Isolation and culture of spermatogonial stem cells (SSCs) are attractive for production of genetic modified offspring. In the present study, buffalo spermatogonial stem-like cells were isolated, cultured and expression pattern of different germ cell marker genes were determined. To recover spermatogonia, testes from age 3 to 7 months of buffalo were decapsulated, and seminiferous tubules were enzymatically dissociated. Two types of cells, immature sertoli cell and type A spermatogonia were observed in buffalo testes in this stage. Germ cell marker genes, OCT3/4 (Pou5f1), THY-1, c-kit, PGP9.5 (UCHL-1) and Dolichos biflorus agglutinin, were determined to be expressed both in mRNA and protein level by reverse transcription polymerase chain reaction and immunostaining in buffalo testes and buffalo spermatogonial stem-like cells, respectively. In the following, when the isolated buffalo buffalo spermatogonial stem-like cells were cultured in the medium supplemented 2.5% fetal bovine serum and 40 ng/mL glial cell-derived neurotrophic factor medium, SSCs proliferation efficiency and colony number were significantly improved than those of other groups (p<0.05). These findings may help in isolation and establishing long term in vitro culture system for buffalo spermatogonial stem-like cells, and accelerating the generation of genetic modified buffaloes.

Isolation and culture of spermatogonial stem cells (SSCs) are attractive for production of genetic modified offspring. In the present study, buffalo spermatogonial stem-like cells were isolated, cultured and expression pattern of different germ cell marker genes were determined. To recover spermatogonia, testes from age 3 to 7 months of buffalo were decapsulated, and seminiferous tubules were enzymatically dissociated. Two types of cells, immature sertoli cell and type A spermatogonia were observed in buffalo testes in this stage. Germ cell marker genes, OCT3/4 (Pou5f1), THY-1, c-kit, PGP9.5 (UCHL-1) and Dolichos biflorus agglutinin, were determined to be expressed both in mRNA and protein level by reverse transcription polymerase chain reaction and immunostaining in buffalo testes and buffalo spermatogonial stem-like cells, respectively. In the following, when the isolated buffalo buffalo spermatogonial stem-like cells were cultured in the medium supplemented 2.5% fetal bovine serum and 40 ng/mL glial cell-derived neurotrophic factor medium, SSCs proliferation efficiency and colony number were significantly improved than those of other groups (p<0.05). These findings may help in isolation and establishing long term in vitro culture system for buffalo spermatogonial stem-like cells, and accelerating the generation of genetic modified buffaloes.

Donor cell in cell cycle and cell cycle synchronization are very important for the success of somatic cell nuclear transfer (SCNT). In this study, we examined the effects of serum starvation, culture to confluence on cell cycle characteristics of porcine fibroblast cells. The effect of the various methods of cell cycle synchronization was determined by flow cytomertry. 24~72 h serum starvation increased (P < 0.05) the proportion of cells at the G_0 /G_1 phase (92.2%~93.7%) as compared to the control group (77.8%). A similar increase in the percentage of G_0 /G_1 was obtained in culturing to confluence group (94.4% and 89.6%). The serum starvation cells were fully reversible with 10% FBS (fetal bovine serum) after serum deprivation 24 h. While used these fibroblasts synchronized by serum deprivation or culturing to confluence as SCNT donors, there was no significant difference in the cleavage and blastocyst development rate ofreconstructed oocytes oftwo groups (P > 0.05). In conclusion, porcine fibroblasts were effectively synchronized at G_0 /G_1 stages of the cell cycle by serum starvation or culturing to confluence, and both can be porcine SCNT donors.

The purpose of this study is to compare the growth of buffalo ovarian granulosa cells(GCs) cultured with different culture medium,and research the proliferation,karyotype and apoptosis of GCs to know well the growth characteristic of GCs in vitro,so that to establish a culture system of buffalo ovarian GCs in vitro.The results showed: the viability of GCs was about 60% after isolation.The growth speed and density of GCs were more dominant as culturing in DMEM medium than that of TCM-199 and DMEM/F12 medium.The GCs began to proliferate after cultured for 24 h,and reached a proliferation peak during cultured for 3 to 5 d.Karyotype analysis displayed that beyond 85% of GCs had normal karyotype in the 1,3,5,7 passage,which did not show that significant difference among these passages.The apoptosis ratio of the 5 and 7 passages of GCs was higher as comparing with the 1 passage(P0.05,P0.01).These results indicated that the DMEM medium was more suitable for GCs culturing in vitro,and the GCs could be passaged and keep relatively stable karyotype,but the apoptosis ratio would be elevate with the increase of the passage in vitro culture.

The feeder layer for buffalo EG-like cells was investigated in this study. Firstly,the buffalo fetal fibroblast (BFF) cells from tissue culture were compared with those from trypsin digestion. Secondly,the BFF cells were treated with mitomyein C and then the treating time was investigated. At last,the treated BFF were used to culture buffalo EG-like cells. The research revealed that: (1)The cells derived from tissue culture were better than trypsin digestion,so tissue culture was more suitable for BFF. (2)The 3rd passage cells were in good condition and more than 90% of them had normal karyotype. (3)Treating BFF with 10 mg/L mitomyein C for 3.5 h detecting by MTT and Brdu was best, not only inhibiting BFF proliferation, but also keep them in good condition. (4)The feeder layer established could sustain buffalo EG-like cell for 8 passage.

Ectopically, expression of defined factors could reprogram mammalian somatic cells into induced pluripotent stem cells (iPSCs), which initiates a new strategy to obtain pluripotent stem cell lines. Attempts have been made to generate buffalo pluripotent stem cells by culturing primary germ cells or inner cell mass, but the efficiency is extremely low. Here, we report a successful method to reprogram buffalo fetal fibroblasts (BFFs) into pluripotent stem cells [buffalo induced pluripotent stem cell (biPSCs)] by transduction of buffalo defined factors (Oct4, Sox2, Klf4, and c-Myc) using retroviral vectors. The established biPSCs displayed typical morphological characteristics of pluripotent stem cells, normal karyotype, positive staining of alkaline phosphatase, and expressed pluripotent markers including Oct4, Sox2, Nanog, Lin28, E-Cadherin, SSEA-1, SSEA-4, TRA-1-81, STAT3, and FOXD3. They could form embryoid bodies (EBs) in vitro and teratomas after injecting into the nude BALB/C mice, and 3 germ layers were identified in the EBs and teratomas. Methylation assay revealed that the promoters of Oct4 and Nanog were hypomethylated in biPSCs compared with BFFs and pre-biPSCs, while the promoters of Sox2 and E-Cadherin were hypomethylated in both BFFs and biPSCs. Further, inhibiting p53 expression by coexpression of SV40 large T antigen and buffalo defined factors in BFFs or treating BFFs with p53 inhibitor pifithrin-a (PFT) could increase the efficiency of biPSCs generation up to 3-fold, and nuclear transfer embryos reconstructed with biPSCs could develop to blastocysts. These results indicate that BFFs can be reprogrammed into biPSCs by buffalo defined factors, and the generation efficiency of biPSCs can be increased by inhibition of p53 expression. These efforts will provide a feasible approach for investigating buffalo stem cell signal pathways, establishing buffalo stem cell lines, and producing genetic modification buffaloes in the future.

Inhibin α-subunit gene could feedback suppress synthesis and secretion of FSH, regulate animal reproduc tion trait at its species level. To investigate feasibility of increasing buffalo reproduction trait by silencing inhibin gene, buffalo inhibin-α gene RNAi vectors were constructed and screened in this study. According to CI）S sequence of buffalo INH-α gene cloned in our lab, 5 pair of siRNA oligonucleotides were designed, synthsized, annealed and di redtly cloned into pSilencer4.1-CMV neo expression vector. The constructed vectors were transfected to in vitro culture buffalo granulose cells, relative transcription level of INH-α gene mRNA were determined by Real time PCR 48 h late. Then the high efficiency knock down vector was chosen to detect the variation of inhibition ratios at 24,48,72 and 96 h. The results showed that：5 buffalo inhibin a-subunit RNAi vectors were constructed including pSileneer4.1 145 ,pSilencer4.1-308 ,pSilencer4.1-769, pSilencer4.1-1013 and pSilencer4. 1-1053, which were conformed correctly by restriction and sequencing analysis. The relative transcription levels of inhibin-α mRNA in buffalo granulose cells transfected with pSilencer4.1 308,pSilencer4.1-769,pSilencer4.1-1013 and pSilencer4.1 1053 decreased. The inhibi- tion ratios were 58.8% ,44.9% ,67.4% and 43.9% ,respectively. Then pSilencer4.1-1013 was transfected and qRT PCR were performed at 2448,72 and 96 h, inhibition ratios were 27.1% ,61.0% ,57.0% and 41.2% ,respectively. In conclusion, effective RNAi vectors for buffalo inhibin-α gene have been successfully constructed, which is of great important in producing INH-α silencing transgenic buffalo.