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.     

The immune response and PBMC subsets in canine visceral leishmaniasis before, and after, chemotherapy

Peripheral blood mononuclear cell subsets, in vitro lymphoproliferative response to leishmanial antigen, and Leishmania-specific serum antibody levels were examined in 11 dogs, naturally infected with L. infantum, and 9 healthy control dogs. A decrease in the percentage of CD4+ T-cells and an increase in the proportion of gammadelta T-cells and sIgG+ B-cells were observed during canine visceral leishmaniasis (CVL). These changes may be responsible for the marked humoral response and the absence of in vitro lymphoproliferation to mitogen and specific parasite antigens. This possibility was supported by the analysis of these subsets after treatment with amphotericin B. One month after therapy, a significant increase in the percentage of CD4+ T-cells and a decrease of gammadelta T-cells and sIgG+ B-cells were observed. At the same time, the lymphocyte blastogenesis assay with leishmanial antigen was positive and the levels of specific antibodies to Leishmania were significantly lower than before the treatment. Five months after therapy, lymphocyte proliferative response to LSA disappeared, antibody and lymphocyte subsets levels returned to those observed during CVL. Therapeutic failure in CVL is associated with the inability of antileishmanial drugs to completely revert the profound immunodepression induced by the infection and prevent relapse.     

Ultrasonographic anatomy and biometric analysis of the thoracic and abdominal organs in healthy foals from birth to age 6 months

Knowledge of normal renal parameters, as documented in mature horses, is essential for the accurate evaluation of abnormal kidneys. Although the ultrasonographic appearance and location of the abdominal organs in foals and the renal dimensions in neonates have been reported, there is currently no information available for the assessment of normal organ growth in foals. The objectives of the study were to describe the ultrasonographic characteristics, location and variations of the thoracic and abdominal organs with relation to age, height and weight; and provide a growth table for comparison with diseased foals. The thoracic and abdominal cavities of 10 healthy foals were evaluated at ages 1, 7, 14 and 21 days and 1, 2, 3, 4, 5 and 6 months. The equipment used was an Ausonics Opus Plus ultrasound. For every evaluation, weight and height were obtained, the foals were sedated and the area of study was clipped and cleaned. The ultrasonographic location, appearance and measurements of the different organs were recorded for each examination. The study revealed that foals age >1 month resemble the mature ultrasonographic pattern. Continual growth of the organs was observed from Day 1 to age 6 months, being faster in the first month. Organ growth was closely correlated with age, but not with sex, height or weight in healthy foals up to age 6 months. Our study has provided measurements of longitudinal organ growth in healthy foals, presented in a simple form for easy comparison with diseased individuals.     

In vitro effects of 5-hydroxytryptamine and cisapride on the circular smooth muscle of the jejunum of horses

OBJECTIVE: To determine effects of cisapride and 5-hydroxytryptamine (5-HT) on the jejunum of horses. SAMPLE POPULATION: Jejunal muscle strips from 8 horses. PROCEDURE: Muscle strips were suspended in isolated muscle baths. Isometric stress responses to 5-HT and cisapride, with and without specific antagonists, were determined. RESULTS: Muscle strips incubated with atropine and tetrodotoxin responded to 5-HT and cisapride with an increase in contractile force. The 5-HT caused a concentration-dependent increase in contractile amplitude, with a maximum response (Emax) of 1,151+/-214 g/cm2 and a molar concentration that induces contractile force equal to 50% of maximum response (EC50) of 0.028+/-0.002 microM. Prior incubation with the 5-HT2 antagonist ketanserin decreased the Emax (626 +/-147 g/cm2) and potency (EC50, 0.307+/-0.105 microM) of 5-HT Prior incubation with the 5-HT3 antagonist tropisetron decreased the efficacy (Emax, 894+/-184 g/cm2) to 5-HT Cisapride also caused a concentration-dependent increase in contractile amplitude, with an Emax of 331+/-82 g/cm2 and an EC50 of 0.302+/-0.122 microM. Prior incubation with ketanserin decreased the Emax (55+/-17 g/cm2) and potency (EC50, 0.520+/-0.274 microM) of cisapride. CONCLUSION AND CLINICAL RELEVANCE: Stimulatory effects of 5-HT and cisapride on circular smooth muscle of equine jejunum are mediated primarily through a noncholinergic effect. The effects of 5-HT are mediated, at least partially, by 5-HT2 and 5-HT3 receptors, whereas the effects of cisapride are mediated primarily by 5-HT2 receptors. This may impact treatment of horses with postoperative ileus.     

含莫纳可林K红曲降低鸡蛋胆固醇效果的研究

48只68周龄的海兰褐蛋鸡分成三组，按0.5％和10％比例分别添加含0．4％莫纳可林K红曲于各组日粮内，饲喂22d，观测其对鸡蛋胆固醇变化的影响。结果表明，投药后5—6d，10％添加组和5％添加组蛋黄胆固醇开始下降；15—22d的胆固醇含量与1～5d相比，10％添加组和5％添加组分别下降了9．02％和5．10％，而对照组升高了5．22％。