First pregnancies carried to term after transfer of vitrified buffalo embryos entirely produced in vitro.

Abstract

The application of Ovum Pick-up (OPU) technology, together with multistep embryo production in vitro (IVEP), represents a valid procedure for the recovery of oocytes from live donors and the attainment of a large number of embryos (Looney et al., 1994; Galli et al., 2001). In fact, collection of oocytes by means of ultrasound guided follicular aspiration is routinely performed with success in large prepubescent and adult ruminant species (Galli et al., 2001). In the buffalo species, because of their low response to superovulation treatments (Karaivanov, 1986), OPU technology, together with an improvement in the multistep process of in vitro embryo production, seems to be the best way to enhance genetic progression through the maternal lineage (Galli et al., 2001; Neglia et al., 2003). However, in vitro embryo production in buffalo has always been associated with a lower efficiency as compared to the bovine (Totey et al., 1992), although recently some encouraging results have been reported both from slaughterhouse recovered oocytes (Gasparrini et al., 2000), and from OPUderived oocytes (Neglia et al., 2003). Obviously, in order to optimize the procedure, an efficient method for embryo freezing should be developed. In fact, until now the only pregnancies recorded for the buffalo species were obtained from the transfer of fresh embryos produced in vitro. In 1991 Madan et al. (Madan et al., 1991) reported the birth of the first buffalo calf after the transfer of a fresh blastocyst obtained through in vitro maturation (IVM), in vitro fertilization (IVF) and in co-culture with oviductal cells (IVC). Four other calves were produced in India in 1994, by transfer of fresh blastocysts obtained in vitro (Madan et al., 1994), using the same method. In 1997, Boni et al. (Boni et al., 1997), reported two pregnancies obtained from the transfer of two fresh morulae, produced after OPU/IVEP procedures. Only three calves have been produced from frozen blastocysts (Galli et al., 1998); in this case zygotes (2 days after IVF) were transferred into ligated sheep oviduct before freezing. The possibility of cryopreserving buffalo embryos by vitrification with good efficiency in terms of in vitro survival rate, has recently been reported (Neglia et al., 2001). On