Oxytetracycline Pharmacokinetics After Intramuscular Administration in Cows with Clinical Metritis Associated with Trueperella Pyogenes Infection

Systemic therapy with oxytetracycline is often used for treatment of clinical metritis although data about its penetration into the uterus and uterine secretion are lacking. Uterine secretions and milk from six cows with clinical metritis were collected for microbiological assay. The animals were treated intramuscularly with long-acting oxytetracycline (20 mg/kg) and samples of plasma, milk and uterine secretions were collected for determination of the antibiotic concentrations by HPLC-PDA analysis. Pharmacokinetics of the antibiotic and in silico prediction of its penetration into the uterus were described. Trueperella pyogenes with MIC values of 16–64 µg mL−1 was isolated (n of cows = 4) from uterine secretions. Oxytetracycline showed fast absorption and penetration in the uterine secretions and milk. No change of withdrawal time for milk was necessitated in cows with clinical metritis. Maximum levels in uterine secretions and predicted concentrations of oxytetracycline in the uterus were lower than MIC values. Systemic administration of long-acting oxytetracycline did not guarantee clinical cure and was not a suitable choice for treatment of clinical metritis associated with Trueperella pyogenes. The appropriate approach to antibiotic treatment of uterine infections of cows requires knowledge on penetration of the antibiotics at the site of infection and sensitivity of pathogens.

The biochemistry surrounding bovine conceptus elongation†

Conceptus elongation is a fundamental developmental event coinciding with a period of significant pregnancy loss in cattle. The process has yet to be recapitulated in vitro, whereas in vivo it is directly driven by uterine secretions and indirectly influenced by systemic progesterone. To better understand the environment facilitating this critical reproductive phenomenon, we interrogated the biochemical composition of uterine luminal fluid from heifers with high vs physiological circulating progesterone on days 12–14 of the estrous cycle—the window of conceptus elongation-initiation—by high-throughput untargeted ultrahigh-performance liquid chromatography tandem mass spectroscopy. A total of 233 biochemicals were identified, clustering within 8 superpathways [amino acids (33.9%), lipids (32.2%), carbohydrates (8.6%), nucleotides (8.2%), xenobiotics (6.4%), cofactors and vitamins (5.2%), energy substrates (4.7%), and peptides (0.9%)] and spanning 66 metabolic subpathways. Lipids dominated total progesterone (39.1%) and day (57.1%) effects; however, amino acids (48.5%) and nucleotides (14.8%) accounted for most day by progesterone interactions. Corresponding pathways over-represented in response to day and progesterone include (i) methionine, cysteine, s-adenosylmethionine, and taurine (9.3%); (ii) phospholipid (7.4%); and (iii) (hypo)xanthine and inosine purine metabolism (5.6%). Moreover, under physiological conditions, the uterine lumen undergoes a metabolic shift after day 12, and progesterone supplementation increases total uterine luminal biochemical abundance at a linear rate of 0.41-fold day−1–resulting in a difference (P ≤ 0.0001) by day 14. This global metabolic analysis of uterine fluid during the initiation of conceptus elongation offers new insights into the biochemistry of maternal–embryo communication, with implications for improving ruminant fertility.

Progesterone alters the bovine uterine fluid lipidome during the period of elongation

Successful bovine pregnancy establishment hinges on conceptus elongation, a key reproductive phenomenon coinciding with the period during which most pregnancies fail. Elongation is yet to be recapitulated in vitro, whereas in vivo it is directly driven by uterine secretions and indirectly influenced by prior circulating progesterone levels. To better understand the microenvironment evolved to facilitate this fundamental developmental event, uterine fluid was recovered on Days 12–14 of the oestrous cycle – the window of conceptus elongation initiation – from cycling heifers supplemented, or not, with progesterone. Subsequent lipidomic profiling of uterine luminal fluid by advanced high-throughput metabolomics revealed the consistent presence of 75 metabolites, of which 47% were intricately linked to membrane biogenesis, and with seven displaying a day by progesterone interaction (P ≤ 0.05). Four metabolic pathways were correspondingly enriched according to day and P4 – i.e. comprised metabolites whose concentrations differed between groups (normal vs high P4) at different times (Days 12 vs 13 vs 14). These were inositol, phospholipid, glycerolipid and primary bile acid metabolism. Moreover, P4 elevated total uterine luminal fluid lipid content on Day 14 (P < 0.0001) relative to all other comparisons. The data combined suggest that maternal lipid supply during the elongation-initiation window is primarily geared towards conceptus membrane biogenesis. In summary, progesterone supplementation alters the lipidomic profile of bovine uterine fluid during the period of conceptus elongation initiation.

Can uterine secretion of modified histones alter blastocyst implantation, embryo nutrition, and transgenerational phenotype?

Extracellular histones support rodent and human embryo development in at least two ways. First, these molecules in uterine secretions protect embryos from inflammation caused by pathogens that gain access to the reproductive tract. Also, histones in uterine secretions likely support penetration of the uterine epithelium by blastocysts during embryo implantation. Extracellular histones seem to preserve amino acid transport system B0,+ in blastocysts by inhibiting its activity. Preservation of system B0,+ is needed because, at the time of invasion of the uterine epithelium by motile trophoblasts, system B0,+ is likely reactivated to help remove tryptophan from the implantation chamber. If tryptophan is not removed, T-cells proliferate and reject the implanting blastocyst. Epigenetic modification of histones could alter their promotion of normal implantation through, say, incomplete tryptophan removal and, thus, allow partial T-cell rejection of the conceptus. Such partial rejection could impair placental development, embryonal/fetal nutrition, and weight gain prior to birth. Small-for-gestational-age offspring are predisposed to developing metabolic syndrome, obesity, and associated complications as adults. Shifting expression of these phenotypes might contribute to transgenerational variation and evolution. The spectrum of possible extracellular histone targets in early development warrant new research, especially since the effects of epigenetic histone modifications might be transgenerational.

Proteomic changes in uterine flushings after levonorgestrel treatment and effects on sperm function

When levonorgestrel (LNG) is given for emergency contraception during the follicular phase inhibits or delays ovulation, but also induces changes in endometrial secretions that modulate sperm functionality. In order to characterize the female reproductive tract secreted molecules that may affect human spermatozoa, we analyzed changes in the protein content of uterine flushings obtained from women during the periovulatory phase of a control and a LNG-treated menstrual cycle. Lectin affinity analysis and two-dimensional gel electrophoresis of uterine samples showed changes in protein glycosylation pattern and the presence of 31 differentially expressed proteins (8 up- and 23 down-regulated). Mass spectrometry and Western blot analyses of the differential expressed proteins showed lactotransferrin (LTF) as one of the up-regulated molecules by LNG. In this study, LTF exhibited significant dose-related effects on sperm functionality, particularly a decrease of calcium ionophore-induced acrosome reaction and protein tyrosine phosphorylation. Overall, the results indicated that LNG promoted changes in the proteome of uterine secretions that might compromise human sperm capacitation. These data further support the participation of other mechanisms of action of LNG as emergency contraceptive, in addition to those on ovulation.

Comparative effects of dexamethasone on gravid uteri histology and immunoreactivity in sheep and goats

Introduction: Dexamethasone treatment greatly improves foetal and maternal survival. However, it causes a decrease in birth weight which has been attributed to decreasing placental functions. In addition to placental role in foetal nutrition, uterine cells secretions play important role in foetal nutrition. The uterine cell development is regulated by progesterone receptor (PR). This study investigated comparative effects of dexamethasone on gravid uteri histology and immunoreactivity in ovine and caprine species. Methods: Sixteen Sahel does and 2 bucks; and 16 Yankasa ewes and 2 rams were used for this study. Dexamethasone injections were administered at 0.25 mg/kg body weight on days 1, 3 and 5 during first trimester, and day 51, 53 and 55 during second trimester. Uterine biopsies were harvested at days 28 and day 78 of gestation for histological and immunohistochemical analysis. Results: Endometrial glands were down-regulated, while PR were up regulated by dexamethasone in both species with more severe effects in sheep compared to goats. The down-regulation of the endometrial glands contributed, to the decrease in birth weight commonly reported to be associated with dexamethasone treatment. Significance: Dexamethasone decreased uterine epithelial proliferation in both species, but more severe in sheep. The decrease in endometrial glands proliferation has the potential to reduce uterine secretions and influence absorption of stratum functionale. The use of dexamethasone might lead to foetal growth retardation and decrease neonatal weight. The differences in uterine responses between the sheep and goats suggest species difference in response to dexamethasone treatment.