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.     

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％。     

Chemical composition, protein quality, palatability, and digestibility of alternative protein sources for dogs

The chemical composition and protein quality of 11 alternative protein sources (chicken products, blood products, enzyme-hydrolyzed fish protein concentrate, soybean meal, and spray-dried pork liver) were determined, and an experiment was conducted to determine palatability and digestibility of processed red blood cell-containing diets. Chicken protein sources differed in concentrations of CP, acid-hydrolyzed fat, and total AA (TAA) by 20, 31, and 24%, respectively, and GE by 1.7 kcal/g. Blood protein sources varied little in acid-hydrolyzed fat and GE concentrations, but concentrations of CP and TAA differed by 11 and 8%, respectively. Protein solubility of chicken and blood protein source categories averaged 57 and 69%, respectively. Protein solubility of enzyme-hydrolyzed fish protein concentrate, soybean meal, and spray-dried pork liver was 53, 67, and 26%, respectively. Based on calculations from immobilized digestive enzyme assay values, lysine digestibility averaged approximately 80.4 and 81.7% for blood and chicken protein sources, respectively. Lysine digestibility values for soybean meal and spray-dried pork liver were 89 and 77%, respectively. A chick protein efficiency ratio (PER) assay showed that chicken protein sources had high protein quality values, as the PER ranged from 2.7 to 5.3, whereas blood protein sources had poor protein quality (PER values less than 1.5). Enzyme-hydrolyzed fish protein concentrate, spray-dried pork liver, and soybean meal had high protein quality (PER values greater than 2.8). In the dog palatability and digestibility experiments, a corn and chicken-based diet supplemented with either 0 or 3% processed red blood cells was tested. The palatability test showed that dogs consumed more of the diet that contained 0% vs. 3% processed red blood cells. The intake ratio for the 3% processed red blood cells diet was 0.34. Nutrient digestibilities did not differ, except for CP, where the digestibility was greater (P = 0.01) for dogs consuming the 0% processed red blood cells diet. These data suggest that chemical composition and quality of alternative protein sources differ greatly among ingredients within the same category. Palatability data suggest that a processed red blood cells-containing diet is not highly palatable but, when this diet was offered as only one food, dogs demonstrated no aversion response but some decrease in protein digestion.     

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.     

Assessing common bottlenose dolphin (Tursiops truncatus) population structure in Mississippi Sound and coastal waters of the north central Gulf of Mexico

1. Population structure of highly mobile marine organisms can be complex and difficult to study, but it is important to understand how populations partition themselves within their environment for accurate assessment of both natural and anthropogenic impacts and successful management. The 2010 Deepwater Horizon oil spill negatively impacted common bottlenose dolphins (Tursiops truncatus) within Mississippi Sound and the surrounding north central Gulf of Mexico (GOMx); however, little was known about their underlying population structure in these waters. Thus, it was unclear how many demographically independent populations were affected by the spill.
2. Common bottlenose dolphin samples were collected throughout inshore waters of Mississippi Sound and coastal waters of the north-central GOMx. Mitochondrial DNA control region sequence data and 19 nuclear microsatellite loci were analysed to determine how many populations are present and characterize their range throughout these waters.
3. Bayesian clustering and migration analyses identified two genetically distinct and demographically independent populations: one predominantly inhabiting Mississippi Sound and adjacent coastal waters, and a second population extending generally from offshore of Mobile Bay, Alabama, east along the Florida Panhandle. Neither of these populations align with the currently delineated management stocks previously used to estimate impacts from the oil spill on common bottlenose dolphins in this portion of the GOMx.
4. These results suggest that revisions may be necessary so that management stocks accurately represent the demographically independent populations present in these waters. Furthermore, better comprehension of underlying population structure will enhance impact assessments on common bottlenose dolphins and provide more appropriate baseline data to support future restoration and conservation objectives.

     

Volatile fatty acid metabolism by epithelial cells isolated from different areas of the ewe rumen

Cells were harvested from four rumen locations in four 2- to 3-yr-old ewes fed fescue hay to determine whether cell origin has an effect on cellular VFA metabolism. Tissue (approximately 150 cm2) was excised from the anterior cranial pillar, ventral sac floor, caudal pillar surface, and dorsal sac ceiling. Cells were isolated using serial tryptic digestion. One milliliter of isolate was incubated for 2 h in 6 mL of medium containing 25 mM propionate and 10 mM butyrate. Incubations were terminated at 0, 30, 60, 90, and 120 min and analyzed for beta-hydroxybutyrate, acetoacetate, lactate, and pyruvate. Cell yield was 22, 22, 24, and 14 (+/- 6) x 106 cells/mL, and viability was 92, 92, 94, and 87% for anterior cranial pillar, ventral sac floor, caudal pillar surface, and dorsal sac ceiling, respectively. All metabolite concentrations and ratios of redox pairs increased throughout the incubations, indicating continuous cellular activity. Final 2-h concentrations (nmol/10(6) cells) were 123, 113, 163, and 158 (+/- 35) for beta-hydroxybutyrate; 38, 42, 24, and 45 (+/- 10) for acetoacetate; 25.3, 20.6, 10.1, and 20.4 (+/- 5.6) for lactate; and 2.54, 0.98, 1.06, and 1.31 (+/- 0.61) for pyruvate in the anterior cranial pillar, ventral sac floor, caudal pillar surface and dorsal sac ceiling incubations, respectively. Origin of rumen tissue had no significant effect on metabolite production, indicating that cellular location is not a critical factor that affects rate of rumen epithelial cell VFA metabolism under these specific in vitro conditions.