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.     

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.     

Fertilizing Capacity of Frozen Epididymal Sperm Collected from Dogs

The collection of epididymal sperm may be a valuable tool for canine reproduction especially since it can enable collection of cells after death of a valuable dog. The aim of the present study was to evaluate the viability of epididymal sperm after freeze-thawing. Epididymides were obtained from four adult dogs by elective orchiectomy. The caudal portion of the epididymides and part of the deferential ducts were squeezed by means of an anatomic clamp into a Petri dish containing either 0.9% saline solution (Group 1) or Ringer solution without lactate (Group 2). Samples were centrifuged at 800 ×  g for 10 min, the supernatant was removed and the pellet was diluted in one step with a Tris/citric acid/OEP (Orvus Es Paste) extender containing 7% glycerol and subjected to semen freezing. Oocytes were obtained from canine ovaries, after ovariohysterectomy. Only oocytes that were approximately 100 μm in diameter, with a dark ooplasm surrounded by three- or four-well formed cumulus cell layers were used for sperm testing. Frozen semen samples were thawed in a water bath at 70°C for 8 s and analysed at room temperature for sperm motility and velocity. Oocytes were incubated with spermatozoa in humidified atmosphere containing 5% CO₂ at 38°C for 18 h. Morphological and functional characteristics of spermatozoa were similar in both groups. However, the percentage of sperm cells bound to oocytes was significantly higher in Group 2 than in Group 1. This result suggests that the Ringer solution without lactate was a more suitable medium for collecting epididymal canine sperm than 0.9% saline.     

Roles of bootstrap resampling,measure of central tendency,and method for calculating thermal units when developing phenology models for pest management

The successful development of phenology models from field studies depends on many factors, some of which are entirely under the control of pest managers. For example, one such factor is the choice of method for calculating thermal units. In this study, we have demonstrated that four methods for calculating thermal units provided for acceptable predictions of one phenological event of one insect species, while another method for calculating thermal units did not. The measure of central tendency (mean or median) that is used to estimate lower developmental temperatures and required thermal summations is another factor that pest managers can control when developing phenology models from field studies. Here, we show that predictions that were made when using phenology models based on median lower developmental temperatures and median required thermal summations were superior to predictions that were made when using phenology models based on mean lower developmental temperatures and mean required thermal summations. The use of bootstrap vs. non-bootstrap estimates of lower developmental temperatures and required thermal summations is yet another factor that pest managers can control when developing phenology models from field studies. In this study, we found that calculating and using bootstrap estimates of lower developmental temperatures and required thermal summations in phenology models did not improve the predictions of one phenological event for one insect species. The implications of these and other findings are discussed.     

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     

Assessing obesity in adult dogs and cats presenting for routine vaccination appointments in the North Island of New Zealand using electronic medical records data

Aims: To assess the prevalence of obesity in adult dogs and cats presented to first-opinion veterinary clinics in the North Island of New Zealand for routine vaccination appointments, using electronic medical records.

Methods: Ten first-opinion veterinary clinics across the North Island of New Zealand provided electronic medical records for all routine vaccination appointments for adult (>1 year old) dogs and cats between 1 January 2011 and 30 June 2016. Animals with a body condition score (BCS) of 6 or 7 on a 9-point scale and 4 on a 5-point scale were classified as overweight; those with a BCS of 8 or 9 on a 9-point scale and 5 on a 5-point scale were classified as obese. A total of 106,144 records were available over the study period, of which 48,041 (45.2%) had both a recorded weight and BCS.

Results: Of the 24,247 records for dogs with both BCS and weight, 6,324 (26.1%) were classified as overweight, and 551 (2.3%) as obese. The prevalence of dogs classified as overweight or obese was highest in dogs aged between 5–13 years. The odds of desexed dogs being classified as overweight or obese was greater than the odds for intact dogs (OR=1.42 (95% CI=1.29–1.57), p<0.001) adjusting for the effects of age. Of the 23,794 records for cats with a recorded weight and BCS, 5,222 (21.9%) were classified as overweight, and 622 (2.6%) as obese. The prevalence of cats classified as overweight or obese was highest in cats aged between 5–11 years. The odds of desexed cats being classified as overweight or obese tended to be greater than the odds for intact cats (OR=1.14 (95% CI=0.98–1.31); p=0.075), adjusting for the effects of age.

Conclusions: Although there are limitations with using electronic medical records to estimate the prevalence of obesity in companion animal populations, the results highlight that a significant number of animals presenting for routine vaccination appointments were classified as overweight or obese.

Clinical Relevance: It is important for veterinarians to record both patient body condition and weight during routine preventative care appointments to allow accurate ongoing monitoring of trends in obesity at both the patient and population levels.

Abbreviations: BCS: Body condition score     

Induction of anatomically based defense responses in stems of diverse conifers by methyl jasmonate: a phylogenetic perspective

Conifers have evolved constitutive and inducible defense mechanisms to help in both wound healing and defense against attack by bark beetles and other organisms. These defenses include oleoresin, phenolics, and static structures in secondary phloem, such as lignified cells and calcium oxalate crystals, that create physical barriers. We used a phylogenetic approach to investigate the defense anatomy of conifer stems of 13 species from five families following treatment with methyl jasmonate (MJ), a compound that induces defense responses in stems of several Pinaceae species. Methyl jasmonate induced a response similar to wounding except that the response was not accompanied by lesion formation, necrosis or a hypersensitive response. In the Pinaceae species studied, MJ induced polyphenolic parenchyma (PP) cell activation and xylem traumatic resin duct (TD) formation. Members of the Taxodiaceae, which are not known to produce large quantities of resin, showed massive xylem TD formation and surface resinosis following MJ treatment. Treatment with MJ caused members of the Araucariaceae and Cupressaceae to form axial phloem resin ducts but not xylem ducts, whereas Podocarpaceae species showed no induction of resin-producing structures. All species treated with MJ showed phenolic deposition in PP cells, and early lignification of phloem fibers was observed in most of the non-Pinaceae species. We conclude that, although evolution of resin-producing structures occurred independently in conifer lineages, MJ seems to induce resin production regardless of tissue location, as well as inducing deposition of phenolic compounds. Co-evolution of conifer defensive strategies and bark beetle pests is discussed.