Two approaches to improve fertility of subclinical mastitic dairy cows.

Mastitis, particularly in its subclinical form, is a widely spread disease that reduces the fertility of lactating cows. A major cause of poor conception risk has been associated with delayed ovulation of a large subgroup of subclinical mastitic cows. This study examined 2 approaches to improve fertility in this subgroup. Subclinical mastitic cows were defined by somatic cell count elevated above a threshold of 150,000 cells/mL of milk determined in all monthly test day samples collected before AI. Uninfected (control) cows were defined by somatic cell count below threshold. In experiment 1, we examined a hormonal approach aimed to correct the timing of ovulation in mastitic cows in which it would otherwise be delayed. The probability of conception of mastitic and uninfected groups following Ovsynch (OVS) and timed AI versus AI following detected estrus (E) was examined (n=1,553 AI) and analyzed by a multivariable, logistic model statement using the GLIMMIX procedure of SAS. The OVS protocol significantly elevated the probability of conception of mastitic cows to a level similar to that of their uninfected counterparts. Actual mean conception risks for uninfected-E, subclinical-E, uninfected-OVS, and subclinical-OVS groups were 41.8, 26.4, 39.3, and 40.5%, respectively. The OVS protocol did not improve probability of conception in cows diagnosed with uterine disease postpartum. In experiment 2, a management approach aimed to better synchronize timing of ovulation with timing of AI in subclinical mastitic cows was examined. A second AI was added 24h after the first (routine) AI, following detection of natural estrus. Probability of conception did not differ between subclinical mastitic cows inseminated once or twice. Lack of improvement in conception risk might be related to low preovulatory LH surge in mastitic cows, which is likely to induce not only delayed ovulation but also disruption of oocyte maturation. Thus the OVS protocol can improve fertility of subclinical mastitic cows, probably due to "corrected" timing of ovulation in cows in which it would otherwise be delayed.

ACTH administration during formation of preovulatory follicles impairs steroidogenesis and angiogenesis in association with ovulation failure in lactating cows.

Ovulation failure, follicular persistence, and formation of follicular cysts are known to impair dairy cow fertility. Although the underlying mechanism is not entirely clear, stress-induced alteration in adrenal hormone secretion can cause these ovarian pathologies. Six synchronized lactating cows were scanned daily by ultrasound, and plasma samples were taken throughout the estrous cycle. Treatment cows (n = 3) were administered with ACTH analog every 12 h from day 15 to day 21 of the cycle to induce formation of follicular cysts. Ovaries were collected at the slaughterhouse on day 23 of the cycle before appearance of follicular pathologies. Control cows (n = 3) were administered placebo, resynchronized, and administered PGF2α on day 6 of the new cycle to induce development of a preovulatory follicle. Follicular fluid was aspirated from the preovulatory follicles of each group to determine their steroid milieu. Slices were taken from the follicular wall for total messenger (m) RNA isolation and semiquantitative reverse transcription polymerase chain reaction (RT-PCR). Administration of ACTH increased (P < 0.02) plasma cortisol concentration and reduced (P < 0.01) milk production. Androstenedione and estradiol concentrations in the follicular fluids were lower (P < 0.05) in ACTH-treated follicles than those in controls. The mRNA expression of luteinizing hormone receptor, 3ß-hydroxysteroid dehydrogenase, cytochrome P450 aromatase (P450arom), and cytochrome P450 17α-hydroxylase (P450c17) were lower (P < 0.02) in the ACTH-treated vs control cows. On the other hand, the expression of steroidogenic acute regulatory protein and cytochrome P450 side-chain cleavage did not differ between groups. In addition, mRNA expression of vascular endothelial growth factor (VEGF)120 and VEGF164 was higher (P < 0.01) in control than in ACTH-treated follicles, but that for angiopoietin-1 and 2 did not differ between groups. Findings indicated that ACTH administration throughout preovulatory follicle development alters follicular steroidogenesis in association with impaired angiogenesis. Such alterations might explain, in part, the mechanism underlying ovulation failure and the formation of persistent or cystic follicles under stress.

Endocrine milieu and developmental dynamics of ovarian cysts and persistent follicles in postpartum dairy cows.

Ovarian follicular cysts and persistent follicles are follicular pathologies involved in reduced fertility of dairy cows. Two separate experiments were performed on high-yielding Holstein cows to characterize ovarian cyclicity and evaluate the developmental dynamics of follicle pathologies postpartum. In experiment 1, 58 cows were monitored by ultrasonography twice weekly from d 18±1 to 69±2 postpartum. First ovulation occurred 38±3, 27±2, 20±1, and 25±3 d postpartum in cows with 1 cycle (n=11), 2 cycles (n=21), 3 cycles (n=13), and 4 cycles (n=7), respectively. Follicular pathologies were developed in cows that were either acyclic (n=6) or had 1 or 2 cycles, but not in cows with more than 2 cycles. In experiment 2, 47 cows were monitored twice weekly from 10 d postpartum to second ovulation. Follicles ≥17 mm in diameter in 2 consecutive scans were aspirated, and concentrations of various hormones were measured. Cows were defined as cyclic (n=30; 64%) or with the potential to develop follicular pathology (n=17; 36%). Aspirated follicles (n=27) were classified into 3 main groups based on follicular growth rate, follicular diameter, and ovarian activity before and after follicular aspiration. Dominant follicles (n=4) were defined as large follicles (20 mm in diameter) with growth rate ≤1 mm/d and normal ovarian activity. Persistent follicles (n=6) had the same growth rate and diameter as the dominant follicles, but persisted at the same diameter for ≥10 d. Ovarian cysts (n=17) were defined as the largest follicular structures (19 to 32 mm in diameter), with abnormal growth rate (>1 mm/d) and abnormal ovarian activity. Single or turnover cysts did not differ in their growth parameters and were therefore combined and further classified according to follicular-fluid hormone concentrations. Estradiol-dominant cysts (n=7) were characterized by normal estradiol (284 to 659 ng/mL) and progesterone (20 to 113 ng/mL) concentrations, similar to those of the dominant follicle (554 to 993 ng/mL and 44 to 106 ng/mL, respectively). Progesterone-dominant cysts (n=5) were characterized by low estradiol (0.06 to 330 ng/mL) and high progesterone (586 to 3,288 ng/mL) concentrations. Low-steroidogenic active cysts (n=5) were characterized by low concentrations of both estradiol (23 to 61 ng/mL) and progesterone (17 to 205 ng/mL). Characterization of spontaneously forming cysts might enable definition of the formation of ovarian follicular pathologies in postpartum cows.

Electrochemical detection of protein-protein interactions using a yeast two hybrid: 17-beta-estradiol as a model.

In this work we present a modified yeast two-hybrid bioassay for the highly sensitive detection of protein-protein interactions, based on the electrochemical monitoring of beta-D-galactosidase reporter gene activity, using p-aminophenyl-beta-D-galactopyranoside (PAPG) as a synthetic substrate. In a model system, the sensitive detection of 17-beta-estradiol was achieved at concentrations as low as 10(-11)M (approx 2 pg/ml) by monitoring 17-beta-estradiol receptor dimerization after exposure to 17-beta-estradiol. The sensitivity of this system was higher than that of standard optical methods by three orders of magnitude.

Combined phage typing and amperometric detection of released enzymatic activity for the specific identification and quantification of bacteria.

Here, we describe a novel electrochemical method for the rapid identification and quantification of pathogenic and polluting bacteria. The design incorporates a bacteriophage, a virus that recognizes, infects, and lyses only one bacterial species among mixed populations, thereby releasing intracellular enzymes that can be monitored by the amperometic measurement of enzymatic activity. As a model system, we used virulent phage typing and cell-marker enzyme activity (beta-D-galactosidase), a combination that is specific for the bacterial strain Escherichia coli (K-12, MG1655). Filtration and preincubation before infecting the bacteria with the phage enabled amperometric detection at a wide range of concentrations, reaching as low as 1 colony-forming unit/100 mL within 6-8 h. In principle, this electrochemical method can be applied to any type of bacterium-phage combination by measuring the enzymatic marker released by the lytic cycle of a specific phage.

Control of methionine biosynthesis in Escherichia coli by proteolysis.

Most bacterial proteins are stable, with half-lives considerably longer than the generation time. In Escherichia coli, the few exceptions are unstable regulatory proteins. The results presented here indicate that the first enzyme in methionine biosynthesis - homoserine trans-succinylase (HTS) - is unstable and subject to energy-dependent proteolysis. The enzyme is stable in triple mutants defective in Lon-, HslVU- and ClpP-dependent proteases. The instability of the protein is determined by the amino-terminal part of the protein, and its removal or substitution by the N-terminal part of beta-galactosidase confers stability. The effect of the amino-terminal segment is not caused by the N-end rule, as substitution of the first amino acid does not affect the stability of the protein. HTS is the first biosynthetic E. coli enzyme shown to have a short half-life and may represent a group of biosynthetic enzymes whose expression is controlled by proteolysis. Alternatively, the proteolytic processing of HTS may be unique to this enzyme and could reflect its central role in regulating bacterial growth, especially at elevated temperatures.

Identification of the Omega4499 regulatory region controlling developmental expression of a Myxococcus xanthus cytochrome P-450 system.

Omega4499 is the site of a Tn5 lac insertion in the Myxococcus xanthus chromosome that fuses lacZ expression to a developmentally regulated promoter. Cell-cell interactions that occur during development, including C signaling, are required for normal expression of Tn5 lac Omega4499. The DNA upstream of the Omega4499 insertion has been cloned, and the promoter has been localized. Analysis of the DNA sequence downstream of the promoter revealed one complete open reading frame and a second partial open reading frame that is interrupted by Tn5 lac Omega4499. The predicted products of these open reading frames are highly similar to reductase and oxidase components of bacterial cytochrome P-450 systems, which allow catabolism or anabolism of unusual compounds. However, the function of the gene products of the Omega4499 locus remains unclear because M. xanthus containing Tn5 lac Omega4499 exhibits no apparent defect in growth, developmental aggregation, fruiting body formation, or sporulation. Deletion analysis of the Omega4499 regulatory region showed that multiple DNA elements spanning more than 500 bp upstream of the transcriptional start site contribute to developmental promoter activity. At least two DNA elements, one downstream of -49 bp and one between -49 and -218 bp, boosted activity of the promoter in response to intercellular C signaling. Three sequences in the Omega4499 promoter region, centered at -55, -33, and -1 bp, nearly match a 7-bp sequence found in other C signal-dependent promoters. We propose that these sequences, matching the consensus sequence 5'-CAYYCCY-3', be called C box sequences, and we speculate that these sequences are cis-acting regulatory elements important for the expression of M. xanthus genes that depend upon intercellular C signaling during development.

In vitro transcription of Myxococcus xanthus genes with RNA polymerase containing sigmaA, the major sigma factor in growing cells.

Myxococcus xanthus is a Gram-negative bacterium that undergoes multicellular development upon starvation. We have developed a simple and rapid procedure for partial purification of RNA polymerase from growing M. xanthus cells, using heparin-agarose and DNA-cellulose chromatographies. In addition to core subunits, the enzyme contains one fairly abundant polypeptide of approximately 105 kDa. We have shown by Western blot analysis and protein sequencing that the 105-kDa polypeptide is sigmaA, the product of the M. xanthus sigA gene. Partially purified sigmaA RNA polymerase, or holoenzyme reconstituted from sigmaA and core RNA polymerase, transcribed in vitro the vegA and aphII genes that are known to be expressed in growing M. xanthus cells. Reconstituted sigmaA RNA polymerase produced vegA mRNA in vitro with the same 5' end as vegA mRNA produced in vivo, demonstrating that initiation of transcription was accurate in vitro. These results provide biochemical evidence that sigmaA is the major vegetative sigma factor of M. xanthus. To our knowledge, this is the first report of in vitro transcription of M. xanthus chromosomal genes, providing a foundation for further biochemical analysis of transcriptional regulatory mechanisms in a microbe that relies extensively on cell-cell interactions.

Heat shock-dependent transcriptional activation of the metA gene of Escherichia coli.

In Escherichia coli, the growth rate at elevated temperatures is controlled by the availability of endogenous methionine, which is limited because of the temperature sensitivity of the metA gene product, homoserine transsuccinylase (HTS). In order to determine the relationship between this control mechanism and the heat shock response, we estimated the cellular levels of HTS during heat shock by Western (immunoblot) analysis and found an increase following induction by temperature shift and by addition of ethanol or cadmium ions. The elevated level of HTS was a result of transcriptional activation of the metA gene. This activation was heat shock dependent, as it did not take place in rpoH mutants, and probably specific to the metA gene, as another gene of the methionine regulon (metE) was not activated. These results suggest a metabolic link between the two systems that control the response of E. coli to elevated temperatures: the metA gene, which codes for the enzyme responsible for regulating cell growth as a function of temperature elevation (HTS), is transcriptionally activated by the heat shock response.