No abstract

This study assessed pronuclear formation, the chromosomal constitution, and the developmental capacity of bovine zygotes formed by intracytoplasmic injection of oocytes with sperm, treated or not with dithiothreitol (DTT). Oocytes were matured in vitro for 22-24 h and then centrifuged so that sperm, prepared by swim-up in the presence or absence of 5 mM DTT, could be injected into the cleared area of the ooplasm. Injected oocytes were activated by treatment with 5 microM ionomycin (5 min) and, after a 3-h interval, with 1.9 mM 6-dimethylaminopurine (DMAP) for 3 h. They were then cocultured with bovine oviductal epithelial cells in M199. Sperm treatment resulted in a significantly higher proportion of male pronucleus formation 16 h after injection (40% vs. 11%; p < 0.0001) and a significantly higher rate of blastocyst development (24% vs. 10%; p < 0.005). Sixty-one percent of blastocysts produced with treated sperm were diploid. Of 12 blastocysts produced with treated sperm and sexed by a polymerase chain reaction, 4 were male and 7 female, and in one a definite diagnosis could not be made. Embryo transfer (2 embryos per heifer) resulted in pregnancies in 6 of 16 recipients at Day 49, but none was carried to term. These results show that the efficiency of bovine intracytoplasmic sperm injection can be improved by sperm pretreatment with DTT and by oocyte activation with ionomycin plus DMAP, although the developmental capacity of the resulting embryos remains limited.

Of 226 donor cattle treated with PMSG to induce superovulation, 76.5% responded with 3 or more ovulations. Flushing at surgery or slaughter 10-16 days after oestrus recovered eggs and embryos that represented 49.3% of the number of ovulations. Of those recovered, 73.3% were embryos, an average yield of 4.0 embryos/treated cow or 4.8 embryo/flushed cow. The location of eggs and embryos was determined in 65 of the donors. Embryos and unfertilized eggs (6.1% of those recovered) were occasionally found in the oviducts. Empty zonae pellucidae were also found in the uterus on all days. The lengths, or diameters, of embryos were extremely variable within days and within donors, but mean values indicated logarithmic growth between Days 10 and 16. Eighty-four synchronous (+/- 1 day) recipients received single embryos, and 51 recipients twin embryos, by surgical transfer. Pregnancies were obtained in recipients up to Day 16 but not on Day 17, indicating the stage by which an embryo must be present to prevent luteolysis. The overall pregnancy rate at Day 42 was 50.4% and further 18.1% of the recipients exhibited extended oestrous cycles. Of 35 recipients that were allowed to go to term, 12 lost their pregnancies, most often between Days 42 and 63.

The classical concept of sex determination in mammals is that a Y chromosomal gene controls the development of the indifferent gonad into a testis. Subsequent divergence of sexual phenotypes is secondary to this gonadal determination. The most likely candidate gene is SRY (sex-determining region Y) in humans, and Sry in mouse. However, several lines of evidence indicate that sexual dimorphism occurs even before the indifferent gonad appears. Here we present evidence that bovine male embryos generally develop to more advanced stages than do females during the first 8 days after insemination in vitro. Corresponding relationships between both cell numbers and mitotic indices and sex were also seen. Although it is not clear whether this phenomenon involves factors originating before or after fertilization, these findings suggest that sex-related gene expression affects the development of embryos soon after activation of the embryonic genome and well before gonadal differentiation.

One thousand eighty-four two-cell bovine embryos produced from 1,574 oocytes matured and fertilized in vitro were cultured as groups separated according to the time when they completed their first cleavage (24, 30, 40, 48, or 62 hr postinsemination; hpi). At 5 days after insemination, the proportions of each group that had progressed to the eight-cell stage or beyond were determined and the 350 that had done so were fixed and examined cytogenetically for cell number, chromosomal abnormalities, and sex. Embryos in the "early" cleaving (24 and 30 hpi) and "late" cleaving (40-62 hpi) groups were compared. Early cleaving embryos were more likely to have developed to the eight-cell stage or beyond (52.2% vs. 20%), contained more cells (22 vs. 17), and were more likely to be male (3.6:1 vs. 0.93:1). It is suggested that these phenotypic differences between the sexes begin before the embryonic genome is generally thought to become activated and are due either to differential processing of X- and Y-bearing sperm within the zygote or to very early differential expression of genes derived from X- and Y-bearing sperm.

Summary. A co-culture system using a suspension of detached bovine oviducal epithelial cells (BOEC)

Strong-cation-exchange, solid-phase extraction of pyrrolizidine alkaloids and their N-oxides from honey samples was followed by reduction of the N-oxides and subsequent analysis of total pyrrolizidine alkaloids using high-performance liquid chromatography-atmospheric pressure chemical ionization mass spectrometry. A limited survey of 63 preprocessing samples of honey, purposefully biased toward honeys attributed to floral sources known to produce pyrrolizidine alkaloids, demonstrated levels of pyrrolizidine alkaloids up to approximately 2000 parts per billion (ppb) in a sample attributed to Echium plantagineum. Up to 800 ppb pyrrolizidine alkaloids was detected in some honeys not attributed by the collector to any pyrrolizidine alkaloid-producing floral source. No pyrrolizidine alkaloids were detected in approximately 30% of the samples in this limited study, while some honeys showed the copresence of pyrrolizidine alkaloids from multiple floral sources such as E. plantagineum and Heliotropium europaeum. In addition, retail samples of blended honeys (with no labeling to suggest that pyrrolizidine alkaloid-producing floral sources were used in the blends) have been shown to contain up to approximately 250 ppb pyrrolizidine alkaloids.

A method for analyzing honey samples was developed that enabled the simultaneous detection and identification of pyrrolizidine alkaloids and their N-oxides. Honey samples were treated with methanol or dilute sulfuric acid and then centrifuged to remove insoluble material. Subsequent strong cation exchange, solid-phase extraction of the supernatant provided a fraction that was analyzed for the presence of pyrrolizidine alkaloids and their N-oxides using high-pressure liquid chromatography coupled to electrospray ionization mass spectrometry. The procedure was validated using extracts of Echium plantagineum and authenticated standards of pyrrolizidine alkaloids and their N-oxides from other plant sources. Of several variations of the solid-phase extraction method assessed in this study, the best combination for generic use involved the dilution of honey with 0.05 M sulfuric acid and the subsequent application of the centrifuged solution to solid-phase extraction columns at the rate of a maximum of 10 g of honey per solid-phase extraction column. The method was applied to the analysis of nine floral honeys, five of which were attributed by the apiarist to Echium vulgare. Seven of the honey samples were positive for pyrrolizidine alkaloids and N-oxides characteristic of E. vulgare.