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The overall in vitro embryo production efficiency in buffalo is hampered by the poor fertilization rate. It is known that the quality of the frozen semen may affect fertilization efficiency. However, it is not possible to rule out that the process of capacitation, required by spermatozoa to acquire the fertilizing ability, is impaired in the in vitro fertilization (IVF) system. Although several agents have been proven to induce sperm capacitation in vitro, heparin treatment is still the most efficient method in most of the domestic species. There is evidence that capacitation is part of an oxidative process and that nitric oxide (NO) acts as a capacitation inducer in human (Herrero et al. 1999 Biol. Reprod. 61, 575–581) and bovine (Rodriguez et al. 2005 Anim. Reprod. Sci. 85, 231–242) spermatozoa. The aim of the present study was to evaluate whether sodium nitroprusside (SNP), a well-known generator of NO in vitro, improves buffalo sperm capacitation in vitro. Frozen–thawed sperm from a bull previously tested for IVF were treated by swim-up in order to select only the motile population. Spermatozoa were incubated in the presence of 0.01 mM heparin (control group) for 1 h (n = 266), 2 h (n = 270), and 3 h (n = 306), and in the presence of 10 M SNP for 1 h (n = 302), 2 h (n = 286), and 3 h (n = 260). The concentration of SNP was chosen on the basis of a preliminary dose-response trial (0.1 M, 1 M, and 10 M). Following incubation with these agents, sperm were exposed for 15 min to 60 g mL-1 of lysophosphatidylcholine, an agent known to induce acrosome reaction only on capacitated spermatozoa. Trypan blue was used first to differentiate live from dead spermatozoa and the dried smears were then fixed in 37% formaldehyde and stained with Giemsa for acrosome evaluation by microscopic examination. The proportion of acrosome-reacted spermatozoa in each group was used to assess the efficiency of capacitation under different incubation conditions. Differences between groups were analyzed by chi-squared test. No dead spermatozoa were found in all groups. Following 1-h sperm treatment with either heparin or SNP, the proportion of acrosome-reacted spermatozoa was similar (35.3% vs. 28.5%, respectively). However, extending the incubation time to 2 h, SNP significantly (P < 0.01) increased the incidence of acrosome reaction compared to heparin (60.1% vs. 44.1%, respectively). Analogously, when the sperm treatment was prolonged to 3 h, SNP gave a significantly (P < 0.01) higher percentage of acrosome reaction compared to the control (68.8% vs. 36.6%, respectively). In conclusion, sperm treatment with SNP for either 2 or 3 h significant improved the efficiency of buffalo sperm capacitation in vitro compared with heparin, that is, the capacitating agent currently used in the IVF system. The promoting effect of SNP indirectly indicates that NO acts as a capacitation inducer in buffalo spermatozoa. Finally, these results suggest the need to evaluate the effect of SNP on the fertilizing capability of buffalo spermatozoa in vitro. [ABSTRACT FROM AUTHOR]

The aim of this study was to compare the efficiency of different combinations of cryoprotectants for vitrification of IVP buffalo (Bubalus bubalis) embryos by the cryotop method (Kuwayama et al. 2005 RBM Online 11, 300–308). In group A, we evaluated the vitrification and warming solutions previously used to vitrify buffalo embryos in French straws (Gasparrini et al. 2001 Theriogenology 55, 307). Embryos were equilibrated in 1.4 M glycerol for 5 min and in 1.4 M glycerol and 3.6 M ethylene glycol (EG) for an additional 5 min. After being transferred into 3.4 M glycerol and 4.6 M EG for 25 s, individual embryos were picked up in an extremely small volume (<0.1 L) of vitrification solution and placed on the top of a very fine polypropylene strip (0.4 mm wide 20 mm long 0.1 mm thick) attached to a hard plastic handle, kindly provided by M. Kuwayama. Each embryo was placed onto the thin strip of the Cryotop and immediately submerged into liquid nitrogen. For warming, the strip of the Cryotop was immersed directly into a 0.5 M sucrose solution; embryos were retrieved and transferred into 0.25 M sucrose for 5 min before culture in SOF medium. In group B, we examined the vitrification and warming solutions previously used for OPS vitrification of buffalo embryos (De Rosa et al. 2006 Reprod. Fertil. Dev. 18, 153). Embryos were equilibrated in 7.5% EG + 7.5% dimethyl sulfoxide (DMSO) for 3 min before transfer into 16.5% EG + 16.5% DMSO + 0.5 M sucrose. After 25 s, they were placed on the cryotop, as previously described, and submerged into liquid nitrogen. For warming, embryos were recovered into a 0.25 M sucrose solution for 1 min, transferred into 0.15 M sucrose for 5 min, and cultured in SOF. IVP buffalo embryos of excellent quality that, by Day 7 of culture (Day 0 = in vitro fertilization), had reached the blastocyst stage (n = 44 and 53 for groups A and B, respectively), over 6 replicates, were vitrified. Embryo survival rate was determined as the percentage of vitrified-warmed embryos undergoing further development during a 24-h in vitro culture period. Differences between methods were analyzed by chi-square test. A significantly higher embryo survival rate was recorded in Group B compared to Group A (67.9 vs. 43.2% respectively; P < 0.05). In conclusion, it was demonstrated that cryotop vitrification, with the combination of cryoprotectants used in group B, is a valid tool to cryopreserve IVP buffalo blastocysts. [ABSTRACT FROM AUTHOR]

The establishment of porcine pluripotent ES cell lines would be an exciting and novel tool for animal biotechnology, such as cloning and transgenesis. Furthermore, it would represent a useful model for biomedical research, cell therapy, xenotransplantation as well as developmental biology research. However, in spite of several studies, no conclusive results have been obtained and a number of technical questions are still to be answered in order to derive genuine ES cells in the pig. Here we report the results obtained in our laboratory aimed at comparing IVF v. parthenogenetic embryos as a source for the establishment of putative ES cells. Oocytes were divided in two groups and subjected to IVF or parthenogenetically activated with ionomicyn and 6-DMAP. They were cultured in NCSU for 7 days and then subjected to immuno-surgery. Inner cell mass were plated onto inactivated STO feeder cells and outgrowth formation was monitored. Cells were passaged to a new STO monolayer every 7 days. Assessment of pluripotency markers was carried out both by RT-PCR and immunocytochemical analysis at every passage for up to 22 passages. Telomerase activity was measured every 5 passages. The results indicate that parthenogenetic embryos, although less resilient than IVF embryos to immuno-surgery, have a significantly greater ability to generate outgrowths and stable cell lines. Moreover, 77% of the 39 parthenogenetic lines derived v. only 33% of the IVF ones expressed pluripotency markers and displayed high telomerase activity. Altogether our findings are consistent with data obtained in the human where the efficiency to derive hES cell lines from parthenogenetic blastocysts appears greater as compared with regular blastocysts from IVF embryos (Cheng L 2008 Cell Research 18, 215–217).Table 1.Supported by: Prin 2005, 2006. [ABSTRACT FROM AUTHOR]

Although removal of cumulus cells improves the efficiency of vitrification of buffalo (Bubalus bubalus) in vitro-matured (IVM) oocytes (Gasparrini et al. 2007 Anim. Reprod. Sci. 98, 335–342), the lack of cells impairs the fertilization process. Therefore, the aim of the present work was to evaluate the influence of a somatic support during in vitro fertilization (IVF) of buffalo vitrified denuded matured oocytes. Since IVF on a cumulus cells monolayer was inefficient, we verified the effects of co-culture with cumulus-enclosed oocytes (COCs). IVM buffalo oocytes (n = 316) were vitrified by the Cryotop method (Kuwayama and Kato 2000, J. Assist. Reprod. Genet. 17, 477 abst) that was recently proven suitable for buffalo oocyte cryopreservation (Attanasio et al. 2006 Reprod. Domest. Anim. 41, 302–310). Denuded buffalo oocytes were equilibrated in 10% ethylene glycol (EG) and 10% dimethyl sulfoxide (DMSO) for 3 min, transferred into 20% EG and 20% of DMSO in TCM199 with 20% fetal calf serum (FCS) + 0.5 m sucrose, loaded on Cryotops, and plunged into liquid nitrogen within 25 s. For warming, oocytes were exposed for 1 min to 1.2 m sucrose and then to decreasing concentrations of the sugar (0.6, 0.4, 0.3 m for 30 s) in TCM199 + 20% FCS. Oocytes were rinsed and allocated to IVM drops for 1.5 h. Survival rate was evaluated at this point and the oocytes that had survived (292/316 = 92.4%) were split into 2 fertilization groups: (A) approximately 5 buffalo oocytes per 50-µL drop of IVF medium, and (B) approximately 3 buffalo oocytes + 3 bovine fresh COCs per 50-µL drop of IVF medium. Since buffalo COCs easily lose their cells following IVF, for better identification we used bovine COCs that have a brighter and more compact cumulus mass. In vitro fertilization and culture were carried out as previously described (Gasparrini et al. 2007). As control, buffalo oocytes (n = 104) were in vitro-matured, fertilized, and cultured up to the blastocyst stage. On Day 1, survival rate was evaluated in the two vitrification groups; cleavage and blastocyst rates were recorded on Days 5 and 7, respectively, in all groups. The experiment was repeated 4 times. Differences in the percentages of survival, cleavage, and blastocyst formation among treatments were analyzed by chi-square test. Within vitrification groups, despite similar survival rates on Day 1 (90.6% v. 93.3%, respectively, in Groups A and B), cleavage rate was significantly improved in Group B compared to Group A (59.2% v. 45.4%, respectively; P < 0.01). Interestingly, the cleavage rate in Group B was not significantly different from that recorded in the control group (71.0%). Although blastocysts were produced in both vitrification groups (3.6% v. 4.1%, respectively, in Groups A and B), the yield was significantly lower than that of the control group (29.0%, P < 0.01). In conclusion, co-culture with bovine COC during fertilization improves the capability of buffalo denuded vitrified oocytes to cleave. [ABSTRACT FROM AUTHOR]

Osteopontin (OPN) is an acidic single-chain phosphorylated glycoprotein found both in the oviduct fluid (ODF) and oviductal epithelium in cattle, which is believed to facilitate fertilization. It was recently reported that addition of a rabbit polyclonal immunoglobulin G antibody against purified bovine milk OPN with sperm oocytes, bovine oocytes, or both decreased (P < 0.05) fertilization compared with the in vitro-fertilized control (Goncalves et al. 2007 Theriogenology 67, 468–74). The aim of the present work was to determine the effect of in vitro addition of OPN to the fertilization medium on both cleavage and postfertilization embryo development in cattle. In the first experiment, in vitro-matured oocytes were fertilized in modified TALP medium in the presence of 0.0, 0.1, 1.0, or 10 µg mL–1 of OPN. In a second experiment, matured oocytes were in vitro-fertilized in modified TALP medium in the presence of 0.0, 10, 20, or 40 µg mL–1 of OPN. In vitro fertilization was carried out with frozen–thawed spermatozoa from a bull previously tested for IVF. After 20 to 22 h of coincubation at 39C, 5% in CO2 in air, presumptive zygotes were vortexed to remove cumulus cells, washed, and transferred, 30 to 50 per well, into 400 µL of SOF modified medium. Zygotes were incubated in a humidified mixture of 5% CO2, 7% O2, and 88% N2 in air at a temperature of 39C. On Day 7 of development (Day 0 = day of insemination), cleavage and development rates into transferable embryos (TE)–tight morulae (TM) and blastocysts (Bl) of superior quality were recorded. Differences in the percentages of both cleavage and blastocyst rates among groups were analyzed by a chi-square test. In experiment 1, numerically higher percentages of TM–Bl (29.5, 29.5, 30.5, and 37.5%, respectively, in the control group and in the groups with 0.1, 1, and 10 µg mL–1 of OPN; P = 0.25) and Bl (28.6, 27.5, 29.1, and 36.7, respectively, in the control group and in the groups with 0.1, 1, and 10 µg mL–1 of OPN; P = 0.24) were observed with 10 µg mL–1 of OPN. In experiment 2, significantly more cleavage (80.0 v. 71.3%; P < 0.05) and higher percentages of TM–Bl (44.6 v. 34.5%; P < 0.05) and Bl (39.2 v. 30.6%; P = 0.06) were observed with 10 µg mL–1 of OPN v. the control. Combined analysis from both experiments showed an overall effect of 10 µg mL–1 of OPN v. the control in the percentages of TM–Bl and Bl (respectively, 41.1 v. 33.3% and 37.7 v. 30.5%; P < 0.05). These results indicate that it is possible to improve the efficiency of bovine in vitro embryo production by adding the oviductal protein OPN. [ABSTRACT FROM AUTHOR]