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Vitrification of in vitro-matured oocytes has important applications in fertility preservation and management of genetic resources. However, despite the increasing interest, the efficiency of oocyte vitrification needs to be improved. It was demonstrated that under stressful conditions of cryopreserving pig oocytes accumulate reactive oxygen species (ROS; Gupta et al. 2010 Fertility and Sterility 93, 2602-2607). Reactive oxygen species are known to exert harmful effects such as mitochondrial damage, ATP (ATP) depletion, altered calcium oscillation during fertilization and consequently their developmental ability may be compromised (Takahashi et al. 2003 Mol. Reprod. Dev. 66, 143-152). The aim of the present study was to evaluate whether the exposure to cryoprotectants and vitrification procedure affect ROS production in bovine in vitro-matured oocytes. Abattoir-derived bovine (n = 360, over 6 replicates) cumulus oocyte complexes (COCs), were in vitro-matured. COCs were mechanically stripped of their cumulus cells by gentle pipetting, washed and divided into 3 groups: control (C; i.e. fresh non treated oocytes), toxicity (T) and vitrification (V) groups. In group V, oocytes were exposed to 10% ethylene glycol (EG) + 10% DMSO for 3 min, then to 20% EG + 20% DMSO and 0.5 M sucrose, loaded on cryotops and plunged into liquid nitrogen within 25 s. Oocytes were warmed into a 1.25 M sucrose solution for 1 min and then to decreasing concentrations of sucrose (0.625 M, 0.42 M and 0.31 M) for 30 s each. In group T, oocytes were simply exposed to the vitrification and warming solutions. ROS determination was carried out by a spectrofluorometer at 495 nm excitation and 525 nm emission. Frozen oocytes were thawed and incubated in 500 μL of TRIS-HCl 40 mM, pH 7.0 in the presence of 5 μmol L-1 of 2′,7′-dichlorfluorescein-diacetate, for 20 min at 37°C into a shaker. After incubation, the extraction was obtained by a syringe and the samples were centrifuged at 3000 rpm for 10 min at 4°C. Data were expressed as arbitrary ROS units per oocyte per min (U) and analysed by ANOVA. The results of this study showed that in bovine oocytes ROS levels tend to increase in the T and V groups compared to group C (76.0 ± 6.4, 249.9 ± 87.3 and 147.6 ± 42.6 in C, T and V groups, respectively). However, there were no statistical differences among groups and this was mainly due to the high variability recorded in both treated groups. In conclusion, these results suggest that both exposure to cryoprotectants and vitrification of in vitro-matured oocytes may influence ROS generation. However, the high variability recorded among replicates recommends further investigations. [ABSTRACT FROM AUTHOR]

Plasminogen activators (PA) are proteolytic enzymes that convert plasminogen into plasmin. Plasmin is involved in physiological processes such as ovulation (Liu 2004 Front. Biosci. 9, 3356-3373), cumulus cell layer expansion (Liu et al. 1986 Endocrinology 119, 1578-1587), oocyte maturation (Dow et al. 2002 Biol. Reprod. 66, 1413-1421) and fertilization (Huarte et al. 1993 Dev. Biol. 157, 539-546). Although the interest is increasing, buffalo oocyte cryopreservation is still inefficient, especially in terms of blastocyst development after IVF. The aim of the present study was to evaluate whether exposure to cryoprotectants and the vitrification procedure affect plasminogen activator activity (PAA) in buffalo in vitro-matured oocytes. A total number of 300 cumulus-oocyte complexes over 5 replicates were selected and in vitro-matured. Cumulus-oocyte complexes were mechanically stripped of their cumulus cells by gentle pipetting, washed and divided into 3 groups (20 oocytes/group, over 5 replicates). The control group consisted of fresh in vitro-matured oocytes. In the vitrification group, denuded oocytes were first exposed to 10% ethylene glycol (EG) + 10% dimethyl sulfoxide (DMSO) for 3 min, then to 20% EG + 20% DMSO and 0.5 M sucrose, loaded on cryotops and plunged into liquid nitrogen within 25 s. Subsequently, oocytes were warmed in a 1.25 M sucrose solution for 1 min and then in decreasing concentrations of sucrose (0.625 M, 0.42 M and 0.31 M) for 30 s each. In order to test cryoprotectant effects, oocytes were simply exposed to the vitrification and warming solutions (toxicity group). Surviving oocytes were extracted by a fine needle, centrifuged at 4000 rpm for 10 min and the supernatant was mixed with the reaction solution: TRIS-HCl 0.1 M, homologous plasminogen, the chromogenic substrate for plasmin S-2251 and incubated at 37°C for 30 min. The PAA levels were measured by a spectrophotometer (405 nm) expressed as Abs/20 oocytes. The data were analysed by the Kruskal-Wallis nonparametric test. Low levels of PAA were detected in the denuded oocytes of the control, toxicity and vitrification groups. No significant differences in mean PAA values were observed among the 3 experimental groups (0.017 ± 0.001, 0.018 ± 0.002 and 0.017 ± 0.001 units, in the control, toxicity and vitrification groups, respectively). In conclusion, cryoprotectants and the vitrification procedure do not affect the proteolytic activity linked to plasmin in in vitro-matured buffalo oocytes. The results show that the vitrification/warming procedure does not exert an effect on in vitro-matured buffalo oocytes in terms of PAA generation, a parameter that plays an important role in fertilization and in vitro embryo development. Further studies are needed to identify factors affecting the efficiency of oocyte cryopreservation. [ABSTRACT FROM AUTHOR]

Removal of cumulus cells is necessary for several technologies such as vitrification, intracytoplasmic sperm injection, and nuclear transfer. However, it is known that the presence of cumulus cells during IVF of buffalo oocytes is fundamental for fertilization and embryo development (Gasparrini et al.2007 Anim. Reprod. Sci. 98, 335–342; Nandi et al.1998 Theriogenology 50, 1251–1262). The aim of this work was to evaluate whether co-culture with intact bovine cumulus–oocyte complexes (COC) during IVF would restore the developmental competence of denuded buffalo oocytes. Due to the scarce availability of buffalo ovaries, the somatic support was provided by bovine cumulus cells. Abattoir-derived COC were matured in vitroaccording to our standard procedures (Gasparrini et al.2006, Theriogenology, 65, 275–287) and randomly distributed in 3 fertilization groups: 1) a control group of COC (n=122), 2) a negative control of denuded oocytes (DO; n=119), and 3) DO co-cultured with in vitromatured bovine COC (DO+COC; n=103) in a 1:1 ratio (3 bovine COC+3 denuded buffalo oocytes/50μL drop). Fertilization was carried out with frozen–thawed spermatozoa from a tested bull in TALP medium supplemented by 0.2mM penicillamine, 0.1mM hypotaurine, and 0.01mM heparin at 38.5°C under a controlled gas atmosphere of 5% CO2in humidified air. After fertilization the zygotes were cultured in SOF medium including essential and nonessential amino acids and 8mgmL-1BSA, at 38.5°C under humidified 5% CO2, 7% O2, and 88% N2, up to the blastocyst stage. On Day 5 and on Day 7 (Day 0=IVF) cleavage and blastocyst rates were respectively recorded. Data were analysed by chi-square test. As expected, cleavage and blastocyst rates were lower (P<0.01) in DO (36.1 and 9.2%, respectively) compared with the control (67.2 and 27.1%, respectively). However, co-culture during IVF (DO+COC) significantly increased (P<0.01) both parameters compared with DO, giving cleavage (70.9%) and blastocyst (27.2%) rates similar to the control. The results of this study demonstrated that co-culture with bovine intact COC during IVF of buffalo denuded oocytes completely restores their fertilizing capability and blastocyst developmental competence. We conclude that this may be a suitable strategy for preserving the developmental competence of oocytes devolved to technologies, such as oocyte vitrification, that require cumulus removal. [ABSTRACT FROM AUTHOR]

The overall in vitroembryo production efficiency in buffalo is hampered by the poor IVF efficiency. The aim of this work was to evaluate whether the fertilizing ability of buffalo sperm is improved by the presence of bovine oviductal cells (BOEC) during IVF. Because of limited availability of buffalo oocytes, this was assessed by heterologous IVF. Bovine oviducts were obtained at a local abattoir from cows that were in the preovulatory phase of a normal estrous cycle. BOEC recovered from 5 oviducts as previously described (Gualtieri and Talevi 2000 Biol. Reprod. 62, 1754-1762) were pooled and plated in 100 μL drops of TCM-199 + 10% FCS, 100 U mL-1penicillin, 100 μg mL-1streptomycin and 0.25 μg mL-1amphotericin B under mineral oil. Medium was changed every 48 h up to Day 6, when cell confluence and cilia activity were optimal. On day of IVF the medium was removed from the drops and replaced with TALP supplemented with 0.2 mMpenicillamine, 0.1 mMhypotaurine, and 0.01 mMheparin (IVF medium). Frozen-thawed sperm from an IVF-tested buffalo bull, treated by Percoll gradients, were used for all IVF groups (2 × 106sperm mL-1). In vitro-matured bovine oocytes (n= 409), over 3 replicates, were distributed in 4 fertilization groups: (A) IVF medium alone (control); (B) BOEC monolayer + IVF medium; (C) sperm preincubated for 6 h in IVF medium; and (D) sperm preincubated for 6 h with BOEC + IVF medium. After 20 h of coincubation at 38.5°C and 5% CO2in air, putative zygotes were denuded, washed, and cultured in SOF medium. Forty-eight hours after IVF, cleavage rate was evaluated, and cleaved and uncleaved oocytes were fixed in 60% methanol and stained with DAPI for nuclei examination under fluorescence microscope. Data were analyzed by chi-square test. Although cleavage rate was not different among groups (46.2, 55.8, 50.0, and 50.0% for A, B, C, and D, respectively), the monospermic penetration rate increased (P4 cells) was higher (P< 0.01) in groups C and D (47.9 and 37.1%, respectively) than in group A (12.1%), whereas group B (21.0%) was only different from group C. We demonstrated that the fertilizing capacity of buffalo sperm, evaluated as oocyte penetration rate after heterologous IVF, is enhanced by the presence of BOEC. This suggests that IVF of buffalo oocytes on BOEC monolayer may improve the IVF efficiency in buffalo. The higher incidence of advanced embryos in both groups with preincubated sperm may be accounted for by an earlier accomplishment of capacitation, leading to anticipated oocyte penetration. However, because the penetration rate in these groups was not improved compared with the control, we hypothesize that sperm viability may have decreased and hence that shorter incubation times should be tested in further studies. [ABSTRACT FROM AUTHOR]