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.     

Dietary carbohydrate/lipid ratios and nutritional condition in juvenile southern rock lobster, Jasus edwardsii

The availability of formulated diets is considered economically imperative if rock lobster aquaculture, based on the collection and ongrowing of puerulus, is to progress. Central to the development of diets is an understanding of the nutritional requirements of the species. This study determined the effect of different dietary carbohydrate/lipid ratios (17:1, 5:1, 2:1, 0.8:1) on the growth and nutritional condition of juvenile southern rock lobsters, Jasus edwardsii, by measuring standard growth parameters, proximate composition of the whole body and digestive gland, and from a histological investigation of the digestive gland. Four replicate groups of eight lobsters (initial weight 5.08±0.98 g (mean±S.D.)) per diet treatment were held in 50 l tanks, in a recirculating system at 18 °C for 84 days. Maximum growth and the highest levels of lipid and dry matter in digestive glands and whole bodies was found in lobsters fed a diet containing 27% carbohydrate and 13.5% lipid (2:1 ratio) suggesting that of the four experimental diets, this diet provided the best balance of lipid and carbohydrate. Digestive gland histology supported this conclusion and lobsters fed low carbohydrate, high lipid diets were in the best nutritional condition, with high lipid accumulation, and structurally sound epithelial cells. Digestive gland epithelial cells of lobsters fed the high carbohydrate, low lipid diets were compressed, of inconsistent shape and size, with low lipid accumulation. Histology is therefore seen as a feasible method, in addition to growth and proximate composition data, to further examine the effect of diets in nutritional studies of crustaceans.     

The Effect of Piglet Expulsion in the Sow on Plasma Cortisol, Adrenocorticotropic Hormone and β-endorphin

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Previous studies have shown an increase in plasma cortisol in gilts over farrowing irrespective of environment suggesting that factor(s) associated with parturition itself cause physiological stress. Factors involved in mediating the hypathalomo–pituitary–adrenal (HPA) axis at parturition are not well understood. This study examines the effect of piglet expulsion on the pituitary–adrenal axis by measurement of plasma cortisol, adrenocorticotropic hormone (ACTH) and β-endorphin. The effect of farrowing environment in modulating the acute response to piglet expulsion is also investigated. Twelve second parity sows, with indwelling jugular catheters, were moved into either a farrowing crate or a straw-bedded pen 5 days before their expected parturition date (EPD). Blood samples were taken from each sow during a prefarrowing baseline period and then rapid samples (2.5 mins) were taken for 10 min following the birth of two piglets. No effect of environment was found on any of the hormonal variables which reinforces the hypothesis that the physiological stress seen in parturient pigs is due to some intrinsic factor of parturition. Plasma cortisol, ACTH and β-endorphin did not change significantly in the period following piglet expulsion suggesting that individual piglet expulsions do not play a major role in 'parturition stress'. There was however, an increase in plasma cortisol, ACTH and β-endorphin in response to increasing piglet number which is consistent with previous studies of general farrowing in which cortisol increased as farrowing progressed. Therefore this study reinforces the hypothesis that physiological stress increases with ongoing parturition although this does not appear to be a result of piglet expulsion. The potential role of other factors which may be involved in causing 'parturition stress' should be investigated.     

Interactions of aflatoxin and the antioxidant butylated hydroxytoluene in two-week-old chicks

Two-week dietary administration of 2500 ppb aflatoxin was sufficient to cause a decrease in bursal weights and a reduction in the number of splenic leukocytes in chicks. No significant effects on weight gain or feed efficiency were evident. The chicks also had elevated heterophil-to-lymphocyte ratios, suggesting a heightened reaction to stress. This effect could be blocked by dietary administration of the antioxidant butylated hydroxytoluene (BHT) at a concentration eight-fold over that normally present to preserve control feed. The BHT treatment increased the activities of the enzymes glutathione-S-transferase, aniline hydroxylase and O-demethylase, which metabolize aflatoxins in the liver.     

Survival of 4 dogs with persistent atrial standstill treated by pacemaker implantation

Pacemakers were implanted in 4 client-owned female dogs which had persistent atrial standstill. Three dogs were alive after 14 to 39 months and 1 dog was euthanized after 10.5 years. This report demonstrates that some dogs with persistent atrial standstill can survive for extended time periods.     

Pneumonitis associated with occult heartworm disease in dogs

Nine of 69 dogs with occult heartworm disease (13%) had allergic pneumonitis characterized by consistent clinical and radiographic signs. Although the clinical signs were severe, the degree of radiographic pulmonary arterial abnormalities was mild. Corticosteroid therapy resulted in rapid resolution of clinical and radiographic signs; thiacetarsamide therapy was then given without complications. This syndrome may not be recognized as heartworm-associated and may be confused with other disorders, some being associated with a poor prognosis and requiring different therapy.     

Effect of a major gene for growth on protein synthesis in mice

Rates of protein synthesis of mice with a major gene (hg) for rapid postweaning gain (line Ch) and their normal counterparts (line CH) were determined at 21, 31 and 42 d of age with an intraperitoneal injection of a flooding dose of 14C-leucine. A preliminary experiment demonstrated that the relationship between the specific activities of leucine in acid-soluble supernatants and carcass protein corresponded to the theoretical precursor-product relationship, indicating that the method is valid for estimating protein synthesis rates. Using this method at 21, 31 and 42 d of age, whole-body protein fractional synthesis rates (FSR) were 43.7, 32.7 and 29.1%/d and 41.9, 32.6 and 33.1%/d for lines CH and Ch, respectively. Although differences between lines were not significant, FSR decreased with age. Absolute synthesis rate (ASR), where ASR = (FSR) X (whole body protein), was greater (P less than .001) at 21, 31 and 42 d of age in line Ch as compared with CH, and increased (P less than .001) with age. The relative contributions of liver, gastrointestinal tract, heart-kidney-lung and remaining carcass to whole body protein ASR were not affected by line, but did change (P less than .05) with age. Whole body protein fractional breakdown rate (FBR), calculated as FSR minus whole body protein fractional growth, indicates that differences between lines CH and Ch whole-body FSR and(or) FBR exist only between 24 and 33 d of age, and that the maximum value of this difference probably does not exceed 10%.