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The aim of this study was to evaluate the influence of temperature during ovary collection/transportation and that of the time interval between ovary collection and processing in the laboratory, on in vitro embryo production efficiency in buffalo species. A retrospective analysis of data collected over the last 4 years in our lab was carried out on 3461 oocytes, recovered over 120 replicates. No differences in oocytes developmental competence were observed in relation to the time interval between ovary collection and processing in the laboratory (3-6 h). On the contrary, a correlation was found between oocyte developmental competence, evaluated in terms of cleavage and blastocyst rate, and temperature during ovary collection/transportation. In particular, lowering temperature during ovary transportation significantly improved both cleavage (74.6 % vs 65.8 and 63.1 % for temperature ranges of 25-30°C, 30-34°C and 34-37°C, respectively ; P

The aim of this study was to screen for Brucella spp. buffalo embryos produced in- vitro, by using cumulus oocytes complexes (COCs) recovered from ovaries of slaughtered buffaloes naturally infected with Brucella spp. Ovaries were collected from 5 female pluriparous buffaloes slaughtered in a local abattoir. EDTA-blood samples and nasal swabs collected from each animal were used for Brucella spp. DNA detection by real-time PCR. Buffalo ovaries (n = 10) were transported to the laboratory and maintained strictly separated throughout laboratory processing. Recovered COCs were matured, fertilized and cultured in vitro until day 7. Some immature COCs, all uncleaved COCs, all blocked cleaved embryos (2 to 16 cells) and all transferable embryos (tight morulae and blastocysts) were separately analysed by real-time PCR assay. Brucella spp. DNA was detected in both blood and nasal mucus of all subjects, whereas no trace of DNA of Brucella spp. was found on either COCs or embryos. Currently, the infected or seropositive buffaloes have to be slaughtered for sanitary reasons. Interestingly, the results of this preliminary trial suggest a possible utilization of the COCs from the infected subjects of high genetic value to obtain safe embryos.

The aim of this study was to evaluate the possibility to vitrify in vitro produced (IVP) buffalo and bovine embryos at different stages of development by an advanced version of the “minimal volume approaches”: the Cryotop method. In both experiments, the embryos were vitrified at the tight morula (TM), early blastocyst (eBl), blastocyst (Bl), expanded blastocyst (xBl) and, only for buffalo, at the hatched blastocyst (hBl) stage. After warming, the embryos were cultured in vitro for 24 hours. Stage of development affected the freezability of IVP embryos of both species with the highest embryo survival rates at advanced stages (xBl=76% and hBl=75% for buffalos and xBl=75% for bovine). These results suggest that Cryotop vitrification is an efficient method for buffalo and bovine IVP embryo cryopreservation.

The aim of this work was to verify the feasibility of an easy, quick double staining technique for evaluation of frozen-thawed semen to predict the fertilizing capability in vitro of buffalo bulls. In Experiment 1, frozen-thawed semen from 6 bulls was stained with double Trypan blue/ Giemsa and the incidence of acrosome-intact live (AIL), acrosome-intact dead (AID), acrosome-lost live (ALL) and acrosome-lost dead (ALD) sperm was recorded. In Experiment 2, sperm from the same bulls were used to fertilize in vitro matured oocytes. The data obtained confirm that there is a strong “bull effect” in buffalo species, with differences in the percentage of AIL sperm at thawing, in cleavage and blastocyst rates among bulls. Interestingly, it was found that this staining technique can be used for a preliminary screening to select semen to use for IVF, as shown by the correlation existent between the percentages of acrosome-intact viable sperm cells at thawing and the blastocyst yields for 4/6 bulls.

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

Although great importance has been recently given to the in vitro embryo production (IVEP) techniques in buffalo species, the overall efficiency is still low because many physiological aspects of oocytes/embryos have not been properly investigated. It has been speculated that buffalo oocytes/embryos, due to their high lipid content (Boni et al., 1992), are particularly sensitive to the increased oxidative stress, that occurs under in vitro conditions (Gasparrini et al., 2003).

The aim of the present study was to evaluate the effect of melatonin supplementation during in vitro maturation on fertilization and embryo development in cattle. Bovine cumulus-oocyte-complexes (COC), recovered from abattoir ovaries, were matured in vitro in the absence (control) and in the presence of 10 μM, 100 μM and 1 mM of melatonin. Matured oocytes were fertilized in vitro with frozen-thawed sperm and cultured up to the blastocyst stage. The results of this work demonstrated that melatonin enrichment of the in vitro maturation (IVM) medium does not affect both cleavage (71.0, 72.8, 72.5 and 72.7 % in the control group and in the groups supplemented with 10 μM, 100 μM and 1 mM of melatonin respectively) and blastocyst rates (41.3, 33.8, 39.4 and 38.3 % respectively) in cattle.