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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.

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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. Thirtysix 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 collagenrelated pathological features in MSTN -/pigs. Hence, MSTN could be used as a therapeutic target for treating UH and tendon abnormalities.

Myostatin (MSTN) is a growth and differentiation factor that regulates proliferation and differentiation of myoblasts, which in turn controls skeletal muscle growth. It may regulate myoblast differentiation by influencing miRNA expression, and the present study aimed to clarify its precise mechanism of action. Here, we found that MSTN −/− pigs showed an overgrowth of skeletal muscle and upregulated miR-455-3p level. Intervention of MSTN expression using siMSTN in C2C12 myoblasts also showed that siMSTN significantly increased the expression of miR-455-3p. It was found that miR-455-3p directly targeted the 3′-untranslated region of Smad2 by dual-luciferase assay. qRT-PCR, Western blotting, and immunofluorescence analyses indicated that miR-455-3p overexpression or Smad2 silencing in C2C12 myoblasts significantly promoted myoblast differentiation. Furthermore, siMSTN significantly increased the expression of GATA3. The levels of miR-455-3p were considerably reduced in C2C12 myoblasts following GATA3 knockdown. Consistently, GATA3 knockdown also reduced the enhanced miR-455-3p expression caused by siMSTN. Finally, we illustrated that GATA3 has a role in myoblast differentiation regulation. Taken together, we identified the expression profiles of miRNAs in MSTN −/− pigs and found that miR-455-3p positively regulates myoblast differentiation. In addition, we revealed that MSTN acts through the GATA3/miR-455-3p/Smad2 cascade to regulate muscle development.