Ultrasonography for detection of pregnancy and study of embryonic development in heifers

Real-time B-mode ultrasonography was used to evaluate uterine changes associated with the estrous cycle in 22 ovulatory periods in 12 nulliparous heifers. Irregular, nonechogenic (black) areas were seen on the images of uterine horns during the periovulatory period. These nonechogenic areas were presumably due to intraluminal fluids since they coincided with the discharge of clear, viscous mucus preceding ovulation and blood-tinged mucus after ovulation. Eight heifers were bred until five pregnant heifers were obtained for study of the ultrasonic morphology of the conceptus. Ultrasound examinations were done daily to day 50 of pregnancy. Discrete, nonechogenic areas were first visible within the uterus between days 12 and 14, when they were approximately 2 mm in diameter. These discrete nonechogenic structures were identified as the embryonic vesicle, since they were observed only in heifers later confirmed to be pregnant and were always in the uterine horn ipsilateral to the corpus luteum. The presence of an embryo within the embryonic vesicle was confirmed by observing an echogenic (white) area with rhythmic pulsations (heartbeat). The embryonic vesicle gradually increased in length from the day of first observation until day 26 when it extended past the curvature of the horn and began to encroach into the contralateral horn. In all heifers, by day 32 the vesicle extended to the tip of the contralateral horn. The embryo was first visible between days 26 and 29 when the mean length was 10 mm. The embryo increased in length an average of 1.1 mm per day. A heartbeat was detectable in the embryo on the first day observed. In one superovulated heifer, five vesicles were visible in the uterine horns by day 14 and by day 33 seven embryos were observed; two of the seven embryos apparently resorbed by day 43.     

Effect of estradiol valerate on ovarian follicles, emergence of follicular waves and circulating gonadotropins in heifers

An experiment was designed to examine the effect of estradiol valerate (EV) on the growth and regression of follicles of a wave and on the emergence of the next follicular wave. Twenty-six beef heifers were xamined daily by ultrasonography and randomly allocated to 1 of 4 treatment groups at the time of ovulation (Day 0): unterated control heifers and those that received 5 mg EV intramuscularly on Day 1, Day 3 or Day 6. Maximum diameter of the dominant follicle was greater (P<0.05) in control heifers than in heifers treated on Day 1 or Day 3. Mean day of onset of regression of the dominant follicle was later (P<0.05) in control heifers than in heifers treated on Day 1 but was not different from heifers treated on Day 3. In heifers treated on Day 6, cessation of growth, maximum diameter and onset of regression were not different from that of control heifers. The emergence of the next follicular wave was earlier (P<0.05) in heifers treated on Day 1 than in control heifers, whereas wave emergence was delayed (P<0.05) in heifers treated on Day 3 or Day 6. The mean day of maximum concentration of FSH prior to the emergence of the next wave was earlier in heifers treated with EV on Day 1 and later in heifers treated on Day 3 or Day 6 compared with that of the controls (P<0.05). Treatment on Day 1 or Day 3 resulted in a significant LH surge in 8 13 heifers, whereas no LH surges were detected in control heifers or in heifers treated on Day 6. The hypothesis that EV suppresses the growth of the dominant follicle, was supported. Estradiol valerate treatment resulted in early emergence of the next follicular wave in heifers treated on Day 1, but treatment on Day 3 or Day 6 resulted in delayed emergence of the next follicular wave.     

Molecular phylogenetics of Stenodermatini bat genera: congruence of data from nuclear and mitochondrial DNA

Within the tribe Stenodermatini the systematics of the complex of species allied with the genus Artibeus has generated several alternative phylogenetic hypotheses. The most recent treatment recognized four genera (Artibeus, Dermanura, Enchisthenes, and Koopmania) and suggested that the most recent common ancestor of these four genera would include the common ancestor of all other currently recognized Stenodermatini genera except Sturnira. To test this hypothesis, we examined an EcoRI-defined nuclear satellite DNA repeat and 402 bp of DNA sequence variation from the mitochondrial cytochrome b gene. Phylogenetic conclusions based on Southern blot analyses, in situ hybridization, and mitochondrial DNA sequence data indicate that Enchisthenes is not closely related to Dermanura, Artibeus, or Koopmania and that Dermanura, Artibeus, and Koopmania shared a common ancestor after diverging from the remainder of the Stenodermatini. If our conclusions are correct, then justification for recognizing Dermanura and Koopmania as generically distinct from Artibeus must be based on the magnitude of difference that distinguishes each rather than on the conclusion that to place them as congeneric with Artibeus creates a paraphyletic taxon.      

Accuracy of ultrasonography in early pregnancy diagnosis in the ewe

Nonbred and pregnant ewes were examined ultrasonographically at intervals of 4 to 6 days on Days 17 to 34 after estrus. Each ewe was diagnosed as pregnant or nonpregnant, and a score for degree of certainty in the diagnosis was recorded. The goal of the study was to define criteria that could be used for identification and accuracy of diagnosis of an early conceptus and to ascertain the confidence which the operator had in makeing the diagnosis. Pregnancy was retrospectively confirmed by ultrasonographic detection of an embryo proper and by embryonic heartbeat on Days 21 to 34, and later judged against the number of lambs born to each ewe. The percentage of ewes accurately diagnosed pregnant by ultrasonography was not significantly higher than that by guessing (50%) before Day 24, but reached 85% on Days 32 and 34. However, the ability to detect nonpregnant ewes by ultrasonography was higher (P<0.01), with a greater specificity starting on Days 21 to 23 (80%) and reaching 98% by Days 32 to 34. Before Day 24, the diagnosis of pregnancy in many cases was based primarily upon the ultrasonographic appearance of the uterine lumen and location of the uterus in relation to the bladder rather than upon detection of the conceptus. For the certainty score there was a main effect of day (P<0.01) but not for the reproductive status (pregnant vs nonpregnant). The certainty score increased in all ewes among days, and was highest on Days 32 to 34. It was concluded that real time transrectal ultrasonographic scanning of sheep between Days 24 and 34 of gestation offers a safe, accurate and practical means for diagnosing pregnancy.     

Mutations affecting lipopolysaccharide enhance ail-mediated entry of Yersinia enterocolitica into mammalian cells.

Two genes of Yersinia enterocolitica, inv and ail, have been identified as having a role in the bacterial adherence to and entry into mammalian cells in vitro. Expression of both genes is regulated by temperature. In stationary phase, ail gene expression is detectable only in bacteria at 37 degrees C, not at lower temperatures. An inv mutant derivative of Y. enterocolitica, which cannot enter mammalian cells when grown at 30 degrees C because of the lack of both inv and ail gene products, was mutagenized with the transposons mini-Tn10 and Tn5B50 to look for an increase in Ail-mediated cell entry. Sixteen mutants that could enter tissue culture cells after growth at 30 degrees C were selected. All of the mutants had increased cell surface Ail levels as detected by an Ail-specific monoclonal antibody. All of the ten Tn5B50 and one of the six mini-Tn10 mutants showed no increase in ail expression, but they had alterations in their lipopolysaccharide (LPS) such that no O side chains were detectable in bacteria grown at 30 degrees C. Thus, these mutants that are increased in their ability to enter cells appear to be so as a result of a change in the LPS on the surface resulting in increased levels of Ail protein able to interact with the mammalian cell surface. In the remaining mini-Tn10 mutants, LPS is normal, and the increase in cell surface Ail levels appears to be due to an increase in ail mRNA present in the cell. These mutants may therefore be affecting a repressor of ail gene expression.     

New conformational constraints in isotopically (13C) enriched oligosaccharides

Multidimensional heteronuclear NMR studies have been applied to the resonance assignment and conformational analysis of 13C-enriched Neu5Acalpha2-3Galbeta1-4Glc. It is demonstrated that three-dimensional ROESY-HSQC experiments provide through-space distance restraints which cannot be observed with conventional homonuclear 1H techniques due to resonance overlap. In particular, connectivities demonstrating the existence of the "anti" conformation about the Galbeta1-4Glc glycosidic linkage are unambiguously observed. It is shown that 13C isotopic enrichment of the trisaccharide at a level >95% enables straightforward measurement of trans-glycosidic 1H-13C and 13C-13C coupling constants and a Karplus-type relation is derived for the latter. In total 15 conformational restraints were obtained for the trisaccharide in aqueous solution, all of which were in excellent agreement with theoretical parameters computed from a 5 ns molecular dynamics simulation of the glycan.      

Molecular characterization and phylogenetic relationships of a protein with potential oxygen-binding capabilities in the grasshopper embryo. A hemocyanin in insects?

Arthropodan hemocyanins, prophenoloxidases (PPOs), and insect hexamerins form a superfamily of hemolymph proteins that we propose to call the AHPH superfamily. The evolutionary and functional relationships of these proteins are illuminated by a new embryonic hemolymph protein (EHP) that is expressed during early stages of development in the grasshopper embryo. EHP is a 78-kDa soluble protein present initially in the yolk sac content, and later in the embryonic hemolymph. Protein purification and peptide sequencing were used to identify an embryonic cDNA clone coding for EHP. In situ hybridization identifies hemocytes as EHP-expressing cells. As deduced from the cDNA clone, EHP is a secreted protein with two potential glycosylation sites. Sequence analysis defines EHP as a member of the AHPH superfamily. Phylogenetic analyses with all the currently available AHPH proteins, including EHP, were performed to ascertain the evolutionary history of this protein superfamily. We used both the entire protein sequence and each of the three domains present in the AHPH proteins. The phylogenies inferred for each of the domains suggest a mosaic evolution of these protein modules. Phylogenetic and multivariate analyses consistently group EHP with crustacean hemocyanins and, less closely, with insect hexamerins, relative to cheliceratan hemocyanins and PPOs. The grasshopper protein rigorously preserves the residues involved in oxygen binding, oligomerization, and allosteric regulation of the oxygen transport proteins. Although insects were thought not to have hemocyanins, we propose that EHP functions as an oxygen transport or storage protein during embryonic development.      

Contribution of phenazine antibiotic biosynthesis to the ecological competence of fluorescent pseudomonads in soil habitats.

Phenazine antibiotics produced by Pseudomonas fluorescens 2-79 and Pseudomonas aureofaciens 30-84, previously shown to be the principal factors enabling these bacteria to suppress take-all of wheat caused by Gaeumannomyces graminis var. tritici, also contribute to the ecological competence of these strains in soil and in the rhizosphere of wheat. Strains 2-79 and 30-84, their Tn5 mutants defective in phenazine production (Phz-), or the mutant strains genetically restored for phenazine production (Phz+) were introduced into Thatuna silt loam (TSL) or TSL amended with G. graminis var. tritici. Soils were planted with three or five successive 20-day plant-harvest cycles of wheat. Population sizes of Phz- derivatives declined more rapidly than did population sizes of the corresponding parental or restored Phz+ strains. Antibiotic biosynthesis was particularly critical to survival of these strains during the fourth and fifth cycles of wheat in the presence of G. graminis var. tritici and during all five cycles of wheat in the absence of take-all. In pasteurized TSL, a Phz- derivative of strain 30-84 colonized the rhizosphere of wheat to the same extent that the parental strain did. The results indicate that production of phenazine antibiotics by strains 2-79 and 30-84 can contribute to the ecological competence of these strains and that the reduced survival of the Phz- strains is due to a diminished ability to compete with the resident microflora.     

Evaluation of the Biolog automated microbial identification system.

Biolog's identification system was used to identify 39 American Type Culture Collection reference taxa and 45 gram-negative isolates from water samples. Of the reference strains, 98% were identified to genus level and 76% to species level within 4 to 24 h. Identification of some authentic strains of Enterobacter, Klebsiella, and Serratia was unreliable. A total of 93% of the water isolates were identified.     

Contribution of phenazine antibiotic biosynthesis to the ecological competence of fluorescent pseudomonads in soil habitats.

Phenazine antibiotics produced by Pseudomonas fluorescens 2-79 and Pseudomonas aureofaciens 30-84, previously shown to be the principal factors enabling these bacteria to suppress take-all of wheat caused by Gaeumannomyces graminis var. tritici, also contribute to the ecological competence of these strains in soil and in the rhizosphere of wheat. Strains 2-79 and 30-84, their Tn5 mutants defective in phenazine production (Phz-), or the mutant strains genetically restored for phenazine production (Phz+) were introduced into Thatuna silt loam (TSL) or TSL amended with G. graminis var. tritici. Soils were planted with three or five successive 20-day plant-harvest cycles of wheat. Population sizes of Phz- derivatives declined more rapidly than did population sizes of the corresponding parental or restored Phz+ strains. Antibiotic biosynthesis was particularly critical to survival of these strains during the fourth and fifth cycles of wheat in the presence of G. graminis var. tritici and during all five cycles of wheat in the absence of take-all. In pasteurized TSL, a Phz- derivative of strain 30-84 colonized the rhizosphere of wheat to the same extent that the parental strain did. The results indicate that production of phenazine antibiotics by strains 2-79 and 30-84 can contribute to the ecological competence of these strains and that the reduced survival of the Phz- strains is due to a diminished ability to compete with the resident microflora.