Ovum pick-up and in vitro embryo production in buffalo species: an update

The present review intends to describe the state of the art of IVEP in buffalo species, emphasizing the advances achieved in the sequential steps of in vitro maturation (IVM), in vitro fertilization (IVF) and in vitro culture (IVC), and the limitations still to overcome. The IVM system has been greatly improved over the years, leading to 90 % maturation rate. In particular, the enrichment of the IVM medium with thiol compounds, known to act as antioxidants factors, has highly improved the IVEP efficiency. The important role of antioxidants factors during IVM has been confirmed by the improved blastocyst development recorded by supplementing the IVM medium with taurine and melatonin. A major factor impairing IVF in buffalo is the bull effect, consisting in the high degree of variation between buffalo bulls in the fertilizing capability in vitro. It has also been demonstrated that marked differences in the kinetics of sperm penetration exist among buffalo bulls and that this parameter can be a useful marker to predict the in vitro fertilizing ability. Although heparin is the in vitro capacitating agent most widely used, a significant improvement of sperm capacitation has been obtained by incubating sperm with biological fluids, such as buffalo estrus serum and the follicular fluid recovered from a pool of dominant follicle. We have recently demonstrated that osteopontin (OPN), a protein present in the oviduct fluid and in the semen, improves capacitation and enhances both cleavage rate and blastocyst yields. The great improvement of blastocyst yields (35-40%) achieved in the following years is, according to our experience, due to the optimization of the IVM and the IVF systems rather than to modifications applied to the IVC system. In fact, at present the original version of SOF remains the most suitable medium for embryo culture in buffalo. Nevertheless, we have demonstrated that energy requirements of buffalo embryos are different from those of sheep and cattle. These findings highlight the importance of identifying species-specific requirements in order to optimize the in vitro culture conditions for buffalo embryos. The major limitation to the diffusion of IVEP in buffalo is the low number of recruitable oocytes, arising from peculiarities of the reproductive physiology of the species. Another limitation is given by the high susceptibility of buffalo IVP embryos to cryopreservation, in part due to their greater lipid content, but also to their poor viability, likely determined by suboptimal in vitro culture conditions. In conclusion, the optimization of culture conditions is fundamental to increase the viability of IVEP embryos for advanced reproductive strategies to become a routine procedure in buffalo breeding. At the same time efforts should be addressed to identify the best management of the recipients, in order to increase pregnancy maintenance after ET of cryopreserved IVP embryos.