Caspase Activation,Hydrogen Peroxide Production and Akt Dephosphorylation Occur During Stallion Sperm Senescence

To investigate the mechanisms inducing sperm death after ejaculation, stallion ejaculates were incubated in BWW media during 6 h at 37°C. At the beginning of the incubation period and after 1, 2, 4 and 6 h sperm motility and kinematics (CASA), mitochondrial membrane potential and membrane permeability and integrity were evaluated (flow cytometry). Also, at the same time intervals, active caspase 3, hydrogen peroxide, superoxide anion (flow cytometry) and Akt phosphorylation (flow cytometry) were evaluated. Major decreases in sperm function occurred after 6 h of incubation, although after 1 h decrease in the percentages of motile and progressive motile sperm occurred. The decrease observed in sperm functionality after 6 h of incubation was accompanied by a significant increase in the production of hydrogen peroxide and the greatest increase in caspase 3 activity. Additionally, the percentage of phosphorylated Akt reached a minimum after 6 h of incubation. These results provide evidences that sperm death during in vitro incubation is largely an apoptotic phenomena, probably stimulated by endogenous production of hydrogen peroxide and the lack of prosurvival factors maintaining Akt in a phosphorylated status. Disclosing molecular mechanisms leading to sperm death may help to develop new strategies for stallion sperm conservation.     

Cluster Membership, Centrality, Structural Holes and Innovation: A study on the Telecommunication Industry in the Republic of Korea

The research focuses on strategic alliances for innovation in the telecommunication industry in the Republic of Korea. The author reviews relevant literature and specifically investigates how cluster membership affects innovative performance. Later, he combines it with an endogenous factor - centrality. The main motivation is to investigate if the existing theories and theories, which are largely based in the Western context, applies in a collectivistic South Korean context.     

Blood calcium,glucose and haematology profiles of parturient bitches diagnosed with uterine inertia or obstructive dystocia

Bitches with dystocia most often present with clinical signs of uterine inertia (UI). The aetiology of myometrial dysfunction in most of these cases is still not elucidated. We compared blood ionized calcium (iCa) and glucose concentrations in bitches diagnosed with primary UI (PUI, n = 14), secondary UI (SUI, n = 6) or obstructive dystocia (OD, n = 6), and we described their haematology profiles. Bitches diagnosed with UI had a patent birth canal and delivered no puppies yet (PUI) or only part of the whole litter (SUI). The OD group had no UI and showed strong abdominal contractions. Blood iCa did not differ between the PUI, SUI and OD groups and was not influenced by litter size. There was a significant positive relationship (R² = .241, p = .013) between iCa concentrations and the dam's body weight. Glucose concentrations were also not significantly different between dystocia groups or influenced by body weight and litter size. Hypocalcaemia was detected in 11 bitches, and hypoglycaemia in two bitches. Pregnancy‐associated anaemia was seen in about one‐third of the bitches. Eight of 12 dogs had increased platelet counts, and ten had leukocytosis with mature neutrophilia. Although iCa did not differ between dystocia groups, low concentrations may have contributed to the development of UI in some of the small size bitches. Hypoglycaemia was uncommon, and therefore, we consider low glucose concentrations not to have played an important role in the pathogenesis of UI in our study population. Pregnancy‐associated anaemia, thrombocytosis, leukocytosis and mature neutrophilia were common findings in otherwise healthy bitches diagnosed with different forms of dystocia.     

Endometrial Explant Culture to Study the Response of Equine Endometrium to Insemination

Mating‐induced endometritis (MIE) is ubiquitous in the horse after natural mating and artificial insemination with frozen/thawed semen causing the most aggressive response. The majority of mares eliminate MIE 24–48 h after insemination. An endometrial explant culture was tested as a potential in vitro exemplar for sperm‐induced MIE. Endometrial prostaglandin F_2α (PGF_2α) secretion and expression of interleukin‐8 (IL‐8) were used as markers of inflammation. Endometrial explants were cultured from uteri collected from follicular phase mares. Explants were challenged with 1 or 10 × 10⁶ sperm/ml frozen/thawed semen, chilled semen, washed sperm or seminal plasma. Medium was collected 24 and 72 h after challenge and assayed for PGF_2α by radioimmunoassay. Treatment of endometrial explants with frozen/thawed, chilled semen or washed sperm did not change the secretion of PGF_2α compared with untreated controls. However, 24 h after challenge cultured explants expressed IL‐8. The in vitro endometrial explant system did not represent the in vivo response to semen when PGF_2α was used as a marker of inflammation, yet the use of gene expression as an inflammatory marker warrants further investigation.     

Developing high throughput quantitative PCR assays for diagnosing Ikeda and other Theileria orientalis types common to New Zealand in bovine blood samples

AIMS: To develop rapid, quantitative PCR (qPCR) assays using high resolution melt (HRM) analysis and type-specific TaqMan assays for identifying the prevalent types of Theileria orientalis found in New Zealand cattle; and to evaluate their analytical and diagnostic characteristics compared with other assays for T. orientalis.

METHODS: Nucleotide sequences aligned with T. orientalis Buffeli, Chitose and Ikeda types, obtained from DNA extracted from blood samples from infected cattle, were used to design HRM and type-specific probe-based qPCR assays. The three type-specific assays were also incorporated into a single-tube multiplex qPCR assay. These assays were validated using DNA extracted from blood samples from cattle in herds with or without clinical signs of T. orientalis infection, other veterinary laboratory samples, as well as plasmids containing T. orientalis type-specific sequences. Diagnostic specificity (DSp) and sensitivity (DSe) estimates for the qPCR assays were compared to blood smear piroplasm results, and other PCR assays for T. orientalis. Copy number estimates of Ikeda DNA in blood were determined from cattle exhibiting anaemia using the Ikeda-specific qPCR assay.

RESULTS: The T. orientalis type-specific and the HRM qPCR assays displayed 100% analytical specificity. The Ikeda-specific qPCR assay exhibited linearity (R²?=?0.997) with an efficiency of 94.3%. Intra-assay CV were ≤0.08 and inter-assay CV were ≤0.095. For blood samples from cows with signs of infection with T. orientalis, the DSp and DSe of the multiplex probe qPCR assay were 93 and 96%, respectively compared with blood smears, and 97 and 100%, respectively compared with conventional PCR assays. For the Ikeda-specific qPCR assay, the number of positive samples (n=66) was slightly higher than a conventional PCR assay (n=64). The concentration of Ikeda genomes in blood samples from 41 dairy cows with signs of infection with T. orientalis ranged between 5.6?×?10⁴ and 3.3?×?10⁶ genomes per µL of blood.

CONCLUSIONS: All qPCR assays had improved specificity and sensitivity over existing conventional PCR assays for diagnosis of T. orientalis Ikeda. The burden of Ikeda DNA in blood was demonstrated using an Ikeda-specific qPCR assay with titrated synthetic gene target.

CLINICAL RELEVANCE: Adoption of high-throughput DNA extraction and qPCR reduced T. orientalis and Ikeda diagnosis times. The Ikeda-specific qPCR assay provides a specific diagnosis for Ikeda in animals with signs of infection with T. orientalis and can be used to monitor the parasite load of Ikeda in blood.