A method for preparation of chromosomes from bovine zygotes and blastocysts

Abstract

King, W.A., Linares, T., Gustavsson, I. and Bane, A., 1979. A method for preparation of chromosomes from bovine zygotes and blastocysts. Vet. Sci. Commun., 3:51-56.A simple technique for making chromosome preparations from zygotes and early blastocysts is described. The morphological features of blastocysts and total number of cells greatly influence the quality of the preparation. ~qTRODUCTIONThe problems of loss of embryos, poor spreading of chromosomes and over-scattering of cells have been noted to be major handicaps of embryo chromosome analysis with the two basic methods for making preparations, namely squashing and air drying (Fujimoto et al., 1975). In additio~it is often difficult, when working with embryos of a relatively small number of cells, to obtain a reasonable number of cells which have chromosomes in a stage of contraction suitable for analysis. The availability of suitable methods for collection of bovine blastocysts has aided the development of methods for evaluation of karyotypes of early preimplantation embryos and enhanced the possibility of correlating the karyotypes with morphological features of these embryos. In this communication a technique for the preparation of chromosomes from bovine zygotes and early blastocysts is described. Some observations on the quality of preparations obtained from embryos which had normal and abnormal morphological features are presented. MATERIALS AND METHODSSixteen blastocysts and four 2-celled zygotes were used (Table i). The blastocysts were recovered 6 to 7 days after heat (day of heat=0) by a non-surgical

Required Concentration and Time of Vinblastine Treatment for Chromosome Preparation in Bovine Blastocysts Derived from In Vitro Fertilization.

“…In investigations of chromosomes, with various mitotic inhibitors, many researchers have used short treatment times for bovine embryos fertilized in vitro. King et al [15] have described a method for preparing chromosomes from bovine blastocysts using 200 µg/ ml colchicine for 2 to 9.5 h. They have shown the mitotic indexes to be 14.7-37.5% in morphologically good blastocysts treated for 8.5-9.5 h, and 4.2-14.3% in embryos treated for 2-4 h. Kajihara et al [5] have reported frequencies of bovine embryos having metaphase plates of approximately 80-83%, and the sexing rate of 63.4% in embryos treated with 0.04 µg/ml colcemid for 1.5-3 h. Tokumaru et al [16] have also reported mitotic indexes of about 5-7% in bovine blastocysts treated with 0.04 µg/ml colcemid for 2 h. Avery et al [17] have shown a low mitotic index of 3.5%, in bovine in-vitro fertilized blastocysts treated with 0.01 µg/ml colcemid for 2 h. Iwasaki et al have reported mitotic indexes of approximately 5 to 12% in bovine blastocysts treated for 4 h with a combination of vinblastine sulfate and podophyllotoxin [7], and a mitoic index of approximately 26% in morulae or blastocysts treated for 6 h with 0.4 µg/ ml vinblastine sulfate [8]. If the treatment time is longer, the mitotic index is higher.…”

Chromosome analysis of bovine oocytes cultured in vitro

“…The oocytes used for this study were prepared by the method described by Xu et al (1987) (King et al, 1979).…”

Chromosome analysis of horse oocytes cultured in vitro

“…by treatment with a mixture of trypsin (1/!/m!) and pronase (lpg/ml) and hyaluronidase (I JL g/ml) and the oocytes were fixed individually on slides (King et al, 1979). Control oocyte-cumulus complexes were examined, their cumulus cells dispersed and they were fixed without culture.…”