Identification of Cytokeratins in Bovine Sperm Outer Dense Fibre Fractions

Outer dense fibres (ODF) are important substructures of mammalian sperm tails that are involved in the regulation of sperm motility. In this study, we investigated the identity of several sodium dodecyl sulphate (SDS)‐insoluble ODF proteins. Bovine ODF were purified by separating sperm heads and tails using ultrasound and Percoll^® density gradient centrifugation. Sperm flagella were treated with the detergent cetyltrimethylammonium bromide (CTAB). CTAB‐insoluble material, which reportedly represents the ODF fraction, was collected, and electron microscopy confirmed a highly purified ODF fraction. We found after solubilization of this fraction with SDS that high amounts of insoluble material were retained after centrifugation. SDS‐insoluble material was collected and quantitatively dissolved in 8 M urea. SDS‐gel electrophoresis in the presence of urea revealed polypeptides with apparent molecular masses of approximately 25, 43, and 50 kDa. Subsequent immunoblotting with anti‐cytokeratin antibodies detected two urea‐soluble, SDS‐insoluble proteins with apparent molecular masses of 45 and 66 kDa. The 45‐kDa protein was identified as cytokeratin 19. An antibody reacting with a palette of cytokeratins (CK 1–18 and CK 20), KL1, was the only antibody that reacted with the 66‐kDa polypeptide. We conclude that sperm ODF fractions contain at least one each of type I and type II intermediate filaments. As keratins and intermediate filaments are described as rope‐like structures, we suggest that these intermediate filaments play an important structural or tension‐bearing role in sperm flagella.     

Effects of Confluent, Roscovitine Treatment and Serum Starvation on the Cell-cycle Synchronization of Bovine Foetal Fibroblasts

The present study was designed to examine the effects of cell-cycle synchronization protocols, such as confluent, roscovitine treatment and serum starvation, in bovine foetal fibroblasts on synchronization accuracy at G0/G1, viability, apoptosis, necrosis and ploidy for use as a nuclei donor. The cells in 5-10 passages were randomly allocated into three treated groups. Cells were cultured either in Dulbecco's modified Eagle's medium (DMEM) + 10% foetal bovine serum (FBS) until 90% confluent (group 1, confluent), in DMEM + 10% FBS + 30 microM roscovitine for 12 h (group 2, roscovitine), or in DMEM + 0.5% FBS for 5 days (group 3, serum starvation). Most of the cells (>80%) in all groups were arrested at the G0/G1 stage. Although the rates did not differ, cells in group 1 showed an increased cell population arrested at the G0/G1 phase. Significantly (p < 0.05) higher rates of apoptosis occurred in group 3 than in group 1 and 2 (10% vs 6% and 6%, respectively). No differences in chromosomal abnormality were observed among groups. However, by increasing the number of cell culture passages up to 15, significantly (p < 0.05) higher chromosomal abnormality was observed than in 5 and 10 passages (39% vs 28% and 23%, respectively) in group 1. The results clearly indicated that bovine foetal fibroblasts could be effectively synchronized at G0/G1 stages by all the three different treatments, confluent, roscovitine and serum starvation. However, cells in confluent showed reduced apoptosis and necrosis when they underwent 5-10 passages, exhibiting increased percentage of cells with stable chromosome diversity. Hence, cells in confluent merit further studies before they could be used as nuclear donors.     

Theileria orientalis: a review

Theileria orientalis (also known historically as T. sergenti and T. buffeli) is responsible for benign or non-transforming theileriosis, and exerts its major effect through erythrocyte destruction. The life cycle of T. orientalis is essentially similar to that of other Theileria species, except that the schizonts do not induce transformation and fatal lymphoproliferation. The pathogenesis of anaemia as a result of infection is not clearly established and may be multifaceted. Clinical signs of weakness, reluctance to walk and abortion are early but non-specific indications of disease, particularly if accompanied by a history of cattle being moved. Physical examination may reveal pallor (pale eyes, vaginal mucosa), pyrexia, and elevated heart and respiratory rates. T. orientalis is an economically important parasite of cattle in New Zealand, Australia and Japan, especially where naïve animals are introduced into an endemic area or in animals under stress. Increased awareness of the risks posed by the parasite is required to enable management practices to be implemented to minimise its impact.     

Comparison of two types of CIDR-based timed artificial insemination protocols for repeat breeder dairy cows

This study compared two types of controlled internal drug release (CIDR)-based timed artificial insemination (TAI) protocol for treatment of repeat breeder dairy cows. In the first trial of the experiment, 55 repeat breeder cows were randomly assigned to the following two treatments. (1) In the EB group, a CIDR device was inserted into the cows, and then the cows were administered an injection of 1 mg estradiol benzoate (EB) plus 50 mg progesterone (P4; Day 0). On Day 7, they were given an injection of PGF(2alpha) and the CIDR device was removed. The cows were given an injection of 1 mg EB on Day 8 and were subjected to TAI 30 h later (n=27). (2) In the gonadotrophin releasing hormone (GnRH) group, a CIDR device was inserted into the cows, and then the cows were administered an injection of 250 microg gonadorelin (GnRH; Day 0). On Day 7, they were given an injection of PGF(2alpha) and the CIDR device was removed. The cows were given an injection of 250 microg GnRH on Day 9 and were subjected to TAI 17 h later (n=28). In the second trial, 41 repeat breeder cows that were confirmed as not pregnant in the first trial were randomly assigned to the same two treatments used in the first trial (an EB group of 20 cows and a GnRH group of 21 cows). The ovaries of 15 cows from each group were examined by transrectal ultrasonography in order to observe the changes in ovarian structures, and blood samples were collected for analysis of serum P4 concentrations. The pregnancy rates following TAI in the first (18.5 vs. 32.1%) and second (40.0 vs. 38.1%) trials and the combined rates (27.7 vs. 34.7%) did not differ between the EB and GnRH groups. The proportions of cows with follicular wave emergence within 7 days did not differ between the EB (12/15) and GnRH groups (13/15). The interval to wave emergence was shorter (P<0.01) in the GnRH group than in the EB group, but there was no difference in the mean diameters of dominant follicles on Day 7 between the groups. Moreover, the proportions of cows with synchronized ovulation following a second EB or GnRH treatment did not differ between the groups. In conclusion, treatment with either EB or GnRH in a CIDR-based TAI protocol results in synchronous follicular wave emergence, follicular development, synchronous ovulation, and similar pregnancy rates for TAI in repeat breeder cows.     

Variation and correlation analysis of flavonoids and carotenoids in Korean pigmented rice (Oryza sativa L.) cultivars

Flavonoids and carotenoids of pigmented rice ( Oryza sativa L.), including five black cultivars and two red cultivars, from Korea were characterized to determine the diversity among the phytochemicals and to analyze the relationships among their contents. Black cultivars were higher in flavonoids and carotenoids than the red and white cultivars. The profiles of eight phytochemicals identified from the rice grains were subjected to principal component analysis (PCA) to evaluate the differences among cultivars. PCA could fully distinguish between these cultivars. The Heugjinjubyeo (BR-1) and Heugseolbyeo (BR-2) cultivars were separated from the others based on flavonoid and carotenoid concentrations. Flavonoid contents had a positive correlation with carotenoid contents among all rice grains. The BR-1 and BR-2 cultivars appear to be good candidates for future breeding programs because they have simultaneously high flavonoid and carotenoid contents.     

Revision of the list of aphelinids (Hymenoptera: Aphelinidae) in South Korea and analysis of their relevance as biological control agents for the EPPO region

An updated list of all 27 species of aphelinids (Hymenoptera: Aphelinidae) in nine genera that have been identified or reported from South Korea, along with a dichotomous taxonomic key to separate them, is provided. Ten of the species are included in the EPPO ‘Positive list of biological control agents widely used in the EPPO region’ (which corresponds to Appendix 1 – Commercially used biological control agents – and to Appendix 2 – Successfully introduced classical biological control agents – of EPPO Standard PM 6/3, 2016 version). Five of these species are successfully used in classical biological control. Other species could be used in the EPPO region, or elsewhere in the world, in the future.     

Atmospheric new particle formation enhanced by organic acids

Atmospheric aerosols often contain a substantial fraction of organic matter, but the role of organic compounds in new nanometer-sized particle formation is highly uncertain. Laboratory experiments show that nucleation of sulfuric acid is considerably enhanced in the presence of aromatic acids. Theoretical calculations identify the formation of an unusually stable aromatic acid-sulfuric acid complex, which likely leads to a reduced nucleation barrier. The results imply that the interaction between organic and sulfuric acids promotes efficient formation of organic and sulfate aerosols in the polluted atmosphere because of emissions from burning of fossil fuels, which strongly affect human health and global climate.     

In situ hybridization for the detection and localization of swine Chlamydia trachomatis

Gnotobiotic piglets were inoculated intralaryngeally with swine Chlamydia trachomatis strain R33 or orally with swine C. trachmatis strain R27. Archived formalin-fixed, paraffin-embedded tissues from piglets euthanatized 4-7 days postinoculation were examined by in situ hybridization for C. trachomatis nucleic acid using a nonradioactive digoxigenin-labeled DNA probes that targeted specific ribosomal RNA or omp1 mRNA molecules of the swine C. trachomatis strains. Positive hybridization signals were detected in bronchial epithelial cells, bronchiolar epithelial cells, pneumocytes, alveolar and interstitial macrophages, and jejunal and ileal enterocytes. Chlamydia-infected cells had a strong signal that was confined to the intracytoplasmic inclusions. Positive hybridization signals were not detected in tissue sections from an uninfected control piglet or in C. psittaci-infected sheep placenta. The morphology of host cells was preserved despite the relatively high temperature required in parts of the incubation procedure. The data indicate that in situ hybridization can be used to detect swine C. trachomatis in formalin-fixed, paraffin-embedded tissue specimens.