Application of PCR-RFLP for quick identification of MSTN mutants in MSTN mutant pig breeding.

Knockout of the MSTN gene is linked to the enlarged tongue, and it causes suckling difficulty in animals. The suckling difficulty has a severe effect on animal mortality. Thus, special care was required to ensure their survivability. Here, it is critical to promptly ascertain the genotype of all pigs after birth. The main objective of the present study was to develop the restriction enzyme-mediated PCR-RFLP assay for MSTN mutant pig genotyping. To accomplish this, conserved oligonucleotide primer and restriction site were deduced according to the mutated sequence of the MSTN mutant pigs. PCR amplification yielded a 176 bp band for all homozygous MSTN mutant (MSTN-/-), heterozygous MSTN mutant (MSTN+/-) and wild-type (WT) pigs. However, MSTN+/- samples produced two fragments with 176 and 87 bp, and WT samples produced one fragment with 87 bp after being digested by BstNI. MSTN-/- samples were not digested by BstNI and yielded a 176 bp band. Thus, we were able to determine the genotype of all pigs using BstNI restriction enzyme-mediated PCR-RFLP method. Overall, the present study reported a simple and fast PCR-RFLP genotyping method for MSTN mutant pig breeding. The present study may contribute to the establishment of commercial breeding systems and the production of double muscle pigs.

Myostatin deficiency decreases cardiac extracellular matrix in pigs.

Myostatin (MSTN), a member of the TGF-ß superfamily, negatively regulates muscle growth. MSTN inhibition has been known to cause a double-muscled phenotype in skeletal muscle and fibrosis reduction in the heart. However, the role of MSTN in the cardiac extracellular matrix (ECM) needs more studies in various species of animal models to draw more objective conclusions. The main objective of the present study was to investigate whether loss of MSTN affects the cardiac extracellular matrix in pigs. Three MSTN knockouts (MSTN-/-) and three wild type (WT) male pigs were generated by crossing MSTN ± heterozygous gilts and boars. Cardiac ECM and underlying mechanisms were determined post-mortem. The role of MSTN on collagen expression was investigated by treating cardiac fibroblasts with active MSTN protein in vitro. MSTN protein was detected in WT hearts, while no expression was detected in MSTN-/- hearts. The heart-to-body weight ratio was significantly decreased in MSTN-/- pigs. The morphometric analyses, including picrosirius red staining, immunofluorescent staining, and ultra-structural thickness examination of the endomysium, revealed a significant reduction of connective tissue content in MSTN-/- hearts compared to WT. Hydroxyproline, type I collagen (Col1A), and p-Smad3/Smad3 levels were significantly lower in MSTN-/- hearts in vivo. On the contrary, cardiac fibroblasts treated with exogenous MSTN protein overexpressed Col1A and activated Smad and AKT signaling pathways in vitro. The present study suggests that inhibition of MSTN decreases cardiac extracellular matrix.

Altered fibrinogen level and fibrin clot structure in myostatin homozygous mutant pig.

Obesity is associated with increased serum fibrinogen level. Myostatin (MSTN), a strong inhibitor of skeletal muscle growth, is recognized as a potential target for obesity. However, the effect of MSTN inhibition on fibrinogen is not largely known. The objective of the present study was to explore fibrinogen levels after MSTN inhibition. Fibrinogen levels and the fibrin clot structure of MSTN homozygous knockout (KO) and wild-type (WT) pigs (n = 4 in each group) were investigated. The protein expression of fibrinogen in the serum and liver of KO pigs decreased greatly (1.6-fold loss for serum and 2.5-fold loss for liver). KO pigs showed significantly decreased gene expression of fibrinogen chains: FGA (fibrinogen-α; 11-fold), FGB (fibrinogen-ß; 8-fold) and FGG (fibrinogen-γ; 7.4-fold). The basal transcriptional regulators of fibrinogen, HNF1 (hepatocyte nuclear factor 1) and CEBP-α (CCAAT/Enhancing-binding protein-alpha) were remarkably down-regulated after interruption of MSTN expression by siRNA (small interfering RNA) in cultured hepatocytes (about 2- and 4-fold, respectively). Compared with WT pigs, KO pigs displayed altered fibrin clot structure with thinner fibers, decreased turbidity and increased permeability. The findings indicate that the inhibition of MSTN could affect fibrinogen levels and the fibrin clot structure.

Association of myostatin deficiency with collagen related disease-umbilical hernia and tippy toe standing in pigs.

Herein, we investigate the high incidence of umbilical hernia and tippy-toe standing and their underlying changes in gene expression and proliferation in myostatin knockout (MSTN-/-) pigs. Thirty-six male MSTN-/- pigs were generated by somatic cell nuclear transfer (SCNT). These pigs presented a considerably high incidence of tippy-toe standing and umbilical hernia (69.4% and 61.1%, respectively). The tendon to body weight ratio was significantly lower than wild-type pigs (0.202 ± 0.017 vs 0.250 ± 0.004, respectively). The crimp length of the MSTN-/- tendon was significantly longer than that of wild-type pigs. The expression of MSTN and the activin type IIB (ACVR2B) was detected in the tendon and linea alba of MSTN-/- pigs. MSTN treatment significantly increased the phosphorylation of Smad2/3 in both tendon and linea alba fibroblasts. Type I collagen (Col1A) and Scleraxis (Scx) expression levels in the tendon and linea alba of MSTN-/- pigs were significantly lower than those in wild-type in vivo, whereas and cyclin-dependent kinase inhibitor 1 (p21) expression levels were higher. Treatment of tendon and linea alba fibroblasts with recombinant MSTN increased Col1A and Scx and decreased p21 expression in vivo. Moreover, there was a significant increase in fibroblast proliferation after treatment. The results indicated that MSTN regulates collagen expression and proliferation in tendon and linea alba fibroblasts; thus, MSTN deficiency causes collagen-related pathological features in MSTN-/- pigs. Hence, MSTN could be used as a therapeutic target for treating UH and tendon abnormalities.

Reproduction traits of heterozygous myostatin knockout sows crossbred with homozygous myostatin knockout boars.

Few studies exist on homozygous myostatin gene mutant (MSTN-/- ) pigs, especially on their reproductive ability. We have previously shown that semen quality of homozygous MSTN-/- boars is comparable to that of wild type (WT). However, no data exist on the reproductive ability of heterozygous MSTN gene mutant (MSTN+/ - ) sows. The present study highlights showed that the heterozygous MSTN+/ - sows have delayed pubertal age than WT sows (255.80 ± 6.79 versus 191.10 ± 3.42, respectively). The number of services per pregnancy of heterozygous MSTN+/ - sows is significantly higher than that of WT sows (3.33 ± 0.43 versus 1.60 ± 0.25, respectively). Moreover, although heterozygous MSTN+/ - sows have natural reproduction ability, their litter size was significantly lower than that of WT sows (7.75 ± 0.44 versus 14.25 ± 0.60, respectively). Offsprings generated from heterozygous MSTN+/ - sow and homozygous MSTN-/- boar were genotyped with the PCR and sequencing method to detect myostatin mutation and to identify whether the piglets are homozygous MSTN-/- or heterozygous MSTN+/ - . The proportion of homozygous MSTN-/- piglets was significantly lower than that of heterozygous MSTN+/ - piglets (2.50 ± 0.35 versus 5.25 ± 0.60, respectively). Furthermore, none of the sows presented dystocia, and the phenotype of heterozygous MSTN+/ - piglets was normal. However, 10% homozygous MSTN-/- piglets died of dyspnoea within 2 hr after birth, 60% of homozygous MSTN-/- piglets showed large tongues, and 50% had umbilical hernias. In summary, this study for the first time reports the reproduction traits of heterozygous MSTN+/ - sows crossbred with homozygous MSTN-/- boars. This study will pave the way in a new direction for the breeding and development of super lean meat varieties in the future.

Ginsenoside Rb1 protects porcine oocytes against methylglyoxal damage thus it improves the quality of parthenogenetic activation and in vitro fertilization embryos.

Panax ginseng, a functional food, has been widely used as an edible nourishment and medicinal supplement. Ginsenoside Rb1 is a major bioactive ingredient of ginseng, which shows very specific anti-apoptosis and anti-oxidant activities. Methylglyoxal (MGO) is one of intermediate products of glucose metabolism, which is absorbed easily from high sugar foods or carbonated beverages. It may involve in a variety of detrimental processes in vivo. However, it has not been fully explored the effects of ginsenoside Rb1 on MGO-induced oocytes damage. This study found that MGO-induced DNA damage and mitochondrial dysfunction result in the failure of porcine oocytes maturation and low in vitro development capacity of parthenogenetic activation (PA) and in vitro fertilization (IVF) embryos. Conversely, Rb1 supplementation recovered the rate of maturation, and improved in vitro development capacity of PA and IVF embryos. Rb1 also provided porcine oocytes a lower level of reactive oxygen species production, higher level of ATP content and mitochondrial membrane potential, and stimulated pluripotency gene expression in blastocysts. The findings of this study reveal ginsenoside Rb1 protects porcine oocyte from the cytotoxicity effects of methylglyoxal and provides novel perspectives for the protection of reproduction system by functional food of ginseng.

The ginsenoside Rh2 protects porcine oocytes against aging and oxidative stress by regulating SIRT1 expression and mitochondrial activity.

Post-ovulatory aging, a major problem faced by oocytes cultured in vitro, causes oxidative damage and mitochondrial dysfunction in oocytes. The ginsenoside Rh2 is one of the main monomeric components of ginseng, but its effects on porcine oocytes are unknown. In the present study, in vitro aging (IVA) and accelerated induction of aging using H2O2 resulted in DNA damage and an increased incidence of abnormal spindle formation in porcine oocytes. Rh2 supplementation increased the antioxidant capacity, reduced the occurrence of early apoptosis, and improved the development of in vitro fertilized blastocysts. It also rescued the abnormal aggregation of mitochondria and the decrease of the mitochondrial membrane potential under mitochondrial dysfunction. Meanwhile, Rh2 enhanced mRNA expression of the anti-aging and mitochondrial biogenesis-related genes silent information regulator of transcription 1 (SIRT1) and peroxisome proliferator-activated receptor coactivator 1-α (PGC-1α), and the antioxidant gene superoxide dismutase 1 (SOD1). The protection of porcine oocytes against aging and oxidative stress by Rh2 was confirmed using the SIRT1-specific inhibitor EX-527. Our results reveal that Rh2 upregulates SIRT1/PGC-1α to enhance mitochondrial function in porcine oocytes and improve their quality. Our study indicates that Rh2 can be used to prevent mitochondrial dysfunction in oocytes.

Silencing myostatin increases area fraction of smooth muscle in the corpus cavernosum of pigs.

Myostatin (MSTN), an inhibitor of skeletal muscle growth, is also expressed in penile smooth muscle; however, it is unclear whether MSTN plays an inhibitory role in penile smooth muscle growth. We investigated the role of MSTN in the smooth muscle of the penile corpus cavernosum of pigs using MSTN homozygous mutant knockout (KO) and wild type (WT) pigs (n = 4 in each group). The mean of area fraction (%) of smooth muscle in the penile corpus cavernosum was 65.9 % ± 1.79 in the KO and approximately 41.7 % ± 5.39 in the WT (P < 0.001). KO pigs showed significantly increased expression of smooth muscle-specific genes, including smooth muscle protein 22 (TAGLN) (6.62-fold), smooth muscle myosin heavy chain (MYH11) (2.41-fold), myocardin (MYOCD) (3.05-fold), and serum response factor (SRF) (4.95-fold), and decreased expression of vimentin (VIM) (1.36-fold). Immunofluorescence staining and Western blotting showed smooth muscle-specific expression of α-smooth muscle actin (SMA) and calponin was higher in KO pigs (P < 0.05) than in WT pigs. KO pigs had less fat deposition inside the corpus cavernosum, and showed downregulation of adiponectin (ADIPOQ) and fatty acid synthase (FASN) (2.5-fold and 1.9-fold loss, respectively). In vitro experiments showed MSTN interference promoted corporal smooth muscle cell growth and expression of smooth muscle-specific markers, whereas it downregulated the expression of fat-specific genes, ADIPOQ and FASN. MSTN inhibition could promote smooth muscle growth and decrease fat deposition in the corpus cavernosum. MSTN, thus, could be a possible target for the treatment of smooth muscle dystrophy-related disorders such as erectile dysfunction.

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.

Positive growth of smooth muscle in uterine horns of myostatin homozygous mutant gilt.

Current studies on myostatin (MSTN), a well-known negative regulator of skeletal muscle, studies mainly focus on the its effects on skeletal muscle.However, its effects on smooth muscle are less studied, especially in the uterine horns. To identify the role of MSTN in uterine horn smooth muscle, this study used 6-8-month-old homozygous MSTN mutant (MSTN-/-) gilts in anoestrum as animal models. Histochemical and immunofluorescence staining, western blotting, and RT-qPCR were performed. The results showed that the uteri of the MSTN-/- gilts were morphologically normal, and the uterine horn smooth muscle content was increased (MSTN-/-: 75.19%, Wild type: 51.52%, P < 0.01). In vivo immunofluorescence staining showed that the expression of the uterine horn smooth muscle-specific marker proteins, namely α-smooth muscle actin (ACTA2) and calponin, increased after MSTN knockout (1.41- and 1.21-fold, respectively, P < 0.05). Increased gene expression was also seen in MSTN-/- gilts in vivo for ACTA2 (approximately 2-fold), smooth muscle myosin heavy chain (7.14-fold), myocardin (9.32-fold), and serum response factor (2.17-fold). Protein expression of smooth muscle-specific markers was increased (1.51-fold for ACTA2, 1.57-fold for calponin, P<0.05). MSTN knockout promoted proliferation of the smooth muscle cell and the gene expression of c-kit, a peristaltic marker (2.43-fold, P < 0.05). The results of the in vitro experiments were consistent with those of the in vivo experiments. The present study indicates that MSTN knockout can increase the smooth muscle content of uterine horns, thus providing potential therapeutic targets for pregnancy disorders caused by increased smooth muscle content.

The protective role of ginsenoside compound K in porcine oocyte meiotic maturation failed caused by benzo(a)pyrene during in vitro maturation.

Benzo(a)pyrene (BaP) is a pollutant and carcinogen derived from air pollution. It causes serious damage to reproductive system, especially ovary. Ginseng is always used in food and traditional medicine as a nutraceuticals or herbal medicine. Ginsenoside compound K (CK) is a major bioactive ingredient of ginseng, that shows very specific anti-apoptosis, anti-oxidant, and anti-inflammatory activities and thus, it protects cells from damage. The aim of this study was to investigate the effects of CK on the BaP-induced inhibition of the in vitro maturation of porcine oocytes and their subsequent embryonic development capacity. We found that supplementation with 10 µg mL-1 CK during in vitro maturation significantly increased maturation rate (P < 0.05) and the expression level of related genes after damage induced by 40 µM BaP treatment. In addition, reactive oxygen species (ROS) levels significantly decreased and ATP content and mitochondrial membrane potential (MMP) increased after CK supplementation (P < 0.05). The competence for embryonic development was improved by the induction of pluripotency gene expression and the inhibition of apoptosis after CK supplementation of BaP-treated oocytes. Supplementation with 10 µg mL-1 CK improved porcine oocyte maturation and subsequent embryonic development of parthenogenetic activation (33.01 vs. 20.92, P < 0.05) and in vitro fertilization (24.01 vs. 16.52, P < 0.05) by increasing antioxidant activity and improving mitochondrial function after BaP-induced damage.