The formation of ferric haem during low-temperature photolysis of horseradish peroxidase Compound I.

Illumination at low temperature of the peroxide compound of horseradish peroxidase (HRP-I) causes partial conversion of the haem electronic structure from a ferryl-porphyrin radical species into a low-spin ferric state. Magnetic-c.d. (m.c.d.) and e.p.r. spectral features of the photolysis product are almost identical with those of the alkaline form of ferric HRP, proposed on the basis of its near-i.r. m.c.d. spectrum to be a Fe(III)-OH species. The ferric product of HRP-I photolysis also contains free-radical e.p.r. signals. Conversion of HRP-I into the Fe(III)-OH species, which requires transfer of a proton and two electrons from the protein, is shown to be a two-step process.     

A spectrophotometric procedure for the determination of objective measurements of equine spermatozoan motility

A spectrophotometric procedure was developed and evaluated for the objective measurement of equine spermatozoan motility. A 100 mul sample of a sperm suspension, prepared by the removal of seminal plasma, was layered under a column of optically clear medium in a specially designed spectrophotometric cuvette maintained at 37 degrees C. Changes in light transmittance above the interface of the sperm suspension and medium were recorded on chart paper. As sperm cells swam into the medium, a decrease in light transmittance was recorded as a deflection on the chart paper. Chart recordings were analyzed for the height (cm) and time (min) to the peak deflection. To standardize the procedure, a fixed number of cells (1x10(9)) were used to prepare suspensions of 300x10(6) cells/ml. Coefficients of variation for mean values obtained under these conditions after the evaluation of five ejaculates from a given stallion were estimated at between 10 and 12%. Correlations between swim-up measurements and computer-assisted semen analysis demonstrated that the percentage of motile cells and mean velocity (mum/sec) of motile cells influenced swim-up measurements. Described here is a simple and inexpensive procedure to determine objective measurements of spermatozoan motility that may have application in semen evaluation and fertility testing in the stallion.     

Production of chimeric rabbits from morulae by a simple procedure

Experiments were conducted to develop a simple and reliable technique to produce chimeric rabbits from morula stage embryos. In Experiments 1 and 2, an in-vitro test of viability was initially performed by culturing embryos to the blastocyst stage. Ninety-three percent of the “chimeric” embryos developed to the blastocyst stage compared to 94% for controls when embryos were manipulated soon after collection (Exp. 1). Eighty-one percent chimeric embryos and 78% control embryos developed to blastocyst stage when embryos were held at room temperature for 4 hr (Exp. 2). In Experiment 3, enough morula-stage embryos were available from true breeding Dutch-belted and albino rabbits to form potentially 67 diverse “color” pairs. These micromanipulated pairs of morulae were successfully combined to produce 64 chimeric embryos (96%, 64/67). They were transferred to the uteri of seven recipient does and three became pregnant producing 13 young. Four of the young exhibited substantial overt chimerism (31%) and one more was a possible chimera.     

Targeting deletion (homoeologous chromosome pairing locus) or addition line single copy sequences from cereal genomes.

We describe here a protocol for obtaining clones containing sequences present in low copy-number from genomic DNA where moderately and highly repeated sequences predominate. Specific chromosomal regions can be targeted by using deletion or addition line material. We have used this protocol to identify a sequence which has been deleted in both the tetraploid and hexaploid wheat mutants for the homoeologous chromosome pairing locus.     

Substrate specificity of aspartate transcarbamylase. Interaction of the enzyme with analogs of aspartate and succinate

The ability of aspartate transcarbamylase from Escherichia coli to catalyze carbamylation of amino acids other than the natural substrate, L-aspartate, was examined. Cysteine, cysteate, cysteinesulfinate, and 3-nitroalanine showed kcat values at pH 7 of 0.16, 0.58, 5.2, and 62 s-1, respectively, while kcat with aspartate was 320 s-1. In a parallel study, competitive inhibition constants of 3-nitropropionate, 3-mercaptopropionate, 3-sulfopropionate, and 3-sulfinopropionate were found to be high, about 0.1 M, compared with that of succinate, 0.56 mM. Although cysteinesulfinate had low activity as a substrate, the pH dependences of kcat and kcat/Km in H2O and D2O observed with the compound closely paralleled those of aspartate. The results of these studies suggest that substrate specificity and reactivity are achieved in part by a strong, highly specific interaction of one or more active site residues with the beta-carboxylate of L-aspartate. Unlike the sigmoidal kinetics found with aspartate, saturation of native aspartate transcarbamylase by cysteine sulfinate showed a lack of cooperativity, even under conditions of activation of the reaction by ATP and inhibition by CTP. The cysteinesulfinate reaction was increased 9-fold by the bisubstrate analog N-phosphonacetyl-L-aspartate. These results were interpreted in terms of an inability of cysteinesulfinate to cause the allosteric conformational change promoted by aspartate.     

Improved developmental potential of rabbit oocytes fertilized by sperm microinjection into the perivitelline space enlarged by hypertonic media

The objectives of the present study were: 1) to develop a simple and more efficient technique for sperm microinjection than is currently available, using the rabbit as a model, and 2) to evaluate the development of rabbit oocytes fertilized by single or multiple sperm microinjection. Hyperosmotic sucrose in phosphate-buffered saline (SPBS) was employed to dehydrate oocytes to increase the perivitelline space for sperm microinjection and prevent possible injury to the vitellus. In the first experiment, 58% (n = 29) oocytes treated with 0.5 M SPBS developed to morulae following multiple sperm microinjection compared, respectively, to 47% (n = 34) and 60% (n = 15) for control IVF with or without sucrose exposure (P greater than 0.05). Blastocyst development from microinjected oocytes, however, was much lower (P less than 0.05) than that of controls (14% vs. 42% and 40%, respectively). Sham operation by puncturing the zona pellucida of the sucrose-treated oocytes with the microinjection pipette did not increase parthenogenesis (P greater than 0.05). In Experiment 2 a smaller-size injection pipette and shorter sucrose exposure time after sperm microinjection resulted in 41% (n = 42) of the oocytes developing into blastocysts for the microinjection group, whereas only 21% (n = 24) developed to blastocysts in the control IVF group (P less than 0.05). When relatively older oocytes (17 hr post ovulation injection) were used to test if microinjection could reduce the time to fertilization and cleavage (Expt. 3), an average of 27% (n = 63) blastocysts resulted from microinjection vs. 0% (n = 28) for the control IVF group.     

Comparison of six-day bovine embryo development in uterine tube (oviduct) epithelial cell co-culture versus in vivo development in the cow

A study was designed to evaluate and compare the appearance of embryos recovered from donor cows on Day 6 to embryos from in vivo fertilized cow zygotes developed to Day 6 on uterine tube (oviduct) epithelial cell co-culture using serum-free CZB medium. Embryo stage of development and quality score were assessed. Hoechst 33342 DNA stain was then used to determine the total number of blastomeres, the number of poor nuclei and the number of nuclei in mitosis. Mean cell counts did not differ for the 70 embryos evaluated in each group (65 cells in vivo, 61 cells in vitro). The percentage of transferable emryos (excellent, good or fair quality), in each group also did not differ (57% in vivo, 56% in vitro). There were no significant differences in any of the measured parameters. Our findings suggest that co-culture of in vivo produced cow zygotes can result in embryos comparable in developmental stage and quality to embryos developed in vivo in the cow for 6 d.     

Maturation, fertilization and development of bovine oocytes in vitro using TCM199 and a simple defined medium with co-culture

Bovine oocytes obtained from ovarian follicles (2 to 5 mm in diameter) from slaughtered cattle were cultured in TCM199 with 10% heat-inactivated estrous cow serum (ECS) for 24 to 25 h at 39 degrees C under 5% CO(2) in air. The 10% ECS was selected on the basis of preliminary studies in which in vitro fertilization rates of oocytes with 10, 15 and 20% ECS in the medium were 46, 30 and 31%, respectively (P<0.05). Of 120 oocytes cultured for 24 to 25 h, 63% were classified as being in Metaphase II. The rate of oocytes matured in vitro was 55% (69 125 ), the proportion of penetrated oocytes which contained male and female pronuclei was 94% (65 69 ), and the incidence of polyspermy was very low (0 to 9%). Of 122 oocytes fertilized in vitro and cultured in TCM199 medium with 10% fetal bovine serum for 7 d, 53% were cleaved, but only 2% developed beyond the 16-cell block. However, in simple semi-defined Chatot-Ziomek-Bavister medium co-cultured with bovine oviduct epithelial cells (BOEC), 75% of 138 oocytes cleaved, and 38% of those which cleaved developed into morulae or blastocysts. The results of this study indicate that co-culture with BOEC exerted a pronounced beneficial effect on development of in vitro fertilized bovine oocytes through the 16-cell block. The medium required in the co-culture system was simple and semi-defined.     

Sequential uterine horn versus simultaneous total uterine flush to recover bovine embryos nonsurgically

Beef cows were superovulated with follicle stimulating hormone (FSH) to compare two nonsurgical methods of embryo recovery from the uterus. In the first method each uterine horn was independently flushed with physiological saline solution (PSS) through a Foley catheter passed through the cervix and into the uterine horn. In the second method both uterine horns were simultaneously flushed with PSS by passing the catheter into the uterine body. In both methods, the numbers of ovulations were determined after embryo collection by counting the corpora lutea (CL) on both ovaries of each cow through a flank incision. Independent flushing (n = 19) averaged 6.4 embryos and 16.1 CL per cow for a recovery rate of 40%. Simultaneous flushings (n = 22) averaged 5.4 embryos and 17.7 CL per cow for a recovery rate of 31%. This difference between the recovery rates of the two flushing methods was not significant (P>0.05).