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This article argues, first, that there is an unappreciated and difficult problem of explaining why states have positive obligations to perform certain actions—such as providing minimum protection—for all those persons in their territories and, second, that one possible solution is to locate the source of the obligations in the political power that states assume over their territories. The article analyzes the principal, superficially plausible accounts of state territorial obligations and shows that they each fail. Among the reasons for the failure is that these accounts cannot explain the existence of territorial obligations in important cases where we would expect the state to have such obligations. The article then argues for an account in terms of state political power that both avoids the major problems affecting other explanations and rests on prima facie plausible moral foundations.

The generation of porcine embryonic stem cells (pESC) would potentially have great impact in the biomedical field given the long-standing history of the pig as a prime animal model for pre-clinical biomedical applications. These cells would also be beneficial for the agricultural area, allowing efficient genetic engineering of this animal, to improve health and production traits. Despite numerous reports, no conclusive results have been obtained on the isolation and propagation of pESC lines and the establishment of pluripotent cells from the pig has remained an elusive goal. In the present study we performed a systematic analysis of different culture media for their ability to support the establishment of homogenous outgrowths from in vitro-produced embryos. Furthermore, we investigated which molecular networks are responsive to the factors contained in the most efficient media, since the identification of dominant signaling pathways that regulate porcine stem-cell pluripotency is likely to facilitate the generation of genuine pESC. Finally we compared IVF blastocysts versus parthenotes as a possible source for putative pESC in terms of blastocyst rate, resilience to immunosurgery procedures, ability to attach to the feeder, to generate outgrowths and to establish stable cell lines.

Contents The aim of the work was to evaluate the in vitro developmental competence of in vitro-matured buffalo oocytes after Cryotop vitrification (CTV) and in vitro fertilization (IVF). To optimize parameters, two cryoprotectant (CP) concentrations and two warming–dilution procedures were applied. Oocytes were vitrified in 16.5% ethylene glycol (EG), 16.5% dimethylsulphoxide (DMSO) and 0.5 m sucrose in Groups A and C, and in higher CP concentrations (20% EG, 20% DMSO and 0.5 m sucrose) in Groups B and D. Warming was performed in 1.25 m sucrose for 1 min, then in 0.62, 0.42 and 0.31 m sucrose, 30 s each (Groups A and B), or in 0.25 m sucrose for 1 min and in 0.15 m sucrose for 5 min (Groups C and D). After warming, the oocytes were fertilized and cultured in vitro. Survival rate post-warming was lower in Group D (83.6%) than in Groups A and B (92.4 and 92.8%, respectively), while intermediate values were found in Group C (85.7%). Survival rates at 24 h decreased in Groups C and D (52.0% and 50%, respectively) and remained high in Groups A and B (84.0% and 85.6%, respectively), thus indicating that the dilution of CP after warming is critical for buffalo oocyte cryopreservation. Similar differences were also observed in cleavage rates (42.7%, 55.3%, 28.4% and 36.3% for Groups A, B, C and D, respectively) whereas no differences in blastocyst rates were found among groups (6.4%, 7.8%, 5.9% and 6.9% for Groups A, B, C and D, respectively). Blastocyst production after IVF of vitrified oocytes proves the feasibility of CTV in buffalo species.

The aim of this study was to evaluate the efficiency of the solid surface vitrification (SSV) and the cryoloop vitrification (CLV) methods to cryopreserve in vitro matured buffalo oocytes. Another objective of the work was to investigate whether the presence of cumulus cells affects the efficiency of oocyte vitrification in this species. In the SSV method, oocytes were vitrified in a solution of 35% ethylene glycol, 5% polyvinyl–pyrrolidone and 0.4% trehalose and they were warmed in a 0.3 M trehalose solution. In the CLV method, oocytes were vitrified in 16.5% ethylene glycol and 16.5% dimethyl sulfoxide and warmed in decreasing concentrations of sucrose. The oocytes that survived vitrification were fertilized and cultured in vitro up to the blastocyst stage. Although high survival rates were recorded in all groups, when the oocytes were vitrified by the CLV method in the absence of cumulus cells, the survival rate was significantly (P < 0.05) lower. However, the CLV gave a significantly higher cleavage rate compared to the SSV with the denuded oocytes (45% versus 26%, respectively; P < 0.05), whereas no differences were found between methods with the cumulus-enclosed oocytes (14% versus 15%, respectively). Blastocysts were produced for the first time from in vitro matured oocytes that were vitrified–warmed in buffalo. Nevertheless, vitrification significantly decreased blastocyst yield, regardless of both the method employed and the presence or absence of cumulus cells.

in italiano. Lo scopo del presente lavoro è stato quello di valutare gli effetti dell’uso combinato di cistina e cisteamina durante la maturazione in vitro sulla sintesi intraoocitaria di glutatione e sullo sviluppo embrionale in vitro nel bufalo. Gli oociti, recuperati da ovaie da macello sono stati maturati in vitro, in presenza di 50 mM di cisteamina e con diverse concentrazioni di cistina (0, 0.3, 0.6, 0.9 mM). L’uso di cistina, alla concentrazione inferiore testata, ha migliorato significativamente l’efficienza di produzione embrionale in vitro, mediante stimolazione della sintesi intraoocitaria di glutatione.

The aim of this study was to evaluate whether the developmental speed affects the cryotolerance of in vitro produced buffalo embryos. In Experiment 1, abattoir-derived oocytes were in vitro matured, fertilized and cultured. The embryos produced by Say 7 of culture were vitrified at the tight morula (TM), early blastocyst (EBL), blastocyst (BL), expanded-blastocyst (XBL) and hatched-blastocyst (HBL) stage. The embryos were vitrified by cryotop in 16.5% ethylene glycol (EG) and 16.5% dimethyl sulfoxide (DMSO) and 0.5 M sucrose. Embryos were warmed in 0.25 M sucrose for 1 min and then in 0.15 M sucrose for 5 min and cultured in vitro for 24 h, to evaluate post-culture viability. In Experiment 2, ovum pick-up (OPU) was carried out on lactating buffaloes to produce embryos that were vitrified-warmed and transferred into synchronized recipients. The lowest (Pin vitro produced buffalo embryos.

Regional human rights courts, such as the Inter-American Court of Human Rights, currently apply a threshold requirement for reviewing state failures to protect against serious human rights violations that excludes from review much of the contemporary criminal organization violence in Latin America, such as that committed by drug cartels in Mexico or the new armed groups in Colombia (sometimes called Bacrim). However, this article will argue — through a close examination of the new illegal armed groups in Colombia — that the justiciability principles underlying this restrictive threshold requirement in fact support much broader review of state failures to protect against the widespread violence from these militarized criminal organizations. These principles, which determine the scope of human rights court review of state protection efforts, counsel different degrees of human rights court involvement in state protection failures depending on the particular circumstances at issue. When applied to the violence from contemporary Latin American militarized criminal organizations, review need not be restricted to avoid imposing an unreasonable burden on states, and should be extended in response to the substantial risk of inadequate state protection against such groups. As a result, the problem of these extremely violent organizations should be understood at least in part as one of human rights, since human rights principles not only limit the methods by which states oppose these groups, but also impose an enforceable obligation to protect against them.

The aim of this work was to evaluate whether providing a support of cumulus cells during IVF of buffalo denuded oocytes submitted to vitrification-warming enhances their fertilizing ability. In vitro matured denuded oocytes were vitrified by Cryotop in 20% EG + 20% of DMSO and 0.5 M sucrose and warmed into decreasing concentrations of sucrose (1.25 M-0.3M). Oocytes that survived vitrification were fertilized: 1) in the absence of a somatic support (DOs); 2) in the presence of bovine cumulus cells in suspension (DOs+susp); 3) on a bovine cumulus monolayer (DOs+monol); and 4) with intact bovine COCs in a 1:1 ratio (DOs+COCs). In vitro matured oocytes were fertilized and cultured to the blastocyst stage as a control. An increased cleavage rate was obtained from DOs+COCs (60.9%) compared to DOs, DOs+susp (43.6 and 38.4, respectively; P < 0.01) and DOs+monol (47.5%; P < 0.05). Interestingly, cleavage rate of DOs+COCs was similar to that of fresh control oocytes (67.8%). However, development to blastocysts significantly decreased in all vitrification groups compared to the control (P < 0.01). In conclusion the co-culture with intact COCs during IVF completely restores fertilizing capability of buffalo denuded vitrified oocytes, without improving blastocyst development.

The aim of the work was to evaluate the in vitro developmental competence of in vitro-matured buffalo oocytes after Cryotop vitrification (CTV) and in vitro fertilization (IVF). To optimize parameters, two cryoprotectant (CP) concentrations and two warming-dilution procedures were applied. Oocytes were vitrified in 16.5% ethylene glycol (EG), 16.5% dimethylsulphoxide (DMSO) and 0.5 M sucrose in Groups A and C, and in higher CP concentrations (20% EG, 20% DMSO and 0.5 M sucrose) in Groups B and D. Warming was performed in 1.25 M sucrose for 1 min, then in 0.62, 0.42 and 0.31 M sucrose, 30 s each (Groups A and B), or in 0.25 M sucrose for 1 min and in 0.15 M sucrose for 5 min (Groups C and D). After warming, the oocytes were fertilized and cultured in vitro. Survival rate post-warming was lower in Group D (83.6%) than in Groups A and B (92.4 and 92.8%, respectively), while intermediate values were found in Group C (85.7%). Survival rates at 24 h decreased in Groups C and D (52.0% and 50%, respectively) and remained high in Groups A and B (84.0% and 85.6%, respectively), thus indicating that the dilution of CP after warming is critical for buffalo oocyte cryopreservation. Similar differences were also observed in cleavage rates (42.7%, 55.3%, 28.4% and 36.3% for Groups A, B, C and D, respectively) whereas no differences in blastocyst rates were found among groups (6.4%, 7.8%, 5.9% and 6.9% for Groups A, B, C and D, respectively). Blastocyst production after IVF of vitrified oocytes proves the feasibility of CTV in buffalo species.

Fertility-related phosphoprotein osteopontin (OPN) is present in the bovine oviduct epithelium and fluid. The objectives were to determine the effects of OPN on percentages of cleavage and embryo development in vitro in cattle, and to assess the ability of OPN to induce in vitro capacitation of bovine sperm. In vitro-matured bovine oocytes were fertilized in the presence of 0, 10, 20, or 40 microg/mL OPN. There were greater percentages (P

The aim of this study was to compare the effect of cadexomer on reducing wound surface area of leg ulcers compared to that obtained in a group patients whose ulcers were treated by compression therapy.For each ulcer group, wound surface area was calculated at day 0 and after 28 days of treatment: this allowed to calculate the average wound surface area reduction, the percent reduction in wound size, as well as the weekly wound size reduction index.In the cadexomer-treated ulcers the total wound area reduction was 9.67 cm(2)/week, with a weekly wound size reduction index per patient of 0.96 cm(2); in the controls (compression therapy-treated patients) the total wound area reduction was 6.11 cm(2)/week, with a weekly reduction index per patient of 0.61 cm(2). At the end of treatment, in the group of patients whose ulcers were treated with cadexomer ointment the average wound size reduction was 43%, whereas in the control-treated patient group the average wound size reduction was 28%.These data suggest that cadexomer can play an important role in the healing of chronic leg ulcers.

The aim of this work was to evaluate the effects of different warming procedures on the survivability of buffalo in vitro matured oocytes vitrified by the Cryotop (CT) method. In vitro matured oocytes were vitrified in a final solution of 20 % ethylene glycol (EG), 20 % of dimethyl sulfoxide (DMSO) and 0.5 M sucrose. In Group A oocytes (n = 111) were warmed in 1.25 M sucrose for 1 min and then exposed to decreasing concentrations of the sugar (0.625, 0.42 and 0.32 M for 30 sec). In Group B, oocytes (n = 122) were warmed into a 0.25 M sucrose solution for 1 min, and then exposed to a 0.15 M sucrose solution for 5 min. Oocytes were rinsed and allocated into the in vitro maturation (IVM) drops for 2 h and then fertilized in vitro. The survival rate was significantly higher in Group A compared to Group B both at 2 h post-warming (92.8 vs 83.6 %; P

The aim of this study was to investigate the effect of the duration of oocyte in vitro maturation (IVM) and gamete co-incubation on the in vitro embryo (IVEP) production efficiency in River buffalo. In Experiment 1, abattoir-derived cumulus oocyte complexes were fixed at 0, 3, 6, 9, 12, 15, 18, 21 and 24 h after the start of in vitro maturation to study the kinetics of nuclear maturation. In Experiment 2, cumulus oocyte complexes were fertilized in vitro following in vitro maturation for 18, 21, 24, 27 or 30 h. After 20 h of gamete co-incubation, presumptive zygotes were denuded and cultured in vitro in synthetic oviduct fluid (SOF) medium. In Experiment 3, following in vitro maturation and fertilization, presumptive zygotes were removed from fertilization drops at 8, 12, 16 and 20 h post-insemination (pi) and placed in culture as described above. Representative samples of oocytes were fixed at 4, 8, 12, 16 and 20 h to evaluate the sperm penetration rate and the incidence of polyspermy at different co-incubation times. The main conclusions of the study are that: (1) the majority of buffalo oocytes accomplish nuclear maturation between 21 and 24 h after the start of in vitro maturation; (2) both cleavage and blastocyst rates linearly decrease with increasing duration of in vitro maturation (from 18 to 30 h); (3) sperm–oocyte incubation for at least 16 h is required for maximum blastocyst yields.

The authors analyse the short-term and medium-term effects of iloprost prostanoid derivate on hemostatic status in a group of patients with obliterating vascular disease of the lower limbs. The study included 10 patients (6 males, 4 females; aged 52 + 5 years old) suffering from Fontaine's stage 3 obstructive arterial disease. After a 10-hour fast each patient received a 6-hour iv infusion of iloprost at a dose of 2 ng/kg/min (approx 50 gamma) a venous blood sample was collected before and after infusion. The test was repeated using the same method after 4 weeks of treatment with the same dose of the drug. The following parameters were analysed in serum: fibrinogen (F) (IL coagulometric method), Factor VII (F VII) (IL coagulometric method), antithrombin II (AT III) (IL chromogenic method), protein C (PC) II coagulometric method) and protein S (PS) (IL coagulometric method). After the first infusion a significant increase was observed in AT III (p0.05), whereas other indices showed no significant variations. After treatment for 4 weeks AT III was again enhanced after infusion (p0.05); with regard to the basal values of other parameters, a significant reduction (p0.05) was found in F VII, whereas no other significant changes were observed. In the light of these results the authors suggest an antithrombotic effect of the drug documented by the short-term increase in AT III probably due to lower consumption, and a medium-term reduction in F VII due to trophic effect of the drug at a vasculoparietal level resulting in the depression of FVII tissue activation factors.

The authors carried out an investigation on the "short" and "middle-term" effect of the prostanoid derivate iloprost on some molecular haemostatic markers in a group of peripheral vasculopathic patients with critical limb ischemia. The series consists of 10 patients (6 males, 4 females, age 52 +/- 5) suffering from peripheral obstructive vasculopathy at the III-IV stage by Fontaine. After overnight fasting, each patient was given an intravenous infusion of iloprost lasting six hours at the rate of 2 ng/kg/min and reaching approximately the global dosage of 50 gamma; before and after the infusion a venous blood sample was withdrawn; the experiment was repeated under the same conditions after a four week treatment with the drug administered daily at the same dosage. For each sample the plasma levels of betathromboglobulin (BTG) fibrinopeptide A (FPA), tissue plasminogen activator (tPA), plasminogen activator inhibitor (PAI-I) and D-dimer (D-D) (ELISA, methods, kits Boehringer) were measured. The basal values of BTG, FPA, tPA, PAI-I and D-D were significantly increased compared to those of a control group; after the iloprost infusion (acute effect) significant changes of the BTG, FPA, tPA and PAI-I were not found; D-D only showed a marked reduction (p0.05); after the four week treatment with infusion the basal values of BTG, FPA, tPA and PAI-I resulted almost unchanged; D-D only showed a marked reduction (p0.05) both as regards the basal value and those after the infusion.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

Porcine pluripotent ES cell lines are a promising tool for biotechnology, biomedical and developmental biology studies. However, no conclusive results have been obtained to derive genuine ES cells in the pig. Here we compare derivation efficiency of putative ES cells from IVF versus parthenogenetic pig embryos. We describe proliferation ability and doubling time, we study pluripotency markers and telomerase activity (TA) of the cell lines obtained. Pig oocytes were either fertilized in vitro or parthenogenetically activated. Blastocysts were subjected to immuno-surgery. Inner cell mass were plated and outgrowth expansion was monitored daily. Self renewal molecules were studied by RT-PCR and/or immunocytochemistry for up to 42 passages. TA was measured every five passages. The results obtained indicate that stable cell lines can be generated from IVF and parthenogenetic embryos. The latter appeared less resilient to immuno-surgery but demonstrated a higher ability to produce outgrowths. 77% of the parthenogenetic lines vs only 33% of the IVF ones expressed pluripotency markers and displayed high TA. Regardless to their origin, colonies showed a latency growth period in the 48 hours after plating, they grew exponentially between day 3 and 6 and then, proliferation rate was greatly reduced. Doubling time was estimated to be 31.5 hours. In both IVF and parthenogenetic cell lines, positivity for Oct-4, Nanog, Sox-2, Rex-1, SSEA-4, Alkaline phosphatase, TRA-1-81 and STAT3 was detected; no signal for LIF-Receptor beta and gp130 was shown. These results indicate that the main pluripotency network related molecules are expressed in the porcine species, while a classical LIF-Receptor beta- gp130-STAT3 activation pathway does not appear to be involved in the maintenance of self renewal. Finally, every cell lines expressed high TA, which was turned down once cells were induced to differentiate, indicating a physiologically normal control of TA in these cells.

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.

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 39�C, 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 39�C. 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.

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.

The aim of this study was to compare the efficiency of different combinations of cryoprotectants for vitrification of IVP buffalo (Bubalus bubalis) embryos at different developmental stages by the open pulled straw (OPS) method. In method 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 before being placed into 1.4 M glycerol + 3.6 M ethylene glycol (EG) for 5 min. Then, embryos were transferred into 3.4 M glycerol + 4.6 M EG for 25 s and loaded into the OPSs. For warming, OPSs were briefly immersed in a 0.5 M sucrose solution; the embryos were exposed to 0.25 M sucrose for 5 min before transfer to SOF medium for culture. In Method B, we examined the vitrification and warming solutions previously used for OPS vitrification of cattle embryos (Vajta et al. 1998 Mol. Reprod. Dev. 51, 53-58). Buffalo embryos were equilibrated in 7.5% EG + 7.5% dimethyl sulfoxide (DMSO) for 3 min before transfer into 16.5% EG + 16.5% DMSO and 0.5 M sucrose. After 25 s, they were loaded into the OPSs. For warming, embryos were recovered in a 0.25 M sucrose solution and transferred into a 0.15 M sucrose solution for 5 min before being placed in SOF medium. A total of 293 IVP buffalo embryos (eight replicates) were vitrified at Day 7 of culture (Day 0 = in vitro fertilization). Embryos were vitrified at the following developmental stages: early blastocyst (eBL, n = 26 and 34 with methods A and B, respectively), blastocyst (Bl, n = 31 and 35 for Methods A and B, respectively), expanded blastocyst (XBl, n = 29 and 38 for Methods A and B, respectively), and hatched blastocyst (HBl, n = 46 and 54 for Methods A and B, respectively). 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 ANOVA following arcsine transformation of data. The overall embryo survival rate recorded at 24 h was not significantly different between Methods A and B (70% vs. 62%, respectively). Specifically, no differences were observed in embryos vitrified at the eBL (70% vs. 73%, A and B, respectively), Bl (69% vs. 70%, A and B, respectively), and HBl (46% vs. 36%, A and B, respectively) stages. In contrast, a significantly higher survival rate was recorded for XBl-stage embryos vitrified-warmed by Method A as compared to Method B (90% vs. 53%, respectively; P < 0.01). In Method A, survival rate of XBl was significantly higher than that of HBl (P < 0.05), but it was not different from that of eBl and Bl. Within Method B, the survival efficiency was similar for eBL, BL, and XBl, whereas survival rate of HBl was significantly lower (P < 0.05). In conclusion, although overall embryo survival in vitro was similar between methods, the combination of cryoprotectants used in Method A seemed more suitable for vitrification of IVP buffalo embryos at the XBl stage.

The efficiency of in vitro embryo production (IVEP) in buffalo is hampered by the poor cleavage rate. The quality of the frozen semen may affect fertilization efficiency, due to damages of the male gamete that occur following cryopreservation. 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 IVF system. Although several agents have been proven to induce sperm capacitation in vitro, heparin is still the most efficient method in most of the domestic species. The aim of the study was to evaluate the efficiency of buffalo estrus serum (BES) and follicular fluid (FF) to induce buffalo sperm capacitation in vitro, indirectly assessed by estimating the capability of spermatozoa to acrosome react. Frozen semen from a bull previously tested for IVF, thawed at 37°C for 40 s in water, was treated by swim-up in order to select only the motile population. Spermatozoa (n = 1546) were assessed immediately after swim-up separation, to evaluate the incidence of acrosomal loss in nontreated cells (time 0). The remaining spermatozoa were incubated in the presence of 0.01 mM heparin (control; n = 3531), 20% BES (n = 2442) and 20% FF (n = 1419), the latter recovered from a pool of dominant follicles, for 1, 2, and 3 hours. Sperm was then exposed for 15 min to 60 μg mL−1 of lysophosphatidylcholine, an agent known to induce acrosome reaction only on capacitated spermatozoa. Trypan blue was used 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 χ2. No dead spermatozoa were found in all groups. Acrosomal loss was observed in only 15.3% of the sperm population at time 0; it may be accounted for either by damages preceding cell death or by freezing-induced capacitation. No differences were found between incubation times within each treatment group. Interestingly, sperm treatment with both BES and FF resulted in a significantly higher incidence of acrosome reaction compared with heparin (84.3, 94.5 vs. 50.1%, respectively; P < 0.001), the capacitating agent currently used in the IVF system, and, in particular, FF showed the highest percentage of acrosome reaction at all incubation times, even when compared with BES (P < 0.01). It is likely that factors derived by BES and FF, present in the oviduct environment around fertilization, play a critical role in processing the male gamete in vivo. These preliminary results show the possibility of significantly improving the efficiency of sperm capacitation in vitro in buffalo species with BES and FF and strongly suggest investigating the effects of these factors also on the fertilizing capability of buffalo spermatozoa. The authors thank to Dr. O. Paciello for his technical assistance.