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 cross breed cattle (Holstein x Zebu) as donors of oocytes for in vitro ferti lization (IVF) and for pronuclear gene microinjection into in vitro-pr oduced embryos. In the first part of the experiment oocytes from Bos t aurus (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 usi ng 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%). Holste in cows yielded the highest number of cumulus oocyte complexes (6.8 pe r ovary) for in vitro maturation, differing (P<0.05) from Zebu x Holst ein and Zebu x Holstein x Holstein F1 by 5.1 and 5.8, respectively. Ho wever, the Holstein breed also yielded the lowest percentage of cleava ge (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 effici ent source of oocytes for the production of embryos capable of reachin g 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 r emaining 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) cle aved in culture. Of the microinjected embryos, 1 out of 50 showed D-ga lactosidase activity. These findings indicate that a tropical crossbre ed of cattle (Zebu x Holstein x Holstein) can be used as a source of o ocytes for IVF programs and gene microinjection studies.