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.     

The in vitro efficacy of a quaternary ammonia disinfectant and/or ethylenediaminetetraacetic acid-tris against commercial broiler hatchery isolates of Pseudomonas aeruginosa

Studies have indicated variations in the degree of efficacy of certain commercial disinfectants used in poultry production facilities. We used an adequate method of in vitro testing to compare the efficacy of disinfectants while testing them in conditions similar to those of the poultry facilities. BioSentry 904, ethylenediaminetetracetic acid (EDTA)-Tris, and a combination of the two were tested by this method against five field isolates of Pseudomonas aeruginosa at 10(3), 10(6), and 10(9) colony-forming units (CFU)/ml. At the 10(9) CFU/ml concentration, most compounds failed to achieve a total kill with a contact time of 15 min. When tested at bacterial concentrations of 10(3) CFU/ml, the combination of EDTA-Tris mixed at a 1:1 ratio with BioSentry 904 killed the bacteria upon initial contact (< or = 0.05 min). This disinfectant mixture exhibited antagonistic, indifferent, or synergetic effects when exposed to different bacterial isolates at a concentration of 10(6) CFU/ml.     

Fertility and sperm quality of broiler breeder males infected with subgroup J avian leukosis virus

In order to assess the effects of subgroup J avian leukosis virus (ALV-J) on semen quality, broiler breeder males were separated by ALV-J status (ALV-J positive = POS, ALV-J negative = NEG) at 44 wk of age. Of the 249 males originally placed at 1 day of age, 101 (40.6%) died by 43 wk of age. Observations of tumor expression and high mortality suggest that many of the males that died prior to 44 wk of age were infected with ALV-J. From 47 to 56 wk of age, hens were inseminated every third week with 7.5 x 10(7) sperm. Fertility and hatch data were collected by incubating eggs laid during the 2 wk postinsemination (WPI). The number of sperm that penetrated the perivitelline membrane of the ovum was determined from eggs laid on the eighth day postinsemination. Sperm mobility index (SMI) was determined at 58 and 60 wk of age from all males producing semen. Whereas SMI and sperm hole penetration measurements indicated that the sperm quality from treatments POS and NEG were similar, fertility was significantly greater in the POS treatment during the first (89.0% vs. 79.0%) and second WPI (59.3% vs. 45.0%). However, because of numerically higher hatch of fertile from the NEG group, the percentage of hatch of eggs set was similar between groups. These data suggest that ALV-J status of caged males has no influence on sperm quality or hatchability of eggs.     

Characterization of Pseudomonas aeruginosa isolates associated with mortality in broiler chicks

In mid-2000, a broiler chicken company in Alabama experienced high early mortality rates in chicks from two different hatcheries. Five isolates of Pseudomonas aeruginosa, obtained from these contaminated hatcheries and resulting broiler chicks with omphalitis, were selected to determine virulence of the bacteria. One-day-old specific-pathogen-free white leghorn chicks were placed into positive pressure isolation units (10 chicks per unit); feed and water were provided ad libitum. The five isolates of P. aeruginosa (1 x 10(1) or 1 x 10(1) colony-forming units/bird) were used to challenge two replicates of 10 chicks via yolk sac inoculation. Two control groups were injected with 0.1 ml of phosphate-buffered saline, and two groups received no treatment. Mortality was recorded daily, and the chicks that died were necropsied and liver and yolk sacs were cultured. After 14 days, the remaining chickens were euthanatized and necropsied. Bacterial isolates retrieved from liver and yolk sacs were identified by the API 20 NE typing system to confirm that they were the same as the challenge isolate. Virulence varied greatly among the isolates, resulting in mortality rates from 0 to 90%. The challenge isolates produced different and often distinctive postmortem lesion patterns. Antibiotic sensitivity tests showed that all five isolates were resistant to sulfisoxazole, ceftiofur, penicillin, lincomycin, bacitracin, oxytetracycline, erythromycin, naladixic acid, and tetracycline. The isolates varied in sensitivity to other antibiotics, but all isolates were sensitive to gentamicin.     

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.     

Empirical predictions of plant material C and N mineralization patterns from near infrared spectroscopy, stepwise chemical digestion and C/N ratios

Prediction of carbon (C) and nitrogen (N) mineralization patterns of plant litter is desirable for both agronomic and environmental reasons. Near infrared reflectance (NIR) spectroscopy has recently been introduced in decomposition studies to characterize biochemical composition. The purpose of the current study was to use empirical techniques to predict C and N mineralization patterns of a wide range of plant materials incubated under controlled temperature and moisture conditions. We hypothesized that the richness of information in the NIR spectra would considerably improve predictions compared to traditional stepwise chemical digestion (SCD) or C/N ratios. Initially, we fitted a number of empirical functions to the observed C and N mineralization patterns. The best functions fitted with R²=0.990 and 0.949 to C and N, respectively. The fractions of C and N mineralized at different points in time were then either predicted directly with regression functions or indirectly by prediction of the parameters of the empirical functions fitted to incubation data. In both cases, partial least squares (PLS) regressions were used and predictions were validated by cross-validations. We found that the NIR spectra (best R²=0.925) were able to predict C mineralization patterns marginally better than the SCD fractions (best R²=0.911), but considerably better than the C/N ratios (best R²=0.851). In contrast, N mineralization was better predicted by SCD fractions (best R²=0.533) than the C/N ratio (best R²=0.497), which was better than NIR predictions (best R²=0.446). Although the predictions with the NIR spectra were only slightly better for C and worse for N mineralization compared to SCD fractions, NIR spectroscopy still holds advantages, as it is a much less laborious and cheaper analytical method. Furthermore, exploration of the applications of NIR spectroscopy in decomposition studies has only just begun, and offers new ways to gain insights into the decomposition process.