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.     

Contribution to the morphology and biochemistry of mustelide blood. 1. Blood specimen collection in various mustelides with special reference to the use of the tissue adhesive "Fimomed"]

An account is given of various techniques of blood sampling in the context of certain mustelids, including cardiopuncture, amputation of tail tip, talon cutting, incision of the ear vein, puncture of the jugular or femoral vein, and catheterisation of the abdominal aorta. Reference is made to details of use of all techniques, characteristics, advantages and potential setbacks, and preferable use of some of the tested methods to collect blood from mustela and martes species. Blood collection from the abdominal aorta may be helpful in obtaining no-haemolysis and no-additive plasma for biochemical multi-screening. Biochemical, pharmacological, and toxicological follow-up checks may be feasible under certain conditions following surgical exposure of the external jugular vein. The use of "Fimomed" (n-butylcyano-acrylate), a tissue adhesive, may help to reduce effort in terms of time and material and consequently, rationalise veterinary hygiene action as a whole, provided that the conditions for its application are observed. The skin adhesive is properly applicable to skin lesions of mustelids. A combination of suturing with adhesive should be used to close laparotomy wounds for better mechanical strength of the abdominal wall. Possible applications of "Fimomed" should be tested with other species as well.     

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.     

Effects of testicular biopsy in clinically normal bulls

OBJECTIVE: To determine whether testicular needle biopsy is detrimental to testicular function in clinically normal bulls. DESIGN: Prospective study. ANIMALS: 6 mixed-breed mature bulls. PROCEDURE: A randomly selected testicle from each bull was biopsied with a 14-gauge needle biopsy instrument. Bulls were then evaluated over a 90-day period for changes in scrotal temperature and thermal patterns, ultrasonographic appearance, and quality of spermatozoa. At the end of the 90-day study, bulls were castrated, and testicles were examined grossly and histologically. RESULTS: Changes were detected in scrotal temperatures and thermal patterns and in the breeding soundness examination results during the first 2 weeks of the study. However, there were no long-term changes in semen quality over the course of the experiment. Hyperechoic areas were detected on ultrasonographic examination and corresponded to the areas of penetration by the biopsy instrument. Microscopic lesions that were indicative of testicular dysfunction were not found. CONCLUSIONS AND CLINICAL RELEVANCE: Results indicate that testicular biopsy is a safe procedure in bulls. Testicular biopsy could possibly be used to further examine bulls that have less than satisfactory results for breeding soundness examinations.     

Long-bone fractures in llamas: six cases (1993-1998)

OBJECTIVE: To determine clinical and radiographic findings, treatment, and outcome for llamas with long-bone fractures. DESIGN: Retrospective study. ANIMALS: 6 llamas. PROCEDURE: Medical records of llamas admitted between 1993 and 1998 because of long-bone fractures were reviewed. Data collected included age, sex, type of fracture, method of fracture repair, and postoperative complications. The Fisher exact test was used to compare age and sex of the llamas with long-bone fractures with those of the hospital population of llamas. All owners were contacted by telephone to determine perceived postoperative problems and whether the llamas were able to perform as expected. RESULTS: Mean age was 160.8 days (range, 23 to 365 days). There was 1 male and 5 females. Fractures were more likely to occur in young llamas (< or = 1 year old) than in adults. Five of the fractures were attributed to traumatic episodes. Long bones affected included the tibia (n = 2), radius (2), femur (1), and humerus (1). Internal fixation with lag screws, plating, or both was performed on fractures of all llamas except 1; that llama was treated by use of confinement to a stall. None of the llamas had intraoperative complications, but postoperative complications were reported in 2 llamas. All fractures healed eventually, and clients were pleased with outcomes. CONCLUSIONS AND CLINICAL RELEVANCE: Long-bone fractures in llamas are uncommon. Several types of long bone fractures can be successfully repaired by use of internal fixation, resulting in few complications and minimal convalescent time.