Yingming Wei

Buffalos (Bubalus bubalis) Cloned by Nuclear Transfer of Somatic Cells

Abstract Cloning of buffalos (Bubalus bubalis) through nuclear transfer is a potential alternative approach in genetic improvement of buffalos. However, to our knowledge, cloned offspring of buffalos derived from embryonic, fetal, or somatic cells have not yet been reported. Thus, factors affecting the nuclear transfer of buffalo somatic cells were examined, and the possibility of cloning buffalos was explored in the present study. Treatment of buffalo fibroblasts and granulosa cells with aphidicolin plus serum starvation resulted in more cells being arrested at the G0/G1 phase, the proportion of cells with DNA fragmentation being less, and the number of embryos derived from these cells that developed to blastocysts being greater. In addition, a difference was found in the development of embryos reconstructed with fetal fibroblasts from different individuals (P < 0.001). Forty-two blastocysts derived from granulosa cells and fetal fibroblasts were transferred into 21 recipient swamp buffalos, and 4 recipients were confirmed to be pregnant by rectal palpation on Day 60 of gestation. One recipient received two embryos from fetal fibroblasts aborted on Day 300 of gestation and delivered two female premature calves. Three recipients maintained pregnancy to term and delivered three female cloned calves after Days 338–349 of gestation. These results indicate that buffalo embryos derived from either fetal fibroblasts or granulosa cells can develop to the term of gestation and result in newborn calves.

Immunisation against inhibin enhances follicular development, oocyte maturation and superovulatory response in water buffaloes

This study was carried out to test the feasibility of enhancing embryo production in vivo and in vitro by immunoneutralisation against inhibin or follistatin. In Experiment 1, multi-parity buffaloes were assigned into three groups: High group (n=8), which received one primary (2mg) and two booster (1mg) vaccinations (28-day intervals) with a recombinant inhibin α subunit in 1 mL of white oil adjuvant; Low group (n=8), which received half that dose; and Control group (n=7), which received only adjuvant. Immunisation against inhibin stimulated development of ovarian follicles. Following superovulation and artificial insemination, inhibin-immunised buffaloes had more developing follicles than the Control buffaloes. The average number of embryos and unfertilised ova (4.5±0.6, n=6) in the High group was higher (P<0.05) than in the Control group (2.8±0.6, n=5) and was intermediate (4.1±0.7, n=7) in the Low group. The pooled number of transferable embryos of the High and Low groups (3.2±0.5, n=13) was also higher (P<0.05) than that (1.6±0.7, n=5) of the controls. The immunised groups also had higher plasma concentrations of activin, oestradiol and progesterone. In Experiment 2, the addition of anti-inhibin or anti-follistatin antibodies into buffalo oocyte IVM maturation medium significantly improved oocyte maturation and cleavage rates following parthenogenic activation. Treatment with anti-follistatin antibody also doubled the blastocyst yield from activated embryos. These results demonstrated that immunisation against inhibin stimulated follicular development, enhanced oocyte quality and maturation competence, yielded more and better embryos both in vivo and in vitro.

In Vitro Culture and Differentiation of Buffalo (Bubalus bubalis) Spermatogonia

The objective of this study was to develop a culture system which could support buffalo spermatogonia differentiation into spermatids in vitro. Testes from 3- to 5-month-old buffaloes were decapsulated and seminiferous tubules were enzymatically dissociated to recover spermatogonia and sertoli cells. The cells were cultured in modified Dulbecco modified Eagle medium supplemented with different concentrations of foetal bovine serum, retinol, testosterone for 2 months at 37 degrees C. Spermatogonia and sertoli cells were identified with an antibody against c-kit or GATA4, respectively. The viability of spermatogonia in the media supplemented with different concentrations of serum was all significantly higher (p < 0.05) compared with that in the medium without serum. A-paired or A-aligned spermatogonia and spermatogonial colonies (AP-positive) were observed after 7-10 days of culture and spermatid-like cells with a flagellum (6-8 microm) appeared after 30 days of culture. For cultured conditions, retinol could not significantly promote the formation of spermatid-like cells (p > 0.05), whereas supplementation of testosterone could significantly promote (p < 0.05) the formation of spermatid-like cells after 41 days of culture. The expression of the spermatid-specific marker gene (PRM2) was identified after 30 days of culture by RT-PCR. Yet, the transition protein 1 (TP1, a haploid makers) was not detected. Meanwhile, spermatids developed in vitro were also confirmed by Raman spectroscopy. These results suggest that buffalo spermatogonia could differentiate into spermatids in vitro based on the analysis of their morphology, PRM2 expression and Raman spectroscopy. Yet, the normality of the spermatid-like cells was not supported by TP1 expression.

Effects of Scriptaid on the Histone Acetylation, DNA Methylation and Development of Buffalo Somatic Cell Nuclear Transfer Embryos

The present study was undertaken to examine the effect of Scriptaid treatment on histone acetylation, DNA methylation, expression of genes related to histone acetylation, and development of buffalo somatic cell nuclear transfer (SCNT) embryos. Treatment of buffalo SCNT embryos with 500 nM Scriptaid for 24 h resulted in a significant increase in the blastocyst formation rate (28.2% vs. 13.6%, p<0.05). Meanwhile, treatment of buffalo SCNT embryos with Scriptaid also resulted in higher acetylation levels of H3K18 and lower methylation levels of global DNA at the blastocyst stage, which was similar to fertilized counterparts. The expression levels of CBP, p300, HAT1, Dnmt1, and Dnmt3a in SCNT embryos treated with Scriptaid were significantly lower than the control group at the eight-cell stage (p<0.05), but the expression of HAT1 and Dnmt1a was higher than the control group at the blastocyst stage (p<0.05). When 96 blastocysts developed from Scriptaid-treated SCNT embryos were transferred into 48 recipients, 11 recipients (22.9%) became pregnant, whereas only one recipient (11.1%) became pregnant following transfer of 18 blastocysts developed from untreated SCNT embryos into nine recipients. These results indicate that treatment of buffalo SCNT embryos with Scriptaid can improve their developmental competence, and this action is mediated by resulting in a similar histone acetylation level and global DNA methylation level compared to in vitro-fertilized embryos through regulating the expression pattern of genes related to histone acetylation and DNA methylation.

Effects of recipient oocyte age and interval from fusion to activation on development of buffalo (Bubalus bubalis) nuclear transfer embryos derived from fetal fibroblasts.

The objective was to investigate the effect of recipient oocyte age and the interval from activation to fusion on developmental competence of buffalo nuclear transfer (NT) embryos. Buffalo oocytes matured in vitro for 22 h were enucleated by micromanipulation under the spindle view system, and a fetal fibroblast (pretreated with 0.1 μg/mL aphidicolin for 24 h, followed by culture for 48 h in 0.5% fetal bovine serum) was introduced into the enucleated oocyte, followed by electrofusion. Both oocytes and NT embryos were activated by exposure to 5 μM ionomycin for 5 min, followed by culture in 2 mM 6-dimethyl-aminopurine for 3 h. When oocytes matured in vitro for 28, 29, 30, 31, or 32 h were activated, more oocytes matured in vitro for 30 h developed into blastocysts in comparison with oocytes matured in vitro for 32 h (31.3 vs 19.9%, P < 0.05). When electrofusion was induced 27 h after the onset of oocyte maturation, the cleavage rate (78.0%) was higher than that of electrofusion induced at 28 h (67.2%, P < 0.05), and the blastocyst yield (18.1%) was higher (P < 0.05) than that of electrofusion induced at 25 or 26 h (7.4 and 8.5%, respectively). A higher proportion of NT embryos activated at 3 h after electrofusion developed to the blastocyst stage (18.6%) in comparison with NT embryos activated at 1 h (6.0%), 2 h (8.3%), or 4 h (10.6%) after fusion (P < 0.05). No recipient was pregnant 60 d after transfer of blastocysts developed from NT embryos activated at 1 h (0/8), 2 h (0/10), or 4 h (0/9) after fusion. However, 3 of 16 recipients were pregnant following transfer of blastocysts developed from the NT embryos activated at 3 h after fusion, and two of these recipients maintained pregnancy to term. We concluded that the developmental potential of buffalo NT embryos was related to recipient oocyte age and the interval from fusion to activation.

Efficient Generation of Transgenic Buffalos (Bubalus bubalis) by Nuclear Transfer of Fetal Fibroblasts Expressing Enhanced Green Fluorescent Protein

The possibility of producing transgenic cloned buffalos by nuclear transfer of fetal fibroblasts expressing enhanced green fluorescent protein (EGFP) was explored in this study. When buffalo fetal fibroblasts (BFFs) isolated from a male buffalo fetus were transfected with pEGFP-N1 (EGFP is driven by CMV and Neo is driven by SV-40) by means of electroporation, Lipofectamine-LTX and X-tremeGENE, the transfection efficiency of electroporation (35.5%) was higher than Lipofectamine-LTX (11.7%) and X-tremeGENE (25.4%, P < 0.05). When BFFs were transfected by means of electroporation, more embryos from BFFs transfected with pEGFP-IRES-Neo (EGFP and Neo are driven by promoter of human elongation factor) cleaved and developed to blastocysts (21.6%) compared to BFFs transfected with pEGFP-N1 (16.4%, P < 0.05). A total of 72 blastocysts were transferred into 36 recipients and six recipients became pregnant. In the end of gestation, the pregnant recipients delivered six healthy calves and one stillborn calf. These calves were confirmed to be derived from the transgenic cells by Southern blot and microsatellite analysis. These results indicate that electroporation is more efficient than lipofection in transfecting exogenous DNA into BFFs and transgenic buffalos can be produced effectively by nuclear transfer of BFFs transfected with pEGFP-IRES-Neo.

Cytoplasmic volume of recipient oocytes affects the nucleus reprogramming and the developmental competence of HMC buffalo (Bubalus bubalis) embryos

The present study was undertaken to examine the effects of cytoplasmic volume on nucleus reprogramming and developmental competence of buffalo handmade cloning (HMC) embryos. We found that both HMC embryos derived from ~150% cytoplasm or ~225% cytoplasm resulted in a higher blastocyst rate and total cell number of blastocyst in comparison with those from ~75% cytoplasm (25.4 ± 2.0, 27.9 ± 1.6% vs. 17.9 ± 3.1%; 150 ± 10, 169 ± 12 vs. 85 ± 6, P<0.05). Meanwhile, the proportions of nuclear envelope breakdown (NEBD) and premature chromosome condensation (PCC) were also increased in the embryos derived from ~150 or ~225% enucleated cytoplasm compared to those from ~75% cytoplasm. Moreover, HMC embryos derived from ~225% cytoplasm showed a decrease of global DNA methylation from the 2-cell to the 4-cell stage in comparison with those of ~75% cytoplasm (P<0.05). Furthermore, the expression of embryonic genome activation (EGA) relative genes (eIF1A and U2AF) in HMC embryos derived from ~225% cytoplasm at the 8-cell stages was also found to be enhanced compared with that of the ~75% cytoplasm. Two of seven recipients were confirmed to be pregnant following transfer of blastocysts derived from ~225% cytoplasm, and one healthy cloned calf was delivered at the end of the gestation period, whereas no recipients were pregnant after the transfer of blastocysts derived from ~75% cytoplasm. These results indicate that the cytoplasmic volume of recipient oocytes affects donor nucleus reprogramming, and then further accounted for the developmental ability of the reconstructed embryos.

Isolation and characterization of buffalo (bubalus bubalis) amniotic mesenchymal stem cells derived from amnion from the first trimester pregnancy

Amniotic mesenchymal stem cells (AMSCs) from livestock are valuable resources for animal reproduction and veterinary therapeutic. The purpose of this study is to explore a suitable way to isolate and culture the buffalo AMSCs (bAMSCs), and to identify their biological characteristics. Digestion with a combination of trypsin-EDTA and collagenase type I could obtain pure bAMSCs more effectively than trypsin-EDTA or collagenase type I alone. bAMSCs could proliferate steadily in vitro culture and exhibited fibroblastic-like morphology in vortex-shaped colony. bAMSCs were positive for MSC-specific markers CD44, CD90, CD105, CD73, β-integrin (CD29) and CD166, and pluripotent markers OCT4, SOX2, NANOG, REX-1, SSEA-1, SSEA-4 and TRA-1-81, but negative for hematopoietic markers CD34, CD45 and epithelial cells specific marker Cytokeratin 18. In addition, bAMSCs were capable of differentiating into adipogenic, osteogenic, chondrogenic and neural lineages, with expression of FABP4, Ost, ACAN, COL2A1, Nestin and β III-tubulin. Glycogen synthase kinase 3 inhibitor: kenpaullone promoted bAMSCs to differentiate into neural lineage. This study provides an effective protocol to obtain and characterize bAMSCs, which have proven useful as a cell resource for buffalo cell reprogramming studies and transgenic animal production.

Efficient generation of sFat-1 transgenic rabbits rich in n-3 polyunsaturated fatty acids by intracytoplasmic sperm injection

N-3 polyunsaturated fatty acids (n-3 PUFAs) have their first double bond at the third carbon from the methyl end of the fatty-acid chain and had been proven to be beneficial to human health. However, mammals cannot produce n-3 PUFAs by themselves because they lack the n-3 fatty-acid desaturase (Fat-1) gene. Thus, the possibility of producing sFat-1 transgenic rabbits was explored in this study. The transgenic cassette of pPGK1-sFat-1-CMV-EGFP was constructed and transgenic rabbit embryos were produced by intracytoplasmic sperm injection (ICSI). When 123 EGFP-positive embryos at the 2-8-cell stage were transplanted into the oviduct of four oestrous-synchronised recipients, two of them became pregnant and gave birth to seven pups. However, transfer of embryos into the uterus of oestrous-synchronised recipients and oviduct or uterus of oocyte donor rabbits did not result in pregnancy. The integration of the sFat-1 gene was confirmed in six of the seven live pups by PCR and Southern blot. The expression of the sFat-1 gene in the six transgenic pups was also detected by reverse transcription polymerase chain reaction (RT-PCR). Gas chromatography-mass spectrometry analysis revealed that transgenic rabbits exhibited an ~15-fold decrease in the ratio of n-6:n-3 PUFAs in muscle compared with wild-type rabbits and non-transgenic rabbits. These results demonstrate that sFat-1 transgenic rabbits can be produced by ICSI and display a low ratio of n-6:n-3 PUFAs.