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A series of experiments was conducted to examine the pattern of interferon-tau (IFN-tau) secretion by bovine blastocysts during extended culture in vitro. In the first experiment, blastocysts were cultured individually for three 48-hour periods. The day of blastocyst formation affected how much IFN-tau was produced during the first two culture periods, but not during the third period. The overall secretion of IFN-tau during the 6-day period increased significantly and well beyond what could be accounted for by the concomitant increase in cell numbers. In the second experiment, blastocysts were initially cultured in individual droplets for 48 hr, then plated into 48-well plates. Medium concentrations of IFN-tau were determined after 48 hr and again after 6 and 12 days of culture. Initial IFN-tau secretion did not affect the ability to form outgrowths or their final size, and initial differences in secretion between groups of blastocysts had disappeared by the second and third analyses. In the third experiment, blastocysts were cultured individually for 48 hr in droplets containing the medium that had been flushed through the uteri of non-pregnant sheep on days 10, 12, and 15 of the estrous cycle. Culture in the medium obtained from the Day 15 flush significantly increased the number of cells that blastocysts contained, as well as IFN-tau secretion.

The effects of culturing bovine embryos in groups were investigated. In the first experiment, 1000 oocytes were matured, fertilized and then cultured in groups of 40 in 25 microl of medium. From half of these groups, blastocysts were removed and cultured separately, while in the other half blastocysts were allowed to remain in the group culture microdrop. Blastocysts developed equally well in both groups, although hatching was reduced in those blastocysts removed from the culture droplet. In the second experiment, 1000 zygotes were cultured from the 8-cell stage to the blastocyst stage either individually or in groups of 40. Culture in groups increased the formation of blastocysts, the percentage of hatching blastocysts, the number of cells within blastocysts and the production of interferon-tau. In the final experiment, 1000 zygotes were cultured in groups up to the blastocyst stage. Two-thirds of these blastocysts were then cultured in groups of three, while the remaining blastocysts were cultured individually. Co-culture did not affect hatching or cell number but significantly elevated interferon-tau secretion. These results demonstrate that group culture either before or after blastocyst formation can alter the expression of a specific gene important for the establishment of pregnancy.

This study was designed to examine the relationship between the speed at which bovine embryos reach the blastocyst stage, their cell number, and interferon-tau production. A total of 800 oocytes were fertilized by frozen-thawed semen. On day 2, 44 hr after exposure to sperm, 78, 320, and 296 embryos were at the two-, four-, and eight-cell stages, respectively, with an overall cleavage rate of 86.8%. Within these three groups 15 (19.2%), 106 (33.1%), and 158 (53.4%) embryos proceeded to the blastocyst stage. Of these 46.7%, 65.1%, and 63.3% hatched in the three groups, respectively. Blastocysts began to appear at day 7, but a few did not form until as late as day 13. Expanded blastocysts (n = 279) were cultured individually for 48 hr in 50-microliter droplets of medium, fixed for cell counts, and the concentration of interferon-tau in the medium was determined. Blastocysts originating from two-cell embryos had significantly fewer cells (46.5 +/- 23.3) than either four-cell- (97.2 +/- 13.5) or eight-cell-derived embryos (113.8 +/- 13.6; P < 0.05). Hatching was accompanied by an increase in cell number (129.8 +/- 15.5 versus 41.9 +/- 14.4; P < 0.01). Blastocysts derived from embryos that had reached the eight- or four-cell stage 44 hr after insemination produced significantly more interferon than embryos derived from two-cell embryos (941.7 +/- 92.1, 930.1 +/- 163.1, versus 232.8 +/- 70.1 pM). In contrast, hatching, ovary batch, the speed of early cleavage, cell number, and quality grade had no effect on interferon-tau secretion. The embryo's age at blastocyst formation was not related to the number of its cells but did have a significant effect (P < 0.001) on interferon-tau production, with mean concentrations in the medium of 294.8 +/- 57.9, 563.3 +/- 82.0, 1126.3 +/- 133.6, 1778.5 +/- 297.2, 512.9 +/- 82.0, 315.0 +/- 157.5, and 157.5 pM among blastocysts appearing from days 7 to 13, respectively. These data suggest that blastocysts that form at days 7 and 8 produce less interferon-tau than those that form on days 9 or 10. Since early-forming blastocysts are generally considered more developmentally competent than those which form late, there may be a negative relationship between early interferon-tau production and competence.

To determine the fate of an episomally expressed transgene, mouse, rat, and cow zygotes were injected into the perivitelline space with approximately 100 pl of buffer containing the replication-defective human adenovirus, AdCMVLacZ/sub360. Viral concentrations ranged from 2.5 to 2.5 x 10 5 plaque-forming units (pfu)/100 pl. As viral titer increased, fewer embryos were able to develop to blastocysts. In the mouse, the percentage of blastocysts formed ranged from 82% in controls to 16% after injection at the highest titer. In the rat and cow, a similar decrease in blastocyst formation was noted (62% to 6% and 26% to 4%, respectively). Reporter gene (galactosidase, LacZ) activity could be detected in mouse embryos after injection at a concentration of only 25 pfu/100 pl, whereas a tenfold higher titer was required in the other two species to observe the blue LacZ reaction product. When examined after 5 (mouse), 6 (rat), or 9 (cow) days of in vitro culture, the proportion of LacZ-positive embryos ranged from 15% to 96%, 6% to 76%, and 18% to 58% in mouse, rat, and cow embryos, respectively, depending upon viral concentration. However, a large percentage of positive embryos proved to be expression mosaics, the degree of which was likewise dependent on titer. While none of the embryos showed LacZ activity at 30 h after injection, 70% of mouse, 8% of rat, and 20% of cow embryos expressed the reporter gene at 42 h. Delaying the timing of injection revealed that the efficiency with which mouse and rat embryos could be infected decreased with increasing degree of differentiation. Only 35% and 18% of mouse embryos expressed the reporter gene after injection at the morula or blastocyst stage, respectively. A similar drop in efficiency was noted in rat embryos when injections took place at the 8-cell, morula, or blastocyst stage, with 70%, 33%, and 9% of embryos, respectively, subsequently showing LacZ activity. Likewise, advanced development resulted in a decrease in the efficiency of viral-mediated gene transfer in cow embryos, with 100%, 78%, and 68% of embryos being positive after injection at the 8-cell, morula, or blastocyst stage, respectively. These results demonstrate that a human adenovirus can be used to express a reporter gene transiently in nonhuman embryos.

A total of 1550 zygotes was used to assess the timing of pronuclear visibility, embryo development following DNA microinjection, and transgene expression in IVM/IVF-generated porcine embryos. After centrifugation, pronuclei could be seen in 61.6% of zygotes. In 55.3% of these only 1 pronucleus was visible. Pronuclear visibility was highest at 20 h post-insemination. Zygotes were microinjected with 1 of 2 LacZ gene constructs driven by either the SV40 early promoter (pSVON) or the human cytoplasmic beta actin promoter (pbActinLacZ). Development and transgene expression were assessed after either 48 h or 7 d in culture. After 48 h, significantly more zygotes with a single visible pronucleus developed to the 8-cell stage than zygotes in which no pronucleus had been seen (43.0 vs 24.8%), while those with 2 pronuclei were intermediate (31.4%). After 7 d, no difference in development to the morula stage was observed between noninjected control embryos (25.5%) and embryos with 1 (21.0%) or 2 pronuclei (22.5%); however, the proportion of embryos reaching the morula stage in the nonpronuclear group was significantly reduced (9.1%). After 48 h in culture, transgene expression was significantly higher in embryos with 2 pronuclei at the time of injection than in those with 1 (36.4 vs 17.9%). After 7 d in culture, 41.5% of morulae derived from zygotes with 2 pronuclei and 29.97% of thsoe derived from zygotes with 1 pronucleus showed signs of transgene expression. At this stage, significantly more morulae expressed the pbActinLacZ than the pSVON transgene (43.8 vs 25.8%). More than 80% of putative transgenic morulae or blastocysts showed evidence of mosaicism. These results demonstrate that IVM/IVF porcine embryos are able to develop in culture and express a microinjected transgene.

Bovine zygotes produced by in vitro maturation-in vitro fertilization (IVM-IVF) were examined for their potential to serve as recipients of transgenes. Pronuclei, which were maximally visible at about 22 h after IVF, were injected with a SV40-LacZ construct (pSVON). Injected zygotes had lower cleavage rates (49.1%, n = 1162, P < 0.01) than did either noninjected controls (87.4%, n = 1420) or noninjected zygotes in which pronuclei were not visible (67.6%, n = 803). Zygotes that were injected into their pronuclei cleaved as well as zygotes injected cytoplasmically. At 48 h after injection, when most embryos had reached the four- and eight-cell stages, more zygotes in the pronuclear group (22.7%, n = 125) stained positively for LacZ than did zygotes in the cytoplasmic group (8.0%, n = 125). A group of zygotes injected into the pronucleus with pSVON was cultured for 9 days. More morulae (10.8%, n = 134) than blastocysts (3.2%, n = 31) expressed the LacZ gene, indicating that silencing of expression occurred as development progressed. Another group of zygotes was injected with a beta-actin-LacZ gene construct (pbActinLacZ) and, of the embryos assayed at 48 h, 10.6% (n = 255) stained positively. At 9 days, 36.3% of morulae (n = 91) and 21% of blastocysts (n = 33) expressed the transgene. Almost all putative transgenic embryos injected with either construct showed a mosaic pattern of LacZ expression, with an average of only 2-3 cells staining at the eight-cell stage and the majority of cells in positive blastocysts showing no evidence of expression.

Sarcocystis neurona is an important cause of equine protozoal myeloencephalitis (EPM) in horses in the Americas. An EPM-like neurological disease also has been reported from other mammals but it is difficult to induce this disease in the laboratory. A 4-month-old male domestic cat developed neurological signs 3 days following castration. The cat was euthanized 12 days later because of paralysis. Encephalomyelitis was the only lesion and was associated with numerous Sarcocystis schizonts and merozoites in the brain and spinal cord. The protozoa reacted positively with S. neurona-specific polyclonal rabbit antibody. Two unidentified sarcocysts were present in the cerebellum. It may be possible that stress of surgery triggered relapse of S. neurona infection in this cat.

Several experiments were conducted to assess the effects of genotype and various culture media on interferon-τ secretion by in vitro-derived bovine blastocysts and to compare these values with interferon released by blastocysts flushed from superovulated cows. In experiment 1, oocytes were inseminated with semen from three different bulls. While paternal genotype had no effect on cleavage rate, the size or hatching ability of blastocysts, it was a significant determinant of the embryo’s ability to develop to the blastocyst stage and of subsequent interferon-τ secretion. In the second experiment, embryos were cultured in synthetic oviductal fluid containing either polyvinyl alcohol, bovine serum albumin or fetal bovine serum. While there was no effect of supplement on the percentage of embryos developing to the blastocyst stage, blastocysts which formed in medium with polyvinyl alcohol had significantly fewer cells, were older at blastocyst formation and produced significantly more interferon-τ. In the third experiment, embryos were cultured to the blastocyst stage in either TCM199 alone or in co-culture with buffalo rat liver, bovine oviductal or bovine uterine epithelial cells. Culture with oviductal or buffalo rat liver cells increased blastocyst cell number, although secretion of interferon-τ was not affected. In the final experiment, bovine blastocysts were flushed from superovulated cows on Day 7 following insemination. Overall, secretion of interferon-τ by in vivo-produced blastocysts did not differ from that of age-matched blastocysts produced in vitro.

(1998). Birth of piglets following DNA microinjection of in vitro‐fertilized porcine zygotes. Animal Biotechnology: Vol. 9, Proceedings of the Food Animal Biotechnology (FAB) Center Symposium on Transgenic Animals and Food Production, pp. 199-200.

Concentration gradients in a vertical column of aqueous supersaturated solution have been measured for a number of solutes. It is assumed that these gradients result from the presence of solute clusters. A theoretical analysis based on the assumption of steady-state quasi-equilibrium allows the size of these clusters to be estimated. Typically they contain of the order of 103 molecules and are between about 4 and 10 nm in size.

Isothermal column experiments were carried out to measure concentration gradients in supersaturated solutions placed in a 40-cm vertical column. Three aqueous solution systems saturated at 30.0° C were used in the study: K 2 SO 4 , Na 2 SO 4 and NaNO 3 . The densities of the solutions at the top and bottom of the column were measured with a density meter that can measure to within 2 × 10 –5 g/ml. The results show that the concentration gradients obtained did not vary significantly with supersaturation. This observation is discussed in terms of solute clustering phenomena.