Combined Treatment with Demecolcine and 6-Dimethylaminopurine during
Postactivation Improves Developmental Competence of Somatic Cell Nuclear
Transfer Embryos in Pigs
Joohyeong Leea,b, Jinyoung You a, Geun-Shik Leea, Seung Tae Leec, Sang-Hwan Hyun d and Eunsong Leea,b
aCollege of Veterinary Medicine, Kangwon National University, Chuncheon, Korea; bInstitute of Veterinary Science, Kangwon National University, Chuncheon, Korea; cCollege of Animal Life Science, Kangwon National University, Chuncheon, Korea; dCollege of Veterinary Medicine, Chungbuk National University, Cheongju, Korea
ABSTRACT
This study determined the effects of postactivation treatment with demecolcine and/or 6- dimethylaminopurine (6-DMAP) on in vivo and in vitro developmental competence of somatic cell nuclear transfer (SCNT) embryos in pigs. SCNT embryos were treated for 4 hours with 0.4 µg/mL demecolcine, 2 mM 6-DMAP, or both after electric activation, then transferred to surrogate pigs or cultured for 7 days. The formation rate of SCNT embryos with a single pronucleus was higher in combined treatment with demecolcine and 6-DMAP (95.2%) than treatment with demecolcine alone (87.1 %). Blastocyst formation of SCNT embryos was significantly increased in combined treatment with demecolcine and 6-DMAP (48.7%) compared with demecolcine (22.2%) or 6-DMAP alone (37.3%). Fluctuation of maturation promoting factor activity showed different patterns among various postactivation treatments. Pregnancy was established in 1 of 5 surrogates after transfer of SCNT embryos that were treated with demecolcine and 6-DMAP. The pregnant surrogate delivered one healthy live piglet. The results of our study demonstrated that postactivation treatment with demecolcine and 6-DMAP together improved preimplantation development and supported normal
in vivo development of SCNT pig embryos, probably influencing MPF activity and nuclear remodeling, including induction of single pronucleus formation after electric activation.
KEYWORDS
Maturation promoting factor; nuclear remodeling; postactivation; somatic cell nuclear transfer
Expression Profiling and Identification of Novel SNPs in CatSper2 Gene and Their
Influence on Sperm Motility Parameters in Bovines
A. Sivakumara, Subodh Kumara, H. M. Yathish b, Chinmoy Mishra c, Rajendra Prasad Modi a, Rajni Chaudharya, Subuhi Khan a, B. Sivamani d, S. K. Ghosh e and Mihir Sarkarf
aDivision of Animal Genetics, Indian Veterinary Research Institute, Izatnagar, India; bDepartment of Animal Genetics and Breeding, Veterinary College, Bidar, India; cDepartment of Animal Breeding and Genetics, OUAT, Bhubaneswar, India; dNutrition, Genetics and Biotechnology Division, Central Institute of Brackishwater Aquaculture, Chennai, India; eDivision of Animal Reproduction, Indian Veterinary Research Institute, Izatnagar, India; fDivision of Physiology & Climatology, Indian Veterinary Research Institute, Izatnagar, India
ABSTRACT
122 randomly selected Vrindavani cattle were studied to detect polymorphism in four fragments of the CatSper2 gene that were comprised of exon 2, 4, 5, and 6 with flanking regions. Using PCR-SSCP and sequencing analysis, three SNPs (T157C, C273A, and A274C) in the first fragment, one SNP (C30G) in the second fragment, and two SNPs (T86G and T292C) in the fourth fragment were identified. The third fragment did not reveal any polymorphism. The SNPs were used for construction of haplotypes and three haplotypes were found. The least square analysis of variance revealed a significant (P < 0.01) effect of haplotype on all three motility parameters. The haplotype II and III were nonsignificantly different from each other while being significantly (P < 0.01) different from haplotype I. The nonsignificant difference of haplotype II with III can lead to a hypothesis that T>G or C>T SNPs may not play a role in sperm motility. However, when the comparison was made between haplotype I and II, it can be inferred that C>T SNP may have a role in sperm motility, as haplotype II has better motility parameters. Expression profiling of Catper2 gene revealed nonsignificant down regulation of CatSper2 gene in poor motility sperm compared to good motility sperm.
KEYWORDS
CatSper2; cattle; expression PCR-SSCP; SNPs
Genomic Regions in Local Endangered Sheep Encode Potentially Favorable Genes
Bianca Moioli , Roberto Steri and Gennaro Catillo
Consiglio per la ricerca in agricoltura e l’analisi dell’economia agraria, Monterotondo, Italy
ABSTRACT
The economic evaluation of farm animal genetic resources plays a key role in developing conservation programs. However, to date, the link between diversity as assessed by neutral genetic markers and the functional diversity is not yet understood. Two genome-wide comparisons, using over 44,000 Single Nucleotide Polymorphisms, identified the markers with the highest difference in allele frequency between the Alpago endangered breed and two clusters, composed of four specialized dairy sheep, and four meat breeds respectively. The genes in proximity of these markers were mapped to known pathways of the Gene Ontology to determine which ones were most represented. Our results indicated that the differences of the Alpago breed from the more productive sheep rely upon genes involved in cellular defense and repair mechanisms. A higher number of different markers and genes were detected in the comparison with the specialized dairy sheep. These genes play a role in complex biological processes: metabolic, homeostatic, neurological system, and macromolecular organization; such processes may possibly explain the evolution of gene function as a result of selection to improve milk yield.
KEYWORDS
Dairy sheep; endangered breeds; farm animal genetic resources; genome-wide analysis; Ovine BeadChip 50K
Molecular Cloning, Identification, and Expression Patterns of Myostatin Gene in
Water Buffalo (Bubalus Bubalis)
Peng Zhu a,b, Haiyang Li a, Guiting Huang a, Jiayu Cuia, Ruimen Zhang a, Kuiqing Cui a, Sufang Yang a and Deshun Shi a
aState Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources, Guangxi University, Nanning, Guangxi, China; bGuangxi Buffalo Research Institute, Chinese Academy of Agricultural Science, Nanning, China
ABSTRACT
Myostatin (MSTN), also named growth differentiation factor 8 (GDF8), is a transforming growth factor-β (TGF-β) family member with a key role in the negative regulation of skeletal muscle growth. However, its role in ovarian folliculogenesis remains unclear. To provide us with a basis for understanding this role, we cloned MSTN and examined its expression patterns in water buffalo (Bubalus bubalis). The complete ORF of the water buffalo MSTN gene is 1,128 nucleotides, which encode a 375 amino acid protein and sharing 99% identity at the deducted amino acid level with that of Bos taurus. Protein sequence analysis showed that MSTN is a weakly acerbic extracellular protein, consisting of signal peptides at 18-19 sites, a TGF-β propeptide, and a TGF-β domain. RTPCR analyses demonstrated that water buffalo MSTN was expressed in multiple tissues but not limited to muscle. Immunohistochemistry staining confirmed the presence of MSTN in oocytes and granulosal cells. To our knowledge, this is the first study to confirm the expression of MSTN in the water buffalo ovary, suggesting an additional role of MSTN in water buffalo folliculogenesis, along with its role in skeletal muscle growth regulation. Further study of the regulatory mechanism of
MSTN in water buffalo reproduction is warranted.
KEYWORDS
Bio-informatics analysis; expression pattern; Myostatin; water buffalo
Multiplex Assay for Identifying Animal Species Found in the Tibetan
Area Using the Mitochondrial 12S rRNA Gene
Jung Nam Leea, MingFeng Jiang a, YongLi Wen a, ShiLin Li b and GuoRong Yuan c
aCollege of Life Science and Technology, Southwest University for Nationalities, Chengdu, Sichuan, China; bLongriba Breeding Farm, Aba, Hongyuan, Sichuan, China; cMaoxian Bureau of Animal Husbandry and Water Boards, Aba, Sichuan, China
ABSTRACT
Southwestern China has an area with unique natural conditions located in alpine regions at altitudes from 2000 to 5000 m; this area is referred to as the Qinghai-Tibetan plateau (QTP). Unique animals, such as yaks (Bos grunniens), are found extensively on the plateau of Southwestern China due to its unique environment. In recent years, the prevalence of fake meat products such as fake jerky has increased in this area. This research was conducted as an attempt to develop a reliable multiplex polymerase chain reaction (mPCR) detection method for identifying nine animal species found in QTP. We developed the mPCR method using the specific sites found in 12S rRNA region of these nine species, which was effective in discriminating between the nine species and was successful in terms of validated reproducibility, detection limit (<6 pg total DNA), discrimination of mixed samples, and specificity (approximately 99%) using real meat samples. Our results show that the mPCR detection method can overcome the limitations of prior detection methods, such as restriction fragment length polymorphism or high-resolution melting analysis methods.
KEYWORDS
Identification; multiplex PCR; yak (Bos grunniens)
Myogenic Differentiation Potential of Mesenchymal Stem Cells Derived from
Fetal Bovine Bone Marrow
Lucas Hidenori Okamuraa,b, Paloma Corderob, Jaime Palominob, Victor Hugo Parraguezc, Cristian Gabriel Torresd and Oscar Alejandro Peralta b,e
aDepartamento de Apoio, Produção e Saúde Animal, Faculdade de Medicina Veterinária, Universidade Estadual Paulista “Júlio de Mesquita Filho”, Araçatuba, São Paulo, Brasil; bDepartamento de Fomento de la Producción Animal, Facultad de Ciencias Veterinarias y Pecuarias, Universidad de Chile, Santiago, Chile; cDepartamento de Ciencias Biológicas, Facultad de Ciencias Veterinarias y Pecuarias, Universidad de Chile, Santiago, Chile; dDepartamento de Ciencias Clínicas, Facultad de Ciencias Veterinarias y Pecuarias, Universidad de Chile, Santiago, Chile; eDepartment of Biomedical Sciences and Pathobiology, Virginia-Maryland Regional College of Veterinary Medicine, Virginia Tech, Blacksburg, Virginia, USA
ABSTRACT
The myogenic potential of bovine fetal MSC (bfMSC) derived from bone marrow (BM) remains unknown; despite its potential application for the study of myogenesis and its implications for livestock production. In the present study, three protocols for in vitro myogenic differentiation of bfMSC based on the use of DNA methyltransferase inhibitor 5-Aza-2′-deoxycytidine (5-Aza),
myoblast-secreted factor Galectin-1 (Gal-1), and myoblast culture medium SkGM-2 BulletKit were used. Plastic-adherent bfMSC were isolated from fetal BM collected from abattoir-derived fetuses. Post-thaw viability analyses detected 85.6% bfMSC negative for propidium iodine (PI). Levels of muscle regulatory factors (MRF) MYF5, MYF6, MYOD, and DES mRNA were higher (P < 0.05) in bfMSC cultured under 100 µM of 5-Aza compared to 1 and 10 µM. Treatment of bfMSC with 10 µM of 5-Aza resulted in down-regulation of MYOD mRNA (Days 7 to 21) and up-regulation of MYF6 (Day 7), MYF5, and DES mRNA (Day 21). Gal-1 and SkGM-2 BulletKit induced sequential downregulation of early MRF (MYF5) and up-regulation of intermediate (MYOD) and late MRF (DES) mRNA. Moreover, DES and MYF5 were immunodetected in differentiated bfMSC. In conclusion, protocols evaluated in bfMSC induced progress into myogenic differentiation until certain extent evidenced by changes in MRF gene expression.
KEYWORDS
Bone marrow; cattle; mesenchymal stem cells; myogenesis
Short-hairpin Mediated Myostatin Knockdown Resulted in Altered Expression
of Myogenic Regulatory Factors with Enhanced Myoblast Proliferation in Fetal
Myoblast Cells of Goats
Rohit Kumar, Satyendra Pal Singh and Abhijit Mitra
Genome Analysis Laboratory, Animal Genetics Division, ICAR- Indian Veterinary Research Institute, Izatnagar, Bareilly, India
ABSTRACT
Myostatin (MSTN) is a well-known negative regulator of skeletal muscle development. Reduced expression due to natural mutations in the coding region and knockout as well as knockdown of MSTN results in an increase in the muscle mass. In the present study, we demonstrated as high as 60 and 52% downregulation (p < 0.01) of MSTN mRNA and protein in the primary fetal myoblast cells of goats using synthetic shRNAs (n ¼ 3), without any interferon response. We, for the first time, evaluated the effect of MSTN knockdown on the expression of MRFs (namely, MyoD, Myf5), follistatin (FST), and IGFs (IGF-1 & IGF-2) in goat myoblast cells. MSTN knockdown caused an upregulation (p < 0.05) of MyoD and downregulation (p < 0.01) of MYf5 and FST expression. Moreover, we report up to ∼four fold (p < 0.001) enhanced proliferation in myoblasts after four days of culture. The anti-MSTN shRNA demonstrated in the present study could be used for the production of transgenic goats to increase the muscle mass.
KEYWORDS
Anti-myostatin shRNA; caprine fetal myoblasts; IGFs; interferon response; MRFs
Two Novel SNPs of PPARγ Significantly Affect Weaning Growth Traits of Nanyang Cattle
Jieping Huang a,b,c, Ningbo Chen c, Xin Li a, Shanshan An a, Minghui Zhaoa, Taihong Sun a, Ruijie Haoa,b and Yun Maa,b aCollege of Life Sciences, Xinyang Normal University, Xinyang, Henan, China; bInstitute for Conservation and Utilization of Agro-Bioresources in Dabie Mountains, Xinyang, Henan, China; cCollege of Animal Science and Technology, Northwest A & F University, Yangling, Shaanxi, China
ABSTRACT
Peroxisome-proliferator-activated receptor gamma (PPARγ) is a key transcription factor that controls adipocyte differentiation and energy in mammals. Therefore, PPARγ is a potential factor influencing animal growth traits. This study primarily evaluates PPARγ as candidate gene for growth traits of cattle and identifies potential molecular marker for cattle breeding. Per previous studies, PPARγ mRNA was mainly expressed at extremely high levels in adipose tissues as shown by quantitative real-time polymerase chain reaction analysis. Three novel SNPs of the bovine PPARγ gene were identified in 514 individuals from six Chinese cattle breeds: SNP1 (AC_000179.1 g.57386668 C > G) in intron 2 and SNP2 (AC_000179.1 g.57431964 C > T) and SNP3 (AC_000179.1 g.57431994 T > C) in exon 7. The present study also investigated genetic characteristics of these SNP loci in six populations. Association analysis showed that SNP1 and SNP3 loci significantly affect weaning growth traits, especially body weight of Nanyang cattle. These results revealed that SNP1 and SNP3 are potential molecular markers for cattle breeding.
KEYWORDS
Association analysis; cattle; growth traits; PPARc
