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

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. Until now the only pregnancies recorded for the buffalo species were obtained from the transfer of fresh embryos produced in vitro. Only three calves have been produced from frozen blastocysts; 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. On the basis of these results a one month trial was carried out on a buffalo farm in order to verify the efficiency of this freezing method, by transferring vitrified and fresh embryos produced using OPU-IVEP technology. 12 lactating pluriparous buffalo cows underwent repeated transvaginal follicular aspiration twice-weekly for seven sessions. OPU was carried out by using a portable ultrasound unit and a metal guide to fit 17 gauge needles both allocated in a properly designed vaginal guide. The Cumulus Oocyte Complexes (COCs) were searched immediately after filtering the aspirated follicular fluid and aspiration medium. COCs were moved to the lab within 4 to 6 h where they were transferred into 50 ml droplets of a final maturation medium consisting of bicarbonate-buffered TCM 199 (B 199) with hormones and cysteamine under medical oil. The droplets were incubated at 38.5°C for 22–24 h under a controlled gas atmosphere. The day after IVM the oocytes were in vitro fertilized. After 20–22 h presumptive zygotes were cultured in 20 ml droplets of SOFaaBSA in a modulation chamber. On day 5 (day 0=IVF day) the cleavage rate was assessed and embryos were transferred into fresh droplets of the same medium for two further days of culture. Final embryo output (morulae +blastocysts) was evaluated on day 7 of culture. Some embryos were transferred fresh into opportunely synchronized buffalo heifers, by using a double injection of PGF2a 12 days apart. Other embryos were vitrified using a method previously described and transferred after warming. Confirmation of pregnancy was obtained by rectal palpation of the genital apparatus at 45 and 120 days (after embryo transfer). A total number of 15 embryos were transferred: 7 blastocysts were transferred fresh and 8 after vitrification and warming. No pregnancies were assessed from fresh transferred embryos, whereas three pregnancies were found at 45 and 120 days after transfer of vitrified embryos (37.5%). Unfortunately one pregnancy was interrupted at 180 days, while the other two reached full term. In February 2003 the first two buffalo calves from vitrified embryos entirely produced in vitro were born. We underline the lack of pregnancies established by fresh embryos: this surprising finding may be explained by the higher sensitivity of buffalo embryos to environmental stress as compared with bovine. However, the results obtained in this trial, demonstrate the possibility of using vitrified embryos produced in vitro in order to produce buffalo calves. This may in turn allow the application of OPU/IVEP technology to accelerate genetic progression through maternal lineage.