Spermatic and oxidative profile of domestic cat (Felis catus) epididymal sperm subjected to different cooling times (24, 48 and 72 hours)

Cooling stored epididymal samples for several days allows facilities to transport and process genetic material post‐mortem. Improvements to this practice allow the preservation of sperm from domestic cats, which are the ideal study model for wild felids. However, the modifications in spermatic features and the oxidative profile are not fully understood in cats. This information is necessary for the development of biotechniques, such as new extenders for cryopreservation. Therefore, the purpose of this study was to evaluate the spermatic and oxidative profile in samples from the epididymal cauda of domestic cats cooled at 5°C for 24, 48 and 72 hr. Spermatozoa were collected from the epididymis cauda. Evaluations consisted of computer‐assisted sperm analysis (CASA), plasma membrane integrity (eosin/nigrosin), acrosome integrity (fast green/rose bengal), sperm morphology, sperm DNA integrity (toluidine blue), mitochondrial activity (3′3 diaminobenzidine), activity of the antioxidant enzymes glutathione peroxidase (GPx) and superoxide dismutase (SOD), measurement of lipid peroxidation (TBARS) and protein oxidation. A decrease in sperm motility parameters was observed after 72 hr of cooling (i.e. total and progressive) with a higher percentage of minor (37.7 ± 6.3%) and total defects (53.4 ± 6.3%). Additionally, a decrease in high mitochondrial activity (Class I: 16.6 ± 2.2%) occurred after 72 hr. The decrease in motility rates after a long cooling time probably was caused by the increase in sperm abnormalities. A long cooling time causes cold shock and mitochondrial exhaustion, but there was no observed change with the oxidative stress condition. Therefore, cat epididymal sperm stored at 5°C appear to maintain a high quality for up to 48 hr of cooling time.     

A fast,low‐cost and efficient method for the diagnosis of sperm DNA fragmentation in several species

Sperm DNA fragmentation is a condition that interferes directly in the reproductive efficiency. Currently, there are several methods for assessing the sperm DNA integrity, such as Alkaline Comet, TUNEL and Sperm Chromatin Structure Assay. However, many of these techniques are laborious and require high‐precision equipment. Thus, the development of new techniques can optimize the evaluation of sperm DNA damage. Therefore, the aim of this study was to standardize the toluidine blue (TB) stain technique for the analysis of DNA fragmentation of dog, cat, bull, stallion and ram spermatozoa. For this purpose, we used six animals of each specie (n = 30), in reproductive age. Sperm was collected by different methods according to the particularities of each species, and such samples were divided into two aliquots: a sperm sample was kept at 5°C (considered as intact sperm DNA), and the remaining samples were submitted to the induction of DNA fragmentation by exposure to ultraviolet light for 4 hr. Samples were then mixed with the intact sample to obtain known and progressive proportions of sperm with fragmented DNA (0%, 25%, 50%, 75% and 100%). Semen smears were performed and subjected to staining with TB. Blue‐stained spermatozoa were considered to have DNA fragmentation. We observed high linear regression coefficients between the expected proportion of damaged DNA and the results of TB for dog, cat, ram, bull and stallion samples. In conclusion, TB stain was considered a fast and effective technique for the study of spermatozoa DNA in several species.     

A 3-year assessment of nitrous oxide emission factors for urine and dung of grazing sheep in a subtropical ecosystem

Purpose

Grazing livestock has strong impact on global nitrous oxide (N₂O) emissions by providing N sources through excreta. The scarcity of information on factors influencing N₂O emissions from sheep excreta in subtropical ecosystems such as those of Southern Brazil led us to conduct field trials in three different winter pasture seasons on an integrated crop–livestock system (ICL) in order to assess N₂O emission factors (EF-N₂O) in response to variable rates of urine and dung.

Materials and methods

The equivalent urine-N loading rates for the three winter seasons (2009, 2010, and 2013) ranged from 96 to 478 kg ha^?1, and the dung-N rates applied in 2009 and 2010 were 81 and 76 kg ha^?1, respectively. Air was sampled from closed static chambers (0.20 m in diameter) for approximately 40 days after excreta application and analyzed for N₂O by gas chromatography.

Results and discussion

Soil N₂O-N fluxes spanned the ranges 4 to 353 μg m^?2 h^?1 in 2009, ??47 to 976 μg m^?2 h^?1 in 2010, and 46 to 339 μg m^?2 h^?1 in 2013. Urine addition resulted in N₂O-N peaks within for up to 20–30 days after application in the 3 years, and the strength of the peaks was linearly related to the N rate used. Emission factors of N₂O (EF-N₂O, % of N applied that is emitted as N₂O) of urine ranged from 0.06 to 0.34% and were essentially independent of N rate applied. By considering a ratio of N excreted by urine and dung of 60:40, a single combined excretal EF-N₂O of 0.14% was estimated.

Conclusions

Our findings showed higher mean EF-N₂O for sheep urine than that for dung (0.21% vs 0.03%), irrespective of the occurrence or not of urine patches overlap. This value is much lower than default value of 1% of IPCC’s Tier 1 and reinforces the needs of its revision.

     

Method of determination of nitrosamines in sausages by CO2 supercritical fluid extraction (SFE) and micellar electrokinetic chromatography (MEKC)

In this paper, the use of supercritical fluid extraction (SFE) and micellar electrokinetic capillary chromatography (MEKC) is proposed for the complete analysis of volatile nitrosamines in sausages. The extraction fluid used was CO2 and variables such as density, temperature of thimbles, extraction time, modifier, fluid flow, and kind of traps were investigated. Several experiments were carried out to obtain the most favorable conditions for analysis of volatile nitrosamines in sausages. The recoveries ranged from 21 to 82% for the five nitrosamines studied. The optimal condition of extraction was 0.2 g of sample fortified with 10 mg/kg, using dynamic extraction during 20 min and with adsorbent Florisil in the trap. The solvent selected for the elution of the analytes was methanol.     

Drivers and risk factors for circulating African swine fever virus in Uganda, 2012–2013

We explored observed risk factors and drivers of infection possibly associated with African swine fever (ASF) epidemiology in Uganda. Representative sub-populations of pig farms and statistics were used in a case-control model. Indiscriminate disposal of pig viscera and waste materials after slaughter, including on open refuse dumps, farm-gate buyers collecting pigs and pig products from within a farm, and retention of survivor pigs were plausible risk factors. Wire mesh-protected windows in pig houses were found to be protective against ASF infection. Sighting engorged ticks on pigs, the presence of a lock for each pig pen and/or a gate at the farm entrance were significantly associated with infection/non-infection; possible explanations were offered. Strict adherence to planned within-farm and community-based biosecurity, and avoidance of identified risk factors is recommended to reduce infection. Training for small-scale and emerging farmers should involve multidimensional and multidisciplinary approaches to reduce human-related risky behaviours driving infection.     

Kisspeptin,c‐Fos and CRFR Type 2 Co‐expression in the Hypothalamus after Insulin‐Induced Hypoglycaemia

Normal reproductive function is dependent upon availability of glucose and insulin‐induced hypoglycaemia is a metabolic stressor known to disrupt the ovine oestrous cycle. We have recently shown that IIH has the ability to delay the LH surge of intact ewes. In the present study, we examined brain tissue to determine: (i) which hypothalamic regions are activated with respect to IIH and (ii) the effect of IIH on kisspeptin cell activation and CRFR type 2 immunoreactivity, all of which may be involved in disruptive mechanisms. Follicular phases were synchronized with progesterone vaginal pessaries and at 28 h after progesterone withdrawal (PW), animals received saline (n = 6) or insulin (4 IU/kg; n = 5) and were subsequently killed at 31 h after PW (i.e., 3 h after insulin administration). Peripheral hormone concentrations were evaluated, and hypothalamic sections were immunostained for either kisspeptin and c‐Fos (a marker of neuronal activation) or CRFR type 2. Within 3 h of treatment, cortisol concentrations had increased whereas plasma oestradiol concentrations decreased in peripheral plasma (p < 0.05 for both). In the arcuate nucleus (ARC), insulin‐treated ewes had an increased expression of c‐Fos. Furthermore, the percentage of kisspeptin cells co‐expressing c‐Fos increased in the ARC (from 11 to 51%; p < 0.05), but there was no change in the medial pre‐optic area (mPOA; 14 vs 19%). CRFR type 2 expression in the lower part of the ARC and the median eminence was not altered by insulin treatment. Thus, disruption of the LH surge after IIH in the follicular phase is not associated with decreased kisspeptin cell activation or an increase in CRFR type 2 in the ARC but may involve other cell types located in the ARC nucleus which are activated in response to IIH.     

Follow-up of mercury levels in fish,human hair and urine in the Madeira and Tapajós basins,Amazon, Brazil

Informal economy of gold mining has contaminated some important river basins in Amazon. Follow-up studies on critical compartments showed some areas with high Hg levels in fish as well as in human hair samples. Average Hg in piscivorous fish in the Madeira river itself was 846 ppb (N=284) with a maximum of 3921 ppb. Mercury in fish from non polluted areas in this basin shows high variability, even for single species. A seasonal variation in Hg content was observed, with higher values at the end of the dry season. In the upper Tapajós basin comparable values were found for fish but with a definite decreasing trend downstream. Average value for piscivorous fish in the whole Tapajós basin is 482 ppb (N=122) with a maximum value of 3770 ppb. Hair Hg was higher in fishing villages in the Tapajós (average: 17 ppm; with N=432 and maximum value of 176 ppm) than in the Madeira (average: 9 ppm; N=169; maximum 71 ppm), and data from some areas of the Tapajós suggest a decrease with time. Mercury was much higher in urine of goldshop workers in Santarém (low Tapajós) than in Alta Floresta (high Tapajbs) and show a decreasing trend in both cases, probably related to the significant decline in gold mining activities during the study period (1986–1994).     

Methoxylated xanthones in the quality control of small centaury (Centaurium erythraea) flowering tops.

In the course of a phytochemical study of the bitter tonic plant, small centaury (Centaurium erythraea), six methoxylated xanthones (1,5-hydroxy-3-methoxyxanthone, 1-hydroxy-3,5,6-trimethoxyxanthone, 1-hydroxy-3,5,6,7-tetramethoxyxanthone, 1-hydroxy-3,5,6,7,8-pentamethoxyxanthone, 1-hydroxy-3,7,8-trimethoxyxanthone and 1,8-dihydroxy-3,5,6,7-tetramethoxyxanthone) were isolated and identified by spectroscopic means (nuclear magnetic resonance, mass spectroscopy, and UV). Subsequently, a high-performance liquid chromatography/diode array detection method was developed for the determination of these and other methoxylated xanthones occurring in the chloroform extract of small centaury aerial parts. The methodology developed was applied to twelve samples, and in all of them, nine xanthones were identified and quantified. This methodology can be considered complimentary to the one proposed by the European Pharmacopoeia.     

Stabilization of soybean oil with heated quercetin and 5-caffeoylquinic acid in the presence of ferric ion

The effect of heated quercetin (400 mg/kg of oil) or 5-caffeoylquinic acid (5-CQA) and the presence of ferric ion (2.2 mg/kg of oil) on the stability of soya oil oxidized in an oxidative stability index (OSI) instrument was investigated. After heating the phenolic at 100 degrees C or 150 degrees C, the OSI values of treated oils were not significantly (p < 0.001) different, whereas, at 200 degrees C, the values decreased significantly with addition of quercetin, but not with 5-CQA. However, the antioxidative activity of quercetin remained significantly greater than that of 5-CQA. The antioxidative potency of quercetin was reduced significantly by addition of ferric palmitate (FP), but that of 5-CQA was not. Reaction between the ortho-dihydroxy groups of the quercetin and ferric ion may reduce the number of hydroxyls available to react with free radicals. Chelating action of 5-CQA might be provided by ortho-dihydroxy grouping of the quinic acid moiety.