Cytochrome-P450 enzymes and autoimmunity: expansion of the relationship and introduction of free radicals as the link

The Cytochrome-P-450 enzymes (CYP) are among the most important xenobiotic-metabolizing enzymes, which produce reactive oxygen species (ROS) as the result of metabolizing xenobiotics.ROS are believed to play important roles in the pathophysiology of autoimmune diseases. ROS can alter the structure of cellular antigens to produce a "neo-antigen" which could mount an autoimmune response against the original antigen through molecular mimicry. ROS are involved in apoptosis, activation of antigen presenting cells and initiation or amplification of diverse immunologic reactions.Taking all these facts together, it could be speculated that CYP may be involved in the initiation and/or amplification of autoimmune phenomena.     

Effects of supplemental dietary vitamin C on quality of semen from Nile tilapia (Oreochromis niloticus) breeders

The objective of this study was to evaluate the effects of vitamin C on growth and quality of semen from Oreochromis niloticus breeders. One hundred and sixty males were fed with different levels of vitamin C (0, 261, 599 and 942 mg/kg diet). The higher weight values were recorded for 599 (166 g) and 942 (175 g) mg of vitamin C/kg diet. Sperm motility, vigour and concentration were higher with 599 and 942 mg of vitamin C/kg diet. The semen volume, gonadosomatic index and plasma protein data from the last week showed a direct relationship with increasing levels of vitamin C. No changes were observed in the hepatosomatic index and blood glucose. The haematocrit and erythrocyte showed higher values estimated by equations derived at 850 and 638 mg vitamin C/kg diet, respectively. The leucocytes were inversely proportional to the increasing levels of vitamin C. After 100 days of feeding, animals fed the diet containing 942 mg vitamin C/kg diet had higher sperm motility, linearity, curvilinear velocity, straight line velocity and average path velocity (p < .05). Higher values of beat cross‐frequency were observed in broodfish fed diets containing 942 and 599 mg vitamin C/kg. The different vitamin C levels did not cause differences in straightness, lateral head displacement and sperm morphology. For Nile tilapia males on intensive rearing and handling conditions, vitamin C levels between 599 and 942 mg/kg may be used for a better performance and quality of semen.     

Comparison of the TBARS Assay and BODIPY C11 Probes for Assessing Lipid Peroxidation in Red Deer Spermatozoa

Several methods are used to measure lipid peroxidation (LPO) in spermatozoa. The objective of this study was comparing the thiobarbituric acid reactive species (TBARS) method and the BODIPY 581/591 C₁₁ (B581) and BODIPY 665/676 C₁₁ (B665) fluorescent probes to measure induced peroxidative damage in thawed epididymal spermatozoa from Iberian red deer. Samples from three males were thawed, pooled, diluted in PBS, incubated at room temperature and assessed at 0, 3, 6 and 24 h under different experimental conditions: Control, hydrogen peroxide (H₂O₂) 0.1 mm or 1 mm , or tert‐butyl hydroperoxide (TBH) 0.1 mm or 1 mm . LPO was assessed by the TBARS assay [malondialdehyde (MDA) detection] and by the fluorescence probes B581 and B665 (microplate fluorimeter and flow cytometry). Increasing MDA levels were only detectable at 1 mm of TBH or H₂O₂. Both fluorescence probes, measured with fluorometer, detected significant increases of LPO with time in all treatments, except Control. Flow cytometry allowed for higher sensitivity, with both probes showing a significant linear relationship of increasing LPO with time for all oxidizing treatments (p < 0.001). All methods showed a good agreement, except TBARS, and flow cytometry showed the highest repeatability. Our results show that both B581 and B665 might be used for LPO analysis in Iberian red deer epididymal spermatozoa, together with fluorometry or flow cytometry. Yet, the TBARS method offered comparatively limited sensitivity, and further research must determine the source of that limitation.     

Synchronizing Cell Cycle of Goat Fibroblasts by Serum Starvation Causes Apoptosis

Cell cycle stage and synchronization of donor cells are important factors influencing the success of somatic cell nuclear transfer. This study examined whether serum starvation has any effect on specific cell death. We also studied the effects of serum starvation, culture to confluence, and full confluency (confluent + 72 h) on cell cycle characteristics and apoptosis of goat dermal fibroblast cells. The cells were obtained from the ear of a 1.5‐year‐old female goat. The following experimental groups were analysed for fibroblast cells: (i) normally growing, (ii) confluent, (iii) full confluency, (iv) cells starved for 48 h and (v) cells starved for 72 h. Analysis of cell cycle distribution by flow cytometry showed that 4.56 and 51.88% of normal cycling cells were at the G0 and G1 phases respectively. In the confluent group, 80% of the cells were arrested in the G0/G1 phase. Serum starvation for 48 and 72 h arrested 84.78% and 90.1% cells at the G0/G1 phase respectively which showed a significant difference when compared with the control group (p < 0.05). Double staining by PI and FITC distinguishes G0 phase from G1 phase. In the full confluency group, 91.53% of cells were at G0/G1 stage, but in contrast to the serum starved group, this high percentage of G0/G1 cells was mainly associated with G1 cells. Under normal culture conditions, 6.39% of cells underwent early apoptosis. In the confluent group 8.93% of cells showed early apoptosis. Serum starvation for 48 and 72 h caused early apoptosis in 8.91 and 39.83% of the cells respectively. Full confluency treatment did not increase the number of apoptotic cells significantly (8.67%). After 72 h, serum starvation significantly increased early apoptosis (p < 0.05). In conclusion, the use of full confluency is suitable for cell cycle synchronization because it arrests cells at the G0/G1 phase and also induces less apoptosis in comparison with the serum starvation group.     

A study of the microbial flora of the anterior vagina of normal sows during different stages of the reproductive cycle

SUMMARY Sterile guarded swabs were used to sample the anterior vaginal and cervical area of 23 normal healthy sows during various stages of the reproductive cycle. The samples were collected one week before farrowing, within 24 hours of farrowing, weekly up to weaning, at mating and at 2 and 3 weeks after mating, and then plated and incubated aerobically and anaerobically. At least one positive sample was obtained from each sow and at each stage of the reproductive cycle. Most positive samples (78.3%) were obtained on the day of farrowing and the least 3 weeks after mating (19.0%). The second highest number of positive samples (45.5%) was found immediately after mating. Although there was no significant difference among sows of different parities, there was a trend for older sows to have more positive samples after farrowing (84.6%). There was a greater decrease in positive samples after farrowing and after mating among younger sows compared with older sows. A wide range of bacteria including aerobic and anaerobic species, were recovered from 142 Isolates. The more representative bacteria were Streptococcus spp (23.2%); Escherichia coll (22.5%); Staphylococcus spp (19.0%) and Corynebacterium spp (13.4%). Of the cultures, 54.7% were pure and 45.3% were mixed. Both the percentage of bacterial isolates as well as the type of culture (pure or mixed) were similar to those frequently reported in clinical cases of vulval discharge syndrome. The results indicate that sows usually develop infections of the reproductive tract at farrowing and mating but these infections do not normally persist.     

Confirmation of epidural needle placement using nerve stimulation in dogs

Caudal epidural anesthesia is useful when anesthesia of the lumbar and sacral dermatomes is needed. Its success relies on the proper placement of the needle in the epidural space. However, accurate positioning of the needle can be difficult in certain patients (i.e.obesity). The purpose of this preliminary study was to document the use of nerve stimulation as a means of confirming accurate needle positioning in the epidural space prior to drug administration. Twenty large breed dogs undergoing hindlimb or perineal surgery were enrolled. Following induction of general anesthesia, patients were prepared for routine epidural drug administration. A 17 ga, 3.5” shielded Tuohy needle was used and was connected to a peripheral nerve stimulator set to deliver a current at 1 Hz, with a pulse width of 0.2 m sec. Initial current was set at 1.2 mA as the needle was advanced into position. Confirmation of epidural needle placement was confirmed when twitches were observed in the hindlimbs and/or tail. Current setting was then decreased incrementally by 0.2 mA until no further twitches were observed. Success of epidural drug placement was confirmed subjectively by motor blockade to the blocked dermatomes and clinical signs of balanced anesthesia (lack of sympathetic response to surgical stimulation while maintained at light plane of anesthesia). Lowest mean (range) current to elicit hindlimb twitches was 0.72 mA (0.4–1.0 mA). Lowest mean (range) current to elicit tail twitches was 0.58 mA (0.4–1.0 mA). Tail twitches were reliably lost at mean current of 0.37 mA (0.2–0.8). Epidural anesthesia was considered to be successful in 19/20 dogs. In only 9/20 dogs, needle placement would have been correct based on using ‘classic’ indicators alone (‘pop’ as enter epidural space, loss of resistance to injection). The results of this study suggest that nerve stimulation may be useful in confirming correct epidural needle placement prior to drug administration.