Y.Q. Lu

Generation and Characterization of Embryonic Stem-Like Cell Lines Derived from In Vitro Fertilization Buffalo (Bubalus bubalis) Embryos

In the present study, buffalo embryonic stem-like (ES-like) cell lines were successfully isolated, cultured and characterized. From a total of 92 normal buffalo embryos obtained by in vitro fertilization, 18 were morulae, 33 were blastocyst and 41 were hatched blastocyst, the inside of morulae or inner cell masses of blastocysts were isolated mechanically and cultured onto mitomocin-C-inactivated buffalo embryonic fibroblasts as feeder layers. Alkaline phosphatase (AP) of ES-like cells, as well as the specific stage embryonic antigen SSEA-1, SSEA-3, SSEA-4 and transcription factor OCT-4, was used to evaluate the characterization of the cells. The spontaneous differentiation of ES-like cells was induced by culturing on leukaemia inhibitory factor-free medium for more than 2 weeks without passage. To evaluate mark gene expression, total RNA was extracted from cells, and specific primers were used for reverse transcriptase-polymerase chain reaction (RT-PCR). After 8-10 days of culture, primary ES-like cell colonies were formed in 0% (0/18) of morulae, 24.24% (8/33) of blastocysts and 60.98% (25/41) of hatched blastocysts, respectively. The forming rate of primary ES-like cells colonies in hatched blastocyst group was significantly (p < 0.05) higher than the obtained for other groups. Two ES-like cell lines could survive to eight passages at least by using the method of mechanical dissociation, but just three passages by using the method of enzymatic dissociation. The cells formed large, multicellular colonies with distinct boundaries, exhibited many important features of ES/ES-like cells, including positive AP, SSEA-1, SSEA-3 and SSEA-4 activity. Undifferentiated buffalo ES-like cells expressed Oct-4, Nanog, Sox2 gene mRNA. In vitro differentiation experiments had demonstrated that those cells were pluripotent.

Membrane progestin receptor beta (mPR-β): A protein related to cumulus expansion that is involved in in vitro maturation of pig cumulus–oocyte complexes

A new group of putative membrane receptors have now been isolated from fish and other vertebrates, including human. These proteins are classified into three groups known as membrane progestin receptor alpha, beta and gamma (mPR-alpha, -beta and -gamma). In the present study we have investigated the role of mPR-beta in regulating in vitro maturation (IVM) of pig cumulus-oocyte complexes (COCs). RT-PCR and Western blot analysis indicated that COCs contain transcripts and proteins for mPR-beta. The levels of both transcripts and proteins increased between 0 and 20h IVM, but then decreased between 20 and 44h. The luteinizing hormone (LH) and follicle-stimulating hormone (FSH) did not affect mPR-beta expression during IVM. Immunofluorescence analysis indicated that the mPR-beta was localized in the plasma membrane of cumulus cell. However, in mouse embryonic fibroblasts (MEFs), mPR-beta was detected at the endoplasmic reticulum (ER) rather than the plasma membrane. Cumulus expansion was impaired significantly (P<0.05) when COCs were incubated in maturation medium containing 10% (v/v) anti-mPR-beta serum during IVM. Bioinformatics analysis predicted that mPR-beta had an ER retention motif and an endocytosis internalization motif. These results suggest that the mPR-beta is a molecule related to cumulus expansion and it might function by regulation of exocytosis. In conclusion, this is the first description of the expression patterns and subcellular localization of mPR-beta in COCs and might shed light on the function of the protein.

In vitro embryo production in buffalo (Bubalus bubalis) using sexed sperm and oocytes from ovum pick up

The objective was to explore the use of sexed sperm and OPU-derived oocytes in an IVP system to produce sex-preselected bubaline embryos. Oocytes were recovered from 20 fertile Murrah and Nili-Ravi buffalo cows by repeated (twice weekly) ultrasound-guided transvaginal ovum pick up (OPU), or by aspiration of abbatoir-derived bubaline ovaries, and subjected to IVF, using frozen-thawed sexed or unsexed bubaline semen. On average, 4.6 oocytes were retrieved per buffalo per session (70.9% were Grades A or B). Following IVF with sexed sperm, oocytes derived from OPU had similar developmental competence as those from abattoir-derived ovaries, in terms of cleavage rate (57.6 vs. 50.4%, P=0.357) and blastocyst development rate (16.0 vs. 23.9%, P=0.237). Furthermore, using frozen-thawed sexed versus unsexed semen did not affect rates of cleavage (50.5 vs. 50.9%, P=0.978) or blastocyst development (15.3 vs. 19.1%, P=0.291) after IVF using OPU-derived oocytes. Of the embryos produced in an OPU-IVP system, 9 of 34 sexed fresh embryos (26.5%) and 5 of 43 sexed frozen embryos (11.6%) transferred to recipients established pregnancies, whereas 7 of 26 unsexed fresh embryos (26.9%) and 6 out of 39 unsexed frozen embryos (15.4%) transferred to recipients established pregnancies. Eleven sex-preselected buffalo calves (10 females and one male) and 10 sexed buffalo calves (six females and four males) were born following embryo transfer. In the present study, OPU, sperm sexing technology, IVP, and embryo transfer, were used to produce sex-preselected buffalo calves. This study provided proof of concept for further research and wider field application of these technologies in buffalo.

Protective Effects of Melatonin against Oxidative Stress in Flow Cytometry-sorted Buffalo Sperm

Previous reports of the ability of melatonin to scavenge a variety of toxic oxygen and nitrogen-based reactants suggest that melatonin could be an effective antioxidant for protecting sperm. In this study, flow cytometry and laser tweezers Raman spectroscopy were used to evaluate the effect of melatonin on buffalo sperm quality to optimize sperm sex-sorting procedures. In fresh sperm incubated in the presence or absence of melatonin (10(-4) u2003m) for 1, 24, 48 h or 72 h at 27°C, the mitochondrial activity was significantly higher than in a non-melatonin control (pu2003<u20030.05). Also, during the flow-sorting process, sperm in melatonin-supplemented groups had higher (pu2003<u20030.05) mitochondrial activity than the control. The intensity of Raman spectra from sperm frozen in media supplemented with melatonin was significantly weaker than that for non-melatonin-treated groups, except for a band at 1302 per cm. Thus, melatonin helps to protect buffalo sperm from reactive oxygen species induced by staining, sorting and freezing and increases semen quality after the freezing-thawing processes. Furthermore, the results indicate the high potential of the laser tweezers Raman spectroscopy technique for rapid, effective and non-invasive assessment of the quality of sperm cells.

Stability of the cytoskeleton of matured buffalo oocytes pretreated with cytochalasin B prior to vitrification

Stabilizing the cytoskeleton system during vitrification can improve the post-thaw survival and development of vitrified oocytes. The cytoskeleton stabilizer cytochalasin B (CB) has been used in cryopreservation to improve the developmental competence of vitrified oocytes. To assess the effect of pretreating matured buffalo oocytes with CB before vitrification, we applied 0, 4, 8, or 12xa0μg/mL CB for 30xa0min. The optimum concentration of CB treatment (8xa0μg/mL for 30xa0min) was then used to evaluate the distribution of microtubules and microfilaments, the expression of the cytoskeleton proteins actin and tubulin, and the developmental potential of matured oocytes that were vitrified-warmed by the Cryotop method. Western blotting demonstrated that vitrification significantly decreased tubulin expression, but that the decrease was attenuated for oocytes pretreated with 8xa0μg/mL CB before vitrification. After warming and intracytoplasmic sperm injection, oocytes that were pretreated with 8xa0μg/mL CB before vitrification yielded significantly higher 8-cell and blastocyst rates than those that were vitrified without CB pretreatment. The values for the vitrified groups in all experiments were significantly lower (Pxa0<xa00.01) than those of the control groups. In conclusion, pretreatment with 8xa0μg/mL CB for 30xa0min significantly improves the cytoskeletal structure, expression of tubulin, and development capacity of vitrified matured buffalo oocytes.

Translocation of Active Mitochondria During Buffalo (Bubalus bubalis) Oocytes In vitro Maturation, Fertilization and Preimplantation Embryo Development

Mitochondria are energy-supplying organelles, whose distribution and functional integrity are necessary for cell survival and development. In this study, the mitochondrial distribution pattern and activity during buffalo oocyte in vitro maturation, fertilization and preimplantation embryo development were revealed using a fluorescent dye and confocal laser scanning microscopy. Distribution of active mitochondria changed during buffalo oocyte in vitro maturation. Active mitochondria were transferred from the outer to inner and perinuclear cytoplasm as oocytes matured in vitro and aggregated around the pronuclei in the fertilized eggs. Active mitochondria were also observed in preimplantation embryos. In the two-cell stage, they were distributed throughout the cytoplasm. From four-cell to the spherical embryonic stages, active mitochondria translocated to the perinuclear and the periphery of the cytoplasm. These results confirm that mitochondria play an important role in oocyte and embryo. The distribution of active mitochondria might be a marked feature of buffalo oocyte maturation, fertilization and preimplantation embryo development in vitro.

Optimization of cryopreservation of buffalo (Bubalus bubalis) blastocysts produced by in vitro fertilization and somatic cell nuclear transfer.

The objective of this study was to optimize cryopreservation conditions for buffalo in vitro produced (IVP) embryos. The in vitro fertilized (IVF) and somatic cell nuclear transferred (SCNT) blastocysts were vitrified with either 40% ethylene glycol (EG), 25% EG + 25% dimethylsulfoxide (DMSO), or 20% EG + 20% DMSO + 0.5 m sucrose, and the IVF blastocysts produced from abattoir-derived ovaries were also slow-frozen with either 10% EG or 0.05 m trehalose dehydrate + 1.8% EG + 0.4% BSA. Cryosurvival rates of blastocysts harvested on various days or at various developmental stages were also examined. In this study: (1) vitrification with 20% EG + 20% DMSO + 0.5 m sucrose had the best cryopreservation efficiency; (2) IVF and SCNT blastocysts had similar cryotolerance (P > 0.05); (3) after thawing, slow-frozen blastocysts reexpanded earlier than the vitrified blastocysts (P < 0.01); (4) cryosurvival rate of expanded blastocysts was higher than that of early blastocysts (P < 0.05); (5) cryosurvival rates of Days 5 to 7 blastocysts (Day 0 = day of IVF or SCNT) were higher than those of Days 8 to 9 blastocysts (P < 0.01); and (6) after embryo transfer, pregnancy rates for fresh and cryopreserved blastocysts were not different (P > 0.05). In conclusion, vitrification of Days 6 to 7 expanded blastocysts with 20% EG + 20% DMSO + 0.5 m sucrose was optimal for cryopreservation of buffalo IVP embryos.

Comparative proteomic analysis of different developmental stages of swamp buffalo testicular seminiferous tubules

With ageing, many protein components change markedly during mammalian spermatogenesis. Most of these proteins have yet to be characterized and verified. Here, we have employed two-dimensional electrophoresis coupled to tandem mass spectrometry to explore the different proteins from pre-pubertal, pubertal and post-pubertal swamp buffalo testicular seminiferous tubules. The results showed that 25 protein spots were differentially expressed among developmental stages, and 13 of them were successfully identified by mass spectrometry. Of which four proteins were up-regulated and three proteins were down-regulated with age, and the remaining six proteins were fluctuated among developmental stages. Bioinformatics analysis indicates that these proteins were probably related to cellular developmental process (53.8%), cell differentiation (53.8%), spermatogenesis (15.4%), apoptotic process and cell death (30.8%). Expression profiles of calumenin (CALU) and galectin-1 (LGALS1) were further verified via Western blotting. In summary, the results help to develop an understanding of molecular mechanisms associated with buffalo spermatogenesis.

268 IDENTIFICATION OF X- AND Y-BEARING SPERMATOZOA IN SORTED BUFFALO (BUBALUS BUBALIS) SEMEN BY FLUORESCENCE IN SITU HYBRIDIZATION

Flow cytometry sorting technology has been successfully used to sort the X- and Y-chromosome bearing sperm. Previous studies showed that fluorescence in situ hybridization (FISH) method was a simple and reliable procedure for assessing the effectiveness of separation of X- and Y-sperm in the swine (Kawarasaki T et al. 1998 Theriogenology 50, 625–635) and the bovine (Rens W et al. 2001 Reproduction 121, 541–546). Reports of sex-preselection by flow-cytometry sorting of the X- and Y-sperm were also seen in the buffalo (Presicce GA et al. 2005 Reprod. Dom. Anim. 40, 73–75; Lu YQ et al. 2006 Anim. Reprod. Sci. 100, 192–196). There was, however, no report to date for using the FISH method to assess the purity of the sorted buffalo sperm. The objective of the present study was to verify the purity of flow cytometrically-sorted buffalo X- and Y-sperm by FISH using bovine X- and Y- chromosome painting probes prepared by microdissection. The X- and Y- chromosomes of bovidea were microdissected respectively from the metaphase spreads of Holstein blood cells with a glass needle controlled by a micromanipulator and amplified by degenerate oligo-nucleotide primer-PCR (DOP-PCR) (Mariela N et al. 2005 Genet. Mol. Res. 4, 675–683). The DOP-PCR products of X- and Y- chromosome were labeled with CY3-dUTP and Biotin-11-dUTP, respectively. The buffalo X- or Y-sperm DNA from unsorted semen and sorted semen were hybridized to the labeled probes, respectively. The results showed that the hybridized signals were clearly visible in the metaphase karyotype of bovine and buffalo semen samples. About 47.7% (594/1246) and 48.9% (683/1396) of the unsorted buffalo sperm emitted strong fluorescent signals when assessed by Y- and X-chromosome painting probes, respectively, which was conformed to the sex ratio in normal buffalo sperm (50%:50%). About 86.1% (1529/1776) hybridization signals of the sperm in the sorted X-semen assessed by X-chromosome painting probes were detected, while 82.2% (2232/2716) of the Y-sorted buffalo sperm emitted strong fluorescent signals when assessed by Y-chromosome painting probe. The results of the flow cytometer re-analysis revealed that the proportions of X- and Y-bearing sperm in the sorted semen were 89.6% and 86.7%, respectively. There were no apparent differences between the two assessment methods of sperm separation by flow cytometry re-analysis and by FISH with the X-Y paint probe. In conclusion, bovine X- and Y-chromosome painting probes prepared using microisolation method could be used to verify the purity of the sorted sperm in the buffalo. This study was supported by the Guangxi Department of Science and Technology (0626001-3-1) and National Key Technology R&D Program, The People’s Republic of China (2006BAD04A18). The authors (M. Zhang, X.J. Zhuang, and Y.Q. Lu) contributed equally to this work.