No abstract

Assisted reproduction technologies have made great progress during the last 15 years in most mammalian species, including humans. Growing evidence indicates that bovine pre-implantation development is a superior model for investigating early human development than the mouse. The purpose of this study was to investigate the effects of two basic culture systems [tissue culture medium (TCM) with 5% CO(2) in air or synthetic oviduct fluid (SOF) with 7% O(2), 88% N(2,) 5% CO(2)] and various protein supplements (serum, bovine serum albumin or polyvinyl alcohol) on the relative abundance of a set of developmentally important gene transcripts in bovine morulae and blastocysts and to compare the results with those for their in-vivo-derived counterparts. The basic culture system including the basic medium composition and oxygen tension had profound effects on the amounts of specific transcripts in bovine embryos, whereas the 'protein source' had only weak effects. Significant differences (P < or = 0.05) in the relative abundance of specific gene transcripts were detected between in-vivo and in-vitro-derived embryos, especially at the morula stage. More differences were found between embryos produced in the TCM system and in-vivo-derived embryos than between SOF-generated embryos and their in-vivo counterparts. No differences were found in the relative abundance of gene transcripts in embryos generated under chemically defined conditions in the two different laboratories. It is concluded that the SOF system provides an environment in which pre-implantation development of bovine embryos is more similar to that occurring in vivo than in the TCM system.

Simian virus 40 (SV40) adsorbs on rabbit spermatozoa but does not penetrate the cells, as indicated by the absence of radioactive material seen on autoradiography of spermatozoa exposed to 13Hithymidine-labeled SV40. In contrast, after exposure of spermatozoa to labeled SV40 DNA, radioactive material was found in the postacrosomal area of the spermatozoa. Furthermore, when spermatozoa exposed to SV40 DNA were fused with cells of the CV-1 line of African green monkey kidney cells, infectious SV40 was isolated. After uterine insemination of rabbits with spermatozoa infected with SV40 DNA, both unfertilized and one-and two-celled fertilized ova were obtained. When The cells were then washed with KRPA and, in some experimnents, treated with rabbit anti-SV40 serum at dilutions of 1: 50-1:300. The spermatozoa were washed with KRPA and maintained in culture medium for 24 and 48 hr at 37-380C.In some experiments, spermatozoa were exposed to SV40 DNA or to [3H ]thymidine-labeled SV40 DNA at a concentration of 1.0-10.0 gg/107 spermatozoa in the presence of 3% (v v) dimethvl sulfoxide. After incubation of the suspension for 2 hr at 37-380C, the spermatozoa were washed in KRPA and some of the samples were treated with 100 ,g/ml deoxyribonuclease I (DNase), dissolved in synthetic medium, for 0.5 hr at 37-380C under 5% CO2 in air.The spermatozoa were washed again and maintained in culture medium for 24 and 48 hr. The culture medium consisted of the synthetic medium with 20% (v/v)

Three approaches were investigated for improvement of in vitro maturation (IVM), in vitro fertilization (IVF), and early embryonic development in cattle. These were: 1) Selection of oocytes, 2) medium supplementation with fetal calf serum (FCS) and cow sera (D0, D1, D10, and D20 to correspond with estrus, metestrus, diestrus, and proestrus, respectively), and 3) addition of follicle-stimulating hormone (FSH), luteinizing hormone (LH), and estradiol-17 beta (E2) during maturation. Greater proportions (percentage) of oocytes initially selected for their compact cumulus cells completed IVM and IVF when compared to unselected oocytes (P less than .05). Proportions (percentage) of selected oocytes that matured and cleaved after in vitro insemination according to serum type used for IVM were: FCS: 110/175 (62.9%) and 37/110 (33.6%) and D0: 130/145 (89.7%) and 52/130 (40.0%); D1 127/130 (97.7%) and 41/127 (32.3%); D10 95/98 (96.9%) and 35/95 (36.8%); D20:113/116 (97.4%) and 49/113 (43.4%). A higher proportion (P less than .05) of embryos resulting from the D20 group reached four- and eight-cell stages. In FCS-supplemented maturation media with no hormones added during maturation (control), results of IVM and IVF were 157/265 (59.2%) and 39/157 (24.8%), respectively. With E2 (1 microgram/ml) and FSH (5 micrograms/ml), comparable results were 189/215 (87.9%) and 71/189 (37.6%); with E2 (1 microgram/ml) plus LH (10 micrograms/ml), 280/327 (85.6%) and 111/280 (39.6%). Added hormones improved IVM results (P less than .05) and, when FSH or LH was added with E2, in vitro development to four- and eight-cell stages was markedly enhanced (P less than .05). Selection of oocytes, D20 serum, and added E2 and FSH or LH for IVM improved in vitro development of bovine embryos after IVF.