Reproductive biotechnology applied to the Bubalus bubalis genetic improvement in Cuba

Abstract

The present work was aimed at establishing a methodology for obtaining Bubalus bubalis embryos under tropical conditions. The study was focused on ovaries of slaughtered buffalo females of 66 ± 6 months of age and 475 ± 25 kg of weight, from which the immature cumulus-oocyte complexes were extracted by the follicular puncture method, having diameters less than 3 mm, between 3mm and 5mm; and those greater than 5 mm using an 18 G needle attached to a 10 mL syringe. In vitro maturation was carried out in TCM199 medium and it was evaluated by chromatin condensation and the presence of the first polar body. In vitro fertilization was performed in Talp-Fertile medium using frozen buffalo semen tablets, at a concentration of 4x10⁶ spz/mL. Embryos were cultured on a monolayer of cumulus cells in SOFaa medium. There were 8.02 follicles/ovary with different diameters: 53.01 %, 28.23 % and 18.75 %. There were 3.93 COC/collection ovaries and, from them, the 58 % was matured. They passed to the fertilization stage in groups of 25 to 35 oocytes per 400 μL of medium, reaching 38.79 % of cleavage and 17 % of buffalo embryos, which developed properly until reaching the blastocyst stage. Based on these results it is concluded that the most appropriate protocol for the genetic improvement of the species is the combination of the follicular puncture method, followed by the maturation of the oocytes for 24 hours under conditions similar to those of cattle, but fertilized with 4x10⁶spz/mL, followed by cultivation in SOFaa medium.