157 PREGNANCIES AND CALVES AFTER TRANSFER OF IN VITRO-PRODUCED RIVER BUFFALO EMBRYOS AFTER CRYOPRESERVATION

Abstract

In the buffalo, the use of embryo-based biotechnologies for breeding and genetic improvement is still very limited because multiple-ovulation embryo transfer delivers poor results compared with cattle and in vitro embryo production has been used mainly for research purposes. At present, very few reports are available on the transfer of in vitro-produced (IVP) and cryopreserved buffalo embryos. Therefore, the scope of this work was to perform a pilot study to evaluate the viability of frozen-thawed IVP embryos by nonsurgical embryo transfer to recipients in an IVF-embryo transfer program on a farm located on the north coast of Colombia, South America. Buffalo oocytes were recovered at the slaughterhouse from selected donors, matured in vitro for 18 to 20 h in TCM-199 + 10% FCS and 0.5 IU of FSH and 0.5 IU of LH in 5% CO₂ at 38.5°C. Four different bulls were used for IVF. After thawing, the semen was separated on a Percoll^® gradient and then diluted into SOF-IVF media supplemented with 1 μg mL^–1 of heparin and phenylalanine. Presumptive zygotes were cultured in modified SOF supplemented with MEM amino acids for 6 days. Half of the medium was replaced on Day 4 and 6. Developing embryos were selected for freezing on Day 6 and 7. Grade 1 embryos were frozen at the blastocyst stage by slow cooling in 10% glycerol or 1.5 M ethylene glycol. Recipients (heifers n = 79 and uniparous cows n = 17) were synchronized using the CIDR-Synch protocol: on Day 0, gonadotropin-releasing hormone was injected and a CIDR was inserted; on Day 7, prostaglandin F_2α was administered; on Day 9, the CIDR was removed; on Day 11, a second injection of gonadotropin-releasing hormone was given; and on Day 17, the embryo was transferred. Each female received, nonsurgically, 1 or 2 embryos in the ipsilateral horn to the functional corpus luteum evaluated by ultrasonography. Pregnancies were evaluated by ultrasonography 30 days after transfer and confirmed by rectal palpation 30 days later. This work was performed in 2 successive experiments during the breeding seasons (January and December, respectively). Overall, 96 recipients were transferred, with 136 embryos obtaining 23 pregnancies (24.2%). There were no statistical differences in pregnancy rate between heifers and cows (25.3 vs 17.7%) and between single (n = 56) and double (n = 39) embryo transfers (21.4 vs 27.5%) by chi square test (P > 0.05). To date, 4 females and 5 males have been born by spontaneous calving (1 stillborn male due to dystocia), 3 pregnancies have been aborted (13%) and 11 pregnancies are ongoing (>7 months). The pregnancy rate obtained in this study in farm conditions (24.2%) is lower than generally obtained with frozen IVP cattle embryos, but it is still a good result in buffalo, where even conventional AI provides a lower success rate as compared with cattle. Finally, this work demonstrates that in vitro embryo production can be successfully implemented in buffalo breeding programs for the exploitation of superior genetics.

This work was supported by Regione Lombardia, Por Fers 2007–2013, n°13827741, InnovaB.