Use of F1 progeny of Holstein X Zebu cross cattle as oocyte donors for in vitro embryo production and gene microinjection

This study was designed to determine the possibility of using F1 crossbreed cattle (Holstein X Zebu) as donors of oocytes for in vitro fertilization (IVF) and for pronuclear gene microinjection into in vitro-produced embryos. In the first part of the experiment oocytes from Bos taurus (Holstein), Bos indicus (Zebu) and F1 crossbred Bos taurus X Bos indicus (Holstein X Zebu) genotypes were inseminated with Bos taurus (Holstein) semen and were allocated for in vitro embryo production using conventional IVF procedures. No differences were observed on the in vitro maturation (IVM) rates between breeds (Holstein X Holstein: 85%, Zebu X Holstein: 84% and Zebu X Holstein X Holstein: 88%). Holstein cows yielded the highest number of cumulus oocyte complexes (6.8 per ovary) for in vitro maturation, differing (P < 0.05) from Zebu X Holstein and Zebu X Holstein X Holstein F1 by 5.1 and 5.8, respectively. However, the Holstein breed also yielded the lowest percentage of cleavage (45.1 vs 71.9% for Zebu X Holstein and 65.1% for Zebu X Holstein X Holstein). Of the 3 genotypes, the hybrid F1 breed was the most efficient source of oocytes for the production of embryos capable of reaching morulae and blastocyst stages (76/250; P < 0.001). In the second part of the study, 599 oocytes from the F1 breed were fertilized in vitro, 1 group of 150 oocytes was used for the determination of the optimal pronuclear visualization period. The highest number of oocytes with 2 pronuclei was observed between 24 to 28 h after IVF (27 to 42%). The remaining 399 oocytes were microinjected with a gene construct bearing the bacterial lacZ gene as the reporter for gene expression. Survival of embryos to microinjection was 73.8%, and 45.5% of them (50/110) cleaved in culture. Of the microinjected embryos, 1 out of 50 showed beta-galactosidase activity. These findings indicate that a tropical crossbreed of cattle (Zebu X Holstein X Holstein) can be used as a source of oocytes for IVF programs and gene microinjection studies.