Incidence of delayed ovulation in Holstein heifers and its effects on fertility and early luteal function

In the first of 3 experiments 134 first-service and 108 repeat-breeder Holstein heifers were palpated at 12-hour intervals starting 24 hours after insemination to compare the incidence of delayed ovulation in the 2 groups. Delayed ovulation was defined as failure to ovulate within the first 24 hours after insemination. Ovulation occurred within 24 hours post insemination in 92.1% of the animals and was delayed in 7.9% of the cases, with no differences between first-service and repeat-breeder heifers, indicating that the subfertility of the repeat-breeder animals was not due to delayed ovulation. The duration of the delay was at most 12 hours since all the animals had ovulated by 36 hours post insemination. Conception rate of the 19 animals with delayed ovulation (42.1%) was not different (P>0.05) from that of the 223 heifers that ovulated on time (44.8%). In a second experiment, no differences were detected between 15 heifers with delayed ovulation and 15 animals that ovulated on time with respect to their progesterone concentrations during the first 8 days post insemination, indicating that delayed ovulation is not associated with delayed luteinization or subnormal early luteal function. In the third experiment, the conception rate of 126 repeat-breeder heifers that were treated with hCG at the time of insemination was 26.7%; the conception rate of 101 repeat-breeder heifers that were inseminated twice, at 12 and 24 hours after the onset of estrus, was 34.6%; and the conception rate of 105 repeat-breeder heifers which were not treated with hCG and which were inseminated only once was 30.5% (P>0.05) It is concluded that delayed ovulation is not an important cause of infertility and does not constitute an important component of the repeat-breeding syndrome in Holstein heifers.     

The effect of the intramammary infusion of Escherichia coli endotoxin on ovulation in lactating dairy cows

The purpose of this experiment was to determine if intramammary inflammation during the periovulatory period affects the occurrence of ovulation in lactating dairy cows. Ten lactating, cyclic, Holstein dairy cows received 2 injections of prostaglandin F2alpha at eleven-day intervals, to synchronize luteolysis. The day of the second injection was designated as day 0. Ovulation was anticipated to occur 3-5 days later (on days 3-5). Beginning at the morning milking on day 1, cows received intramammary infusions of either Escherichia coli endotoxin (10 microg; n=5) or infusion vehicle (pyrogen free Hank's balanced salt solution; n=5) into 2 quarters immediately after milking. The same quarters were infused after each milking through day 4. Venous blood samples were collected daily from day -1 through 13 for determination of progesterone to monitor luteolysis and formation of a new corpus luteum. Blood samples were also collected at 4-h intervals (days -1 to 2), then at 2-h intervals (days 2 to 5) to measure concentrations of luteinizing hormone. Ovaries were examined ultrasonographically on days -1 through 5 and on day 12 to monitor follicular growth and formation of the corpus luteum. Collectively, these observations were used to determine if and when ovulation occurred. Intramammary infusion of E. coli endotoxin induced an immediate increase in the concentration of somatic cells in milk from treated quarters. However, this treatment had no effect on the occurrence or timing of ovulation. Based on ultrasonography and concentrations of progesterone, four of five cows in each treatment group appeared to have ovulated. Preovulatory surges of LH were detected within the intensive bleeding periods for three cows in each treatment group. The magnitude of the LH surge was reduced in cows receiving endotoxin.     

Pattern of circulating luteinizing hormone isoforms during the estrous and luteal phases in Holstein heifers

The pattern of distribution of circulating luteinizing hormone (LH) isoforms in cattle during estrus and the luteal phase was investigated. In each stage, the stage of the estrous cycle was synchronized in seven Holstein heifers with a prostaglandin analogue. After estrus was detected, blood samples were taken at 2-h intervals for 24h. In the luteal phase, animals received 250 microg i.v. of GnRH and blood samples were collected every 15 min for 5h. LH concentration in the samples was determined. Samples with the greatest LH concentration in estrus (pre-ovulatory peak) and those collected 60 min after GnRH administration (luteal phase) were analyzed by chromatofocusing, eluted with a pH gradient from 10.5 to 3.5. Eluted LH was grouped into basic (pH > or = 7.5), neutral (pH 7.4-6.5) and acidic isoforms (pH < or = 6.4) as well as by pH unit. In both phases, basic forms were the most abundant, and these were greater (P < 0.05) during the luteal phase (78.4 +/- 4.2%) as compared with during estrus (57.1 +/- 6.2%); the proportion of neutral and acidic isoforms in estrus (13.7 +/- 2.6%; 28.5 +/- 2.8%) was greater (P < 0.05) as compared with the luteal phase (3.0 +/- 0.7; 18.7 +/- 3.4). These results indicate that the relative proportion of LH isoforms secreted by the adenohypophysis differ by stage of estrous cycle. The addition of excess of NaCl to the column modifies the antigen-antibody binding in the RIA, and the proteins eluted are erroneously quantified as LH; this is an artifact of the technique.     

The biosynthesis of gram-negative endotoxin. Identification and function of UDP-2,3-diacylglucosamine in Escherichia coli

Escherichia coli mutants defective in the pgsB gene are phosphatidylglycerol-deficient in certain genetic settings and accumulate novel, glucosamine-derived phospholipids (Nishijima, M., and Raetz, C. R. H. (1979) J. Biol. Chem. 254, 7837-7844). The simplest of these compounds is 2,3-diacylglucosamine 1-phosphate (2,3-diacyl-GlcN-1-P) ("lipid X" of E. coli), in which beta-hydroxymyristoyl moieties are the sole fatty acid substituents (Takayama, K., Qureshi, N., Mascagni, P., Nashed, M. A., Anderson, L., and Raetz, C. R. H. (1983) J. Biol. Chem. 258, 7379-7385). We now report a sensitive radiochemical method for detection of 2,3-diacyl-GlcN-1-P in wild type E. coli and demonstrate that there are about 4000 molecules/cell (0.02% of the total CHCl3-soluble phosphorus). In mutants bearing the pgsB1 lesion, the levels are 100- to 300-fold higher. In addition, we have discovered a novel liponucleotide, UDP-2,3-diacyl-GlcN, that also accumulates in conjunction with the pgsb1 mutation. This material represents 0.005% of the wild type phospholipid and accumulates 50- to 100-fold in the mutant. The identification of UDP-2,3-diacyl-GlcN in E. coli is based on: 1) migration of a minor 32P-labeled lipid from wild type and mutant cells with a UDP-2,3-diacyl-GlCn standard during two-dimensional thin layer chromatography; 2) susceptibility of this 32P-labeled material to cleavage by a liponucleotide-specific pyrophosphatase; and 3) chromatographic identification of [32P]UMP and [32P]2,3-diacyl-GlcN-1-P (lipid X) as the sole products of the enzymatic degradation. As shown in the accompanying article, this novel nucleotide is crucial for biosynthesis of lipid A disaccharides in extracts of E. coli and Salmonella typhimurium.     

The effect of exogenous oxytocin on luteal function in mares

Daily injections of 150 units oxytocin administered to 6 mares on Days 4, 5, 6, 7 and 8 after ovulation (Day 0 = ovulation) failed to induced luteolysis as indicated by the maintenance of normal plasma progestagen concentrations and the occurrence of normal ovulatory intervals. Three additional mares were given oestrogen injections 24 h before an injection of oxytocin on Day 7 after ovulation, but this treatment also failed to induce luteolysis since plasma progestagen concentrations were maintained in all three mares. Two mares exhibited normal ovulatory intervals, while the third developed a corpus luteum which persisted for 46 days.     

Ultrastructural changes in alveolar macrophages in rats under the effect of Escherichia coli endotoxin

Electron-microscopic investigation has shown that alveolar macrophages phagocyte surfactant in the initial period of the endotoxin shock, necrotized cells--in the intermediate period, fibrin--at the stage of late endotoxemia.     

The function of ubiquinone in Escherichia coli

1. The function of ubiquinone in Escherichia coli was studied by using whole cells and membrane preparations of normal E. coli and of a mutant lacking ubiquinone. 2. The mutant lacking ubiquinone, strain AN59 (Ubi(-)), when grown under aerobic conditions, gave an anaerobic type of growth yield and produced large quantities of lactic acid, indicating that ubiquinone plays a vital role in electron transport. 3. NADH and lactate oxidase activities in membranes from strain AN59 (Ubi(-)) were greatly impaired and activity was restored by the addition of ubiquinone (Q-1). 4. Comparison of the percentage reduction of flavin, cytochrome b(1) and cytochrome a(2) in the aerobic steady state in membranes from the normal strain (AN62) and strain AN59 (Ubi(-)) and the effect of respiratory inhibitors on these percentages in membranes from strain AN62 suggest that ubiquinone functions at more than one site in the electron-transport chain. 5. Membranes from strain AN62, in the absence of substrate, showed an electron-spin-resonance signal attributed to ubisemiquinone. The amount of reduced ubiquinone (50%) found after rapid solvent extraction is consistent with the existence of ubiquinone in membranes as a stabilized ubisemiquinone. 6. The effects of piericidin A on membranes from strain AN62 suggest that this inhibitor acts at the ubiquinone sites: thus inhibition of electron transport is reversed by ubiquinone (Q-1); the aerobic steady-state oxidation-reduction levels of flavins and cytochrome b(1) in the presence of the inhibitor are raised to values approximating those found in the membranes of strain AN59 (Ubi(-)); the inhibitor rapidly eliminates the electron-spin-resonance signal attributed to ubisemiquinone and allows slow oxidation of endogenous ubiquinol in the absence of substrate and prevents reduction of ubiquinone in the presence of substrate. It is concluded that piericidin A separates ubiquinone from the remainder of the electron-transport chain. 7. A scheme is proposed in which ubisemiquinone, complexed to an electron carrier, functions in at least two positions in the electron-transport sequence.     

The function of OmpA in Escherichia coli

Outer membrane protein A (OmpA) is a major protein in the Escherichia coli outer membrane. In this study, the function of OmpA in E. coli stress survival was examined. An E. coli K1 ompA-deletion mutant was significantly more sensitive than that of its parent strain to sodium dodecyl sulfate (SDS), cholate, acidic environment, high osmolarity, and pooled human serum. A number of amino acid changes at the extracellular loops of OmpA did not affect the viability of E. coli, while short peptide insertions in the periplasmic turns of the OmpA beta-barrel decreased E. coli resistance to environmental stresses. Moreover, ompA mutants were found to survive much better within brain microvascular endothelial cells than the wild-type strain, supporting that OmpA is a major target in mammalian host cell defense. These results indicated that OmpA plays a vital structural role in E. coli, and suggested that a perfect beta-barrel structure of OmpA is important for outer membrane stability. Based on these results and the published OmpA structural analyses, I propose that OmpA is composed of three functional domains including a hydrophilic extracellular mass, a beta-barrel transmembrane structure, and a peptidoglycan binding domain.     

Control of endotoxin release in Escherichia coli fed-batch cultures

High amounts of outer membrane (OM) components were released in glucose-limited fed-batch (GLFB) cultures at 37 °C at specific growth rates approaching 0.05 h⁻¹. Endotoxin analyses from a 20 °C GLFB culture gave similar results. An alternative fermentation technique, the temperature-limited fed-batch (TLFB) technique, reduced the endotoxin concentration in a culture with a biomass concentration of 30 g l⁻¹ from the 850 mg l⁻¹ in traditional GLFB cultures to about 20 mg l⁻¹. The TLFB technique uses the temperature to regulate the dissolved oxygen tension, while all substrate components are unregulated. It appears to be severe glucose limitation that triggers the extensive release of endotoxins rather than a low growth rate. Furthermore, it is not the low temperature that stabilizes the OM when using the TLFB technique. Simulations and experimental data show that this technique results in the same biomass productivity as the GLFB technique.     

Steroid hormone feedback on LH in prepubertal Holstein heifers

A significant dose-response relationship between gonadotropin-releasing hormone (GnRH) and time to luteinizing hormone (LH) peak, peak serum LH and total serum LH was obtained in prepubertal Holstein heifers (28 weeks of age) (Experiment 1). For the second experiment, the effect of steroid feedback on the anterior pituitary was determined. A steady infusion of saline, estradiol-17 beta or progesterone was maintained for 24 h while GnRH, in various schemes, was administered 8 h after the beginning of steroid infusion. Estradiol-17 beta infusion (2.08 micrograms/h), although it did not affect peripheral concentrations of estrogen, caused an LH release 24 to 30 h later in 37.5% of the heifers. This amount of exogenous estrogen did not affect the LH response to a single GnRH (4 micrograms) challenge. When the same GnRH dosage (4 micrograms) was administered 6 times at hourly intervals, the heifers infused with estradiol had a lower response after the first 2 injections of GnRH and a greater response after the last 4 injections than heifers infused with saline. When GnRH was infused (4 micrograms/h) for 6 h, beginning 8 h after steroid infusion, estradiol infusion caused a significantly higher peak LH and total LH release than an infusion of either saline or progesterone (7.3 micrograms/h). The progesterone infusion had no effect on the GnRH-stimulated LH release. We conclude that prepubertal dairy heifers have an anterior pituitary capable of responding to the feedback effect of estrogen in a positive manner.     

The effect of spatial structure on adaptation in Escherichia coli

Populations of organisms are generally organized in a given spatial structure. However, the vast majority of population genetic studies are based on populations in which every individual competes globally. Here we use experimental evolution in Escherichia coli to directly test a recently made prediction that spatial structure slows down adaptation and that this cost increases with the mutation rate. This was studied by comparing populations of different mutation rates adapting to a liquid (unstructured) medium with populations that evolved in a Petri dish on solid (structured) medium. We find that mutators adapt faster to both environments and that adaptation is slower if there is spatial structure. We observed no significant difference in the cost of structure between mutator and wild-type populations, which suggests that clonal interference is intense in both genetic backgrounds.     

Effect of Escherichia coli endotoxin on adenine nucleotide translocation in canine heart mitochondria

The in vitro effect of Escherichia coli endotoxin on the translocation of adenine nucleotides in dog heart mitochondria was studied. Mitochondrial adenine nucleotides were labeled with ¹⁴C by incubating mitochondrial preparations in the presence of [¹⁴C]ADP. The exchange reaction was initiated by addition of unlabeled ADP, proceeded for 5 to 60 s at 4 °C, and was terminated by addition of atractyloside. The results showed that preincubation of mitochondria with endotoxin (50 μg/mg protein) for 10 min at 23 °C decreased the exchange reaction by 21.2% (P < 0.05). The inhibitory effect of endotoxin was increased with increasing concentrations of endotoxin with an I₅₀ value of 45 μg/mg protein. The initial rate and the total extent of exchange were both affected. Double reciprocal plots showed that only the V but not the K_m for ADP was affected by endotoxin, indicating that the inhibition was noncompetitive in nature. The exchange of adenine nucleotide remained depressed by endotoxin in the presence of either oligomycin or antimycin A, indicating that the inhibitory effect of endotoxin was independent of the action of endotoxin on oxidative phosphorylation. The leakage of labeled adenine nucleotides from mitochondria at 23 °C was increased by 100% by endotoxin (100 μg/mg protein) in the absence of added unlabeled ADP, and this increase in the leakage could not be blocked by atractyloside. The endotoxin-induced changes in adenine nucleotide exchange and leakage were either partially or completely prevented by hydrocortisone, heparin, dibucaine, or EDTA. Since most of these agents have in common an effect on lipid metabolism, it is suggested that endotoxin-induced alterations in the exchange and leakage of adenine nucleotides in heart mitochondria are protected through a mechanism involving membrane lipid reorganization.