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.     

Inflammation and wound healing in cats with chronic gingivitis/stomatitis after extraction of all premolars and molars were not affected by feeding of two diets with different omega‐6/omega‐3 polyunsaturated fatty acid ratios

Feline chronic gingivitis/stomatitis (FCGS) is a painful inflammatory disease in cats. Extraction of teeth, including all premolars and molars, has been shown to be the therapy of choice in cats not responding sufficiently to home care (e.g. tooth brushing) and/or medical treatment (corticosteroids and/or antibiotics). In this study, we hypothesize that a cat food with an omega‐6 polyunsaturated fatty acid (ω6 PUFA) to ω3 PUFA ratio of 10:1 reduces inflammation of the FCGS and accelerates soft tissue wound healing of the gingiva after dental extractions, compared to a cat food with a ω6:ω3 PUFA ratio of 40:1. The cats were fed diets with chicken fat and fish oil as sources of fatty acids. In one diet, part of the fish oil was replaced by safflower oil, resulting in two diets with ω6:ω3 PUFA ratios of 10:1 and 40:1. This double‐blinded study in two groups of seven cats revealed that dietary fatty acids influence the composition of plasma cholesteryl esters and plasma levels of inflammatory cytokines. The diet with the 10:1 ratio lowered PGD₂, PGE₂ and LTB₄ plasma levels significantly, compared to the diet with the 40:1 ratio (p = 0.05, p = 0.04, and p = 0.02 respectively). However, feeding diets with dietary ω6:ω3 PUFA ratios of 10:1 and 40:1, given to cats with FCGS for 4 weeks after extraction of all premolars and molars, did not alter the degree of inflammation or wound healing.     

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.     

Phosphorus Redistribution in Maize Plants, Zea mays L., in Relation to the Nitrogen Supply

The influence of different nutrition regimes on phosphorus (P) redistribution in maize plants, cv. LG 11, was studied. Nutrient solutions in four combinations of high and low nitrate concentrations and high and low phosphate concentrations were used to grow four different groups of plants. During the development of the plants dry weights and total P contents were measured. It was concluded that P redistribution into reproductive organs was dependent on N supply and was a consequence of N redistribution. The ratio of N and P concentrations was not equal in several parts of the plant, and was not constant during the development. In different parts of a young plant the ratio increased. As soon as redistribution of N and P started the N/P ratio decreased. The minimum N/P ratio of a mature maize plant was about 3 for leaves and 1 or 2 for roots and stems.     

Structure–activity relationships of eugenol derivatives against Aedes aegypti (Diptera: Culicidae) larvae

BACKGROUND: Dengue fever is a severe public health problem for several countries. In order to find effective larvicides to aid control programs, the structure‐activity relationships of eugenol derivatives against Aedes aegypti (Diptera: Culicidae) larvae were evaluated. Additionally, the composition and larvicidal activity of Syzygium aromaticum essential oil was assessed. RESULTS: Four compounds representing 99.05% of S. aromaticum essential oil have been identified. The essential oil was active against Ae. aegypti larvae (LC₅₀ = 62.3 and 77.0 ppm, field‐collected and Rockefeller larvae respectively). The larvicidal activity of eugenol, the major compound of the essential oil, was further evaluated (LC₅₀ = 93.3 and 71.9 ppm, field‐collected and Rockefeller larvae respectively). The larvicidal activity and structure‐activity relationships of synthetic derivatives of eugenol were also assessed. The larvicidal activity of the derivatives varied between 62.3 and 1614.9 ppm. Oxidation of eugenol allylic bond to a primary alcohol and removal of the phenolic proton resulted in decreased potency. However, oxidation of the same double bond in 1‐benzoate‐2‐methoxy‐4‐(2‐propen‐1‐yl)‐phenol resulted in increased potency. CONCLUSION: Structural characteristics were identified that may contribute to the understanding of the larvicidal activity of phenylpropanoids. The present approach may help future work in the search for larvicidal compounds. Copyright © 2012 Society of Chemical Industry     

In vitro and in vivo bioactivity of recombinant canine hepatocyte growth factor

Hepatocyte growth factor (HGF) is crucial for the development and regeneration of the liver and offers a possible new therapeutic strategy for the treatment of canine liver disease. In this study, the in vitro and in vivo bioactivity of recombinant canine HGF (rcHGF) produced with a baculoviral expression system in insect cells was measured. In vitro rcHGF induced mitogenesis, motogenesis, and phosphorylated the HGF receptor c-MET and its downstream mediators PKB and ERK1/2 in two canine epithelial cell lines. After a partial hepatectomy (phx) in dogs, rcHGF increased phosphorylation of c-MET, PKB and ERK1/2. A moderate increase was seen with the cell cycle protein PCNA in rcHGF treated livers, but no HGF-induced increase in liver weight was seen 7 days after phx. Furthermore, rcHGF treated livers showed lower levels of the key mediator of apoptosis, caspase-3, at 7days after phx. It is concluded that rcHGF is a biologically active protein in vitro and in vivo and the baculoviral expression system supplies sufficient amounts of rcHGF for future clinical studies in dogs.