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.     

Characterisation of a maize-infecting potyvirus from Spain

In order to characterise and classify an unknown maize-infecting potyvirus isolated from fields in northeast Spain, the entire coat protein gene and the C-terminal twothirds of the large nuclear inclusion protein (NIb) gene were cloned and sequenced. Protein sequencing enabled the cleavage site between the two proteins to be deduced and also revealed that on storage the viral coat protein undergoes a specific degradation in which the N-terminal 39 amino acids are removed. Comparison of the nucleotide sequence of the 3 non-coding region of the viral RNA and the predicted amino acid sequence of the coat protein with the equivalent regions of other members of the potyvirus group revealed that the Spanish virus is closely related to maize dwarf mosaic virus strain A.     

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.     

In Vivo Embryo Recovery Rate by Laparoscopic Technique from Rabbit Does Selected for Growth Rate

Rabbit does from R line selected for growth rate present a low reproductive performance and this study aimed to evaluate both the recovery efficacy and viability of recovered embryos after vitrification and the reproductive performance of donor does subjected to in vivo recovery. Does were divided into three groups: 28 does without in vivo recovery (control), 25 does in which in vivo recovery was started in the nulliparous state (group 1) and 30 does with at least one litter before in vivo recovery (group 2). Does were superovulated with a single subcutaneous injection of 50 IU of equine chorionic gonadotropin (eCG) per female, and were then artificially inseminated 60 h later and immediately administered an intravenous dose of 75 IU of human chorionic gonadotropin (hCG) per female. Does from group 1 and 2 were recovered in vivo 76-80 h post-insemination by repeated laparoscopies at one to four times and permitted one or two parturitions between recoveries [in vivo (IV) recovery]. At the end of the experiment, about 16 does of all groups were recovered post-mortem (PM recovery). All normal embryos were vitrified, devitrified and then cultivated in vitro to evaluate the viability after thawing. A significant increase in the ovulation rate was found in does recovered PM than in those recovered IV in the nulliparous state. However, no significant differences were observed in the recovery rate, the donor rate, the number of normal embryos recovered with at least one normal embryo per doe and the viability after thawing between the PM and IV groups. A significant decrease in the fertility rate, total born, live born and weaned kids was found for does from group 1 in comparison with does from group 2. Results support the use of repeated laparoscopy to increase the number of recovered embryos per donor doe especially in such R line does, if they are permitted to produce at least one litter before the beginning of in vivo recovery.     

Use of Powdered Egg Yolk vs Fresh Egg Yolk for the Cryopreservation of Ovine Semen

Egg yolk is a common additive to sperm cryopreservation diluents. Because of its animal origin, however, it also represents a potential risk of microbiological contamination in the diluent. This potential contamination can be avoided by using powdered egg yolk, instead of fresh egg yolk, as it is pasteurized. This study was conducted to determine ram sperm cryosurvival was affected by the type of egg yolk used (powdered egg yolk or fresh egg yolk) and by yolk concentration (10, 15 or 20%) in the diluent. Microbiological analyses were also performed to quantify the microbiological contamination in the diluents containing the two types of egg yolk. Sperm cryosurvival was determined by motility and morphology analyses after thawing. Motility parameters were assessed using a computer-assisted sperm analysis (CASA) system, and the percentage of sperm with a normal apical ridge was evaluated using a differential interference contrast microscope. No significant differences were observed between diluents in the percentage of sperm with normal apical ridge. However, higher percentages of total motile cells were observed for samples containing powdered egg yolk (69%) compared to samples containing fresh egg yolk (60%). However, sperm in diluents containing fresh egg yolk, exhibited higher values for average-path velocity, straight-line velocity and beat cross frequency and lower values for amplitude of lateral head displacement (p <0.05), compared to cells in diluents containing powdered egg yolk. Microbiological contamination was similar (<200 CFU/ml) in both diluents, and no bacterial growth was observed in either, when antibiotics were added. Therefore, powdered egg yolk can be effective used in diluents for the freezing of ram semen. However, the in vivo fertility of sperm frozen in diluents containing powdered egg yolk should be tested, as some motility parameters were different for sperm treated with powdered egg yolk compared to fresh egg yolk.     

Histopathological findings in farmed rainbow trout (Oncorhynchus mykiss) naturally infected with 3 different Aeromonas species

This study describes the macroscopic and microscopic lesions in farmed rainbow trout (Oncorhynchus mykiss) naturally infected with genetically identified Aeromonas salmonicida, A. hydrophila, and A. veronii species. The genus Aeromonas includes bacteria that naturally inhabit both waterways and organisms. At least 27 Aeromonas species have been identified to date, some of which can cause significant economic losses in aquaculture. As up to 68.8% of Aeromonas isolates may be misidentified in routine biochemical and phenotypic tests, however, reported cases of Aeromonas infection in fish may be wrongly identified. Our findings confirmed that the 3 Aeromonas species studied are associated with septicemia and dermal lesions in rainbow trout.     

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.     

Blood and haemoglobin meal as protein sources in diets for gilthead sea bream (Sparus aurata): effects on growth, nutritive efficiency and fillet sensory differences

Two parallel experiments were conducted to evaluate the effect of partial substitution of fish meal by two different animal protein sources, blood meal (B) and haemoglobin meal (H) at 0, 50 and 100 g kg⁻¹ of level inclusion in diets for gilthead sea bream, considering a long feeding period for juveniles (Trial 1) and a short feeding period (Trial 2) for on-growing fish. In Trial 1, 33 g juveniles were fed for 242 days and the fish fed with 5% and 10% of haemoglobin dietary inclusion obtained less growth, although feed efficiency, protein efficiency ratio and muscle composition were similar in all diets. In Trial 2, 179 g initial weight fish were fed for 164 days and growth of fish fed H10 showed the lowest growth, although nutrient efficiency and muscle composition were not affected significantly. The results of these experiments demonstrated that blood meal can substitute fish meal (up to 10%) with no effect on performance, but may lead to sensory differences compared with fish fed diet 0, while the inclusion of 5% blood meal had no effect on growth or sensory characteristics. Fish fed 10% haemoglobin inclusion had the poorest growth values.