western-blotting. Changes in cell cycle after transfection were also analyzed with flow cytometry. The results indicated that leader peptide mutation resulted in down-regulation of P21 expression levels and up-regulation of CDK2 expression levels. The flow cytometry results showed that the proportion of cells in the G0/G1-phase was lower and that of cells in the S-phase was higher in bMSTN-mut transfected group than that in the control group. The proliferation rate of bMSTN-mut transfected cells was also significantly higher than that of the control cells. In conclusion, the studies have shown that the pEF1a-IRES-DsRed-Express2-bMSTN-mut recombinant plasmid could effectively promote the proliferation of bovine fibroblast cells. The site-directed mutagenesis of bMSTN gene leader peptide and in vitro expression in bovine fibroblast cells could be helpful to further the studies of bMSTN in regulating bovine muscle cell growth and development.

Expression of Myostatin in Gastrocnemius Muscle of Cancer Cachexia Mice Model / 上海第二医科大学学报

Objective To study the expression level of myostatin in the gastrocnemius muscle of cancer cachexia mice and to observe the improvement of the status and the influence on myostatin expression by use of meloxicam.(Methods The) tumor-bearing cachexia mice model was established by Lovo cell line subcutaneous inoculation.Twenty-four BALB/c mice were randomly divided into 3 groups(n=8 for each group):group A,control;group B,tumor-bearing mice plus saline;group C,tumor-bearing mice plus meloxicam(5 mg/kg).The food intake and body composition were documented.The expression of myostatin in the gastrocnemius muscle was investigated by RT-PCR and immunohistochemistry in all the animals,and the correlation analysis between serum TNF? and myostatin was conducted. Results The body weight of group B was about 60% of group A,and the average food intake was significantly declined(P